TW200535246A - Composite organic-inorganic nanoclusters - Google Patents

Abstract

Composite organic-inorganic nanoclusters (COINs) are provided that produce surface-enhanced Raman signals (SERS) when excited by a laser. The nanoclusters include metal particles and a Raman-active organic compound. The metal required for achieving a suitable SERS signal is inherent in the nanocluster and a wide variety of Raman-active organic compounds and combinations thereof can be incorporated into the nanocluster. In addition, polymeric microspheres containing the nanoclusters and methods of making them are also provided. The nanoclusters and microspheres can be used, for example, in assays for multiplex detection of biological molecules.

Description

200535246 (1) 九、發明說明 【發明所屬之技術領域】 本發明大體上有關包含金屬粒子及有機化合物之奈米 團簇，且有關該等奈米團簇於藉表面增強拉曼光譜偵測分 析物時的應用。 【先前技術】 多重反應係爲自然存在於物理及生物界之並行過程。 當此原理應用來增加生化或臨床分析之效率時，基本挑戰 是發展具有在大量分析物中區分個別分析物之組份的探針 識別系統。高密度DNA晶片及微陣列係爲探針識別系統 ，其中使用位於固體表面上之物理位置來識別核酸或蛋白 質探針。 此外，偵測且識別微量分析物之能力在實質每個科學 範圍內皆變得愈來愈重要，由表面下之水的每十億分之份 數污染物至分析血淸中癌症治療藥物。拉曼光譜是一種提 供豐虽之光學-光譜資料的分析技術，而已證明表面增強 拉曼光譜（SERS )係爲進行定量及定性分析之最靈敏方法 中之一。與紅外線光譜相同地，拉曼光譜係由對應於針對 待分析試樣（分析物）之分子振動的譜帶波長分布所組成 。進行拉曼光譜分析時，來自光源（通常爲雷射）之光束 係聚焦於試樣上，以生成非彈性散射之輻射，其經光學收 集並導入波長分配光譜儀內，偵測器在此將撞擊光子之能 量轉換成電信號強度。 -5- 200535246 (2) 在可用於化學結構分析之許多分析技術中，拉曼光譜 分析引人之處在於其自極小之光學聚焦區域或偵測孔穴提 供豐富之結構資料的能力。與一般具有數十奈米至數百奈 米之半峰寬的單一波峰之螢光光譜比較之下，拉曼光譜具 有多重半峰寬小達數奈米之接合結構相關波峰。此外，表 面增強拉曼散射（SERS )技術使得可得到106至1〇14倍 拉曼信號增強。該巨幅增強倍數主要因爲硬幣形金屬之曲 面上增強的電磁場。雖然電磁增強（ΕΜΕ )顯然與在使用 個別金屬彩體時之金屬表面糙度或粒徑有關，但自聚集膠 體偵測SERS最有效。已知化學增強亦可藉著將分子在特 定取向下放置成緊鄰該表面而得到。 藉SERS對許多化學物質及生物化學物質進行之分析 已使用下列者證明：（1 )電解電池中之活性電極；（2 ) 活性銀及金膠體試劑；及（3 )活性銀及金基材。然而， 前述技術皆無法提供定量測量。結果，SERS無法廣泛使 用。此外，許多生物分子諸如蛋白質及核酸不具有獨特之 拉曼特徵，因爲此等類型之分子通常包含有限數量之共同 單體。 S ERS效應主要係來自電磁場增強及化學增強。經記 載在50至1 〇〇奈米範圍內之銀粒徑對SERS最有效。理論 及實驗硏究亦顯示金屬粒子接合點係爲有效S ER S之部位 【發明內容】 -6 - 200535246 (3) 本發明實施例提供複合型有機-無機奈米團簇（COIN )，其包含數種熔合或聚集之金屬粒子，該粒子形成含有 吸附於聚集粒子表面及初級金屬粒子相遇之接合點中的拉 曼活性有機化合物的金屬團簇。通常，C Ο IN可自各式各 樣之有機化合物合成，以產生增強之拉曼信號及可識別之 拉曼特徵。因爲可合成各式各樣之COIN，故在本發明實 施例中，COIN可作爲生物分析物之多重偵測的編碼系統 〇 通常，本發明粒子之尺寸係小於約1微米，且係於有 機化合物存在下藉著粒子生長而形成。該奈米團簇之製備 利用金屬吸附有機化合物之能力。實際上，因爲拉曼活性 有機化合物係於金屬膠體形成期間吸附於金屬粒子上，故 可有許多拉曼活性有機化合物摻入奈米粒子中，而不需要 特定之附著化學。 一般SERS測量中，分析物係藉著沈積於金屬原子上 或共同聚集金屬原子或膠體與分析物而偵測。如圖1 A所 示，標準 SERS可作爲放大步驟，以藉著將標的分析物 ”A”及”B”吸附於銀粒子上而根據其拉曼特徵來偵測標的分 子” A”及”B”。圖1C之光譜顯示在藉鹽誘發膠體聚集後所 得之SERS信號至少較不添加鹽者強1 0倍，其中難以偵測 之信號係來自標記誘發之膠體聚集。 已發現有機化合物可吸附於金屬膠體上，導致金屬膠 體聚集。此外，已發現所聚集之金屬膠體於高溫下熔合。 有機拉曼標記可摻入聚結金屬粒子內，以產生具有特性 -7- 200535246 (4) SERS活性之複合型有機·無機奈米團簇（COIN )。例如 吾人合成約1 2奈米直徑之銀種晶晶膠體，且混合該銀 體與有機拉曼標記（例如20 // Μ 8-氮雜-腺嘌呤），之 藉著於還原劑存在下加熱該溶液而自AgN03生成附加之 屬銀。溶液顏色自黃變橘，然後變棕，最後變藍。顏色 變係藉吸光度測量來定量（圖2 A )。主要吸收峰於前 分鐘自3 9 5奈米向紅色移動，之後保持約4 2 0奈米。同 ，在500奈米出現小型肩峰（圖2B)。之後，在較高 長（即 700奈米）之吸光度增加至62.5分鐘時間點。 12.5分鐘時間週期間，SERS活性達到最大値（圖2B ) 因爲SERS活性在完成主峰轉變之後且在開始銀聚集體 降之前（在 700奈米波峰降低之前）達最高値，推 SERS活性COIN形成具有兩個時期：粒子增大（熔合 期及後續之粒子叢集期。該兩期過程皆得到電子顯微鏡 究之支持。當銀種晶晶懸浮液在有機拉曼標記不存在下 熱至l〇(TC歷經40分鐘時，該溶液保持淡橘色，而大部 銀粒子保持小於1 〇奈米。拉曼標記添加於銀種晶晶溶 ，溶液加熱以產生橘色。此時，S ERS活性無法偵測， 部分小型銀膠體皆轉變成大於1 〇奈米之相對大型者。 期加熱之後，產生帶棕顏色，其伴隨有強拉曼活性。此 例中，在此階段中，出現包含二或多個初級粒子之粒子 簇。掃描式電子顯微鏡（SEM )分析顯示此實例中 S E R S活性粒子係爲約1 〇 〇奈米之聚集體，包含約2 0至 奈米之初級粒子。 膠 後 金 改 50 時 波 在 〇 沉 論 ) 硏 加 分 液 大 長 實 團 之 -8- 30 200535246 (5) COIN產生特性SERS信號。吾人比較COIN之SERS-活性，數據來自存有各種試劑之典型SERS反應（圖3A 至3D)。典型SERS反應需要添加鹽以誘發強SERS活性 之奈米粒子聚集。圖3A出示拉曼標記（8-氮雜-腺嘌呤） 與銀膠體及單價鹽（+LiCl )混合時之典型拉曼光譜。當 反應省略該鹽（-LiCl )時，SERS信號無法偵測。相反地 ，在未添加鹽之COIN試樣測得強拉曼信號（圖3B )，當 包含鹽時，拉曼信號大幅降低，可能因爲COIN粒子之聚 集及沉降增加。與典型SERS光譜比較下，位在1 100厘 米^及1570厘米μ處之波峰幾乎完全自COIN光譜消失。 其他進行測試之拉曼標記（參照圖8中之實施例）亦發現 光譜差異。例如，COIN粒子對於試驗拉曼標記（1 0 // Μ Ν-苄醯基腺嘌呤，參照圖9Α至Β )具有可忽略之拉曼增 強活性。亦發現SERS信號被0.3 %牛血淸蛋白（BSA )完 全抑制。相反地，COIN之信號在存有添加之BSA時並未 大幅改變，而與鹽存在或不存在無關。Twe en-20®，一種 一般用於生化反應之非離子性界面活性劑，顯然抑制由鹽 誘發之聚集，但導致低度之膠體聚集，如個別實驗所示。 有趣地是發現在存在3 0%乙醇（加鹽）下之sers反應使位 於1550厘米-1處之波峰高度較無乙醇反應增加（圖9G) 。另一方面，COIN信號之光譜及相對波峰強度等於在水 中之COIN (圖3D及圖9H)。此等功能性分析淸楚顯示 COIN具有不同於典型SErs反應所用之以鹽誘發膠體聚 集的化學及物理性質。 -9- 200535246 (6) 附加實施例中，提供製造複合型有機-無機奈米團簇 之方法。該方法可例如藉著於拉曼活性有機化合物存在下 於適於形成金屬膠體之條件下還原金屬離子而進行，以製 得數個熔合或聚集金屬粒子之團簇，拉曼活性有機化合物 係吸附於該金屬粒子上及/或該金屬粒子之接合點中。 本發明奈米團簇可藉著稱爲有機化合物輔助金屬熔合 (OCAMF )之物理化學方法製備。有機化合物可吸附於金 屬膠體上，藉著改變該粒子之表面ζ電位而導致聚集（圖 7Α至Β )，發現所聚集之金屬膠體於高溫下熔合。有機拉 曼標記可摻入該聚結金屬粒子內，以形成安定之團簇，且 產生特性增強之拉曼散射信號。該有機拉曼標記分子與該 金屬膠體之間的相互作用具有多重優點。除了作爲信號來 源之外，有機分子促進且安定化金屬粒子結合，其係有利 於SERS之ΕΜΕ。另一方面，該金屬粒子提供空間來容納 並安定化拉曼標記分子，尤其是團簇接合點中者。此等複 合型有機-無機奈米團簇（COIN )可作爲分子探針之指示 器（reporter )。此觀念係說明於圖1B中，其中可自化合 物” A”及” B”製得2種類型之COIN，之後使用特定親和性 探針加以功能化，以經由該探針連接於所硏究分析物來偵 測分析物”C”及”D”。 使用以OCAMF爲主之COIN合成化學，可藉著混合 有限量之拉曼標記來產生許多不同之C Ο IN特徵。因此， COIN適用於多重檢測。在簡化情況下，標有COIN之試 樣的拉曼光譜可由三個參數來定出特性： -10- 200535246 (7) (a )波峰位置（稱爲L )，其視所使用之拉曼標記 的化學結構及有效標記之數量而定， (b )波峰數量（稱爲Μ )，其視在單一 COIN中一 起使用之標記的數目而定，及 (c )波峰高度（稱爲i )，其視相對波峰強度之範圍 而定。 可能之拉曼特徵之總數目（稱爲T)可自下式計算： {L—ky.k\ 其中P(i,k)=ik-i+l，係爲強度乘數，表示可藉著針對特 定i値結合k ( k == 1至Μ )種標記所生成之不同拉曼光譜 的數目。爲說明可混合多種標記以製得C Ο IN，吾人測試 用於COIN合成之3種拉曼標記組合物（L = 3，M = 3，且 i = 2 )。如圖4所示（亦參照圖1 1 ) ，：I種標記、2種標記 及3種標記之結果皆如同預測。此等光譜特徵證明位置接 近之波峰（介於AA及AN之間1 0厘米·1 )可目測離析。 理論上，藉著使用以COAMF爲主之COIN合成化學，將 多種有機分子摻入COIN中以作爲拉曼標記，可在500至 2000厘米d之拉曼位移範圍內製得超過百萬之COIN特徵 〇 本發明所使用之術語有機化合物係表示含有至少一個 方族環及至少一個氮原子之任何烴分子。有機化合物亦可 含有原子諸如0、S、P及其類者。本發明所使用之拉曼活 性有機化合物係表示因應雷射激發產生獨特之s E R S特徵 200535246 (8) 的有機分子。各種有機化合物（拉曼活性及非拉曼活性兩 者）皆可作爲奈米團簇中之組份。特定實施例中，拉曼活 性有機化合物係爲多環芳族或雜芳族化合物。一般拉曼活 性化合物具有低於約5 0 0道耳呑之分子量。 數個非限制實例中，各種有機拉曼標記（如表i所示 )使用於COIN合成。所測試之化合物可分成數類：（a) 無色且非螢光（例如8 -氮雜-腺嘌呤），（b )彩色染料（ 例如亞甲基藍），（c )螢光染料（例如9 -胺基吖啶）， 及（d )硫醇化合物（例如6 -氫硫基嘌呤）。所有化合物 在低於1 m Μ下皆可溶於水溶液中。應注意來自c Ο IN之 拉受位移峰並非必然符合S E R S者。試驗時，超過4 0種有 機化合物在摻入COIN中時顯示正信號（表1及圖8 )， 其中螢光染料產生最強之COIN信號。200535246 (1) IX. Description of the invention [Technical field to which the invention belongs] The present invention relates generally to nanoclusters containing metal particles and organic compounds, and these nanoclusters are detected and analyzed by surface enhanced Raman spectroscopy. Application. [Previous technology] Multiple reactions are parallel processes that naturally exist in the physical and biological worlds. When this principle is applied to increase the efficiency of a biochemical or clinical analysis, the basic challenge is to develop a probe recognition system with components that distinguish individual analytes across a large number of analytes. High-density DNA wafers and microarrays are probe identification systems in which physical locations on a solid surface are used to identify nucleic acid or protein probes. In addition, the ability to detect and identify micro-analytes has become increasingly important in virtually every scientific context, from contaminants per billion parts of water beneath the surface to the analysis of cancer treatments in blood pupae. Raman spectroscopy is an analytical technique that provides abundant optical-spectral data. Surface-enhanced Raman spectroscopy (SERS) has proven to be one of the most sensitive methods for quantitative and qualitative analysis. Like infrared spectroscopy, Raman spectroscopy consists of a band wavelength distribution corresponding to the molecular vibrations of a sample (analyte) to be analyzed. When performing Raman spectroscopy, a light beam from a light source (usually a laser) is focused on the sample to generate inelastically scattered radiation, which is optically collected and introduced into a wavelength distribution spectrometer where the detector will strike Photon energy is converted into electrical signal strength. -5- 200535246 (2) Among the many analytical techniques that can be used for chemical structure analysis, Raman spectroscopy is attractive because of its ability to provide a wealth of structural information from its extremely small optically focused area or detection holes. Compared with the fluorescence spectrum of a single peak, which generally has a half-peak width of tens of nanometers to hundreds of nanometers, Raman spectra have multiple structure-related peaks with multiple half-widths as small as several nanometers. In addition, Surface Enhanced Raman Scattering (SERS) technology enables 106 to 1014 times Raman signal enhancement. This huge enhancement factor is mainly due to the enhanced electromagnetic field on the curved surface of the coin-shaped metal. Although electromagnetic enhancement (ΕΜΕ) is obviously related to the metal surface roughness or particle size when using individual metal chromosomes, self-aggregating colloids are most effective for detecting SERS. It is known that chemical enhancement can also be obtained by placing molecules in a specific orientation next to the surface. The analysis of many chemical and biochemical substances by SERS has been proven using: (1) active electrodes in electrolytic cells; (2) active silver and gold colloid reagents; and (3) active silver and gold substrates. However, none of the aforementioned techniques can provide quantitative measurement. As a result, SERS cannot be widely used. In addition, many biological molecules such as proteins and nucleic acids do not have unique Raman characteristics, because these types of molecules often contain a limited number of common monomers. The S ERS effect mainly comes from electromagnetic field enhancement and chemical enhancement. Silver particle diameters recorded in the range of 50 to 1000 nanometers are most effective for SERS. Theoretical and experimental investigations have also shown that the junction of metal particles is a site of effective S ER S [Content of the Invention] -6-200535246 (3) The embodiment of the present invention provides a composite organic-inorganic nano-cluster (COIN), which includes Several kinds of fused or agglomerated metal particles that form metal clusters containing Raman-active organic compounds adsorbed on the surface of the agglomerated particles and the junction where the primary metal particles meet. Generally, CO IN can be synthesized from a wide variety of organic compounds to produce enhanced Raman signals and identifiable Raman characteristics. Because a variety of COINs can be synthesized, in the embodiments of the present invention, COIN can be used as a coding system for multiple detection of biological analytes. Generally, the size of the particles of the present invention is less than about 1 micron, and is based on organic compounds. Formed by particle growth in the presence. The preparation of the nano-cluster utilizes the ability of metals to adsorb organic compounds. In fact, because Raman-active organic compounds are adsorbed on metal particles during the formation of metal colloids, many Raman-active organic compounds can be incorporated into nano-particles without the need for specific adhesion chemistry. In general SERS measurements, analytes are detected by depositing on metal atoms or co-aggregating metal atoms or colloids and analytes. As shown in Figure 1A, standard SERS can be used as an amplification step to detect the target molecules "A" and "B by adsorbing the target analytes" A "and" B "on the silver particles based on their Raman characteristics. ". The spectrum in FIG. 1C shows that the SERS signal obtained after salt-induced colloidal aggregation is at least 10 times stronger than that without salt addition, and the signal that is difficult to detect is from label-induced colloidal aggregation. It has been found that organic compounds can be adsorbed on metal colloids, causing the metal colloids to aggregate. In addition, the aggregated metal colloids have been found to fuse at high temperatures. Organic Raman tags can be incorporated into agglomerated metal particles to produce composite organic-inorganic nanoclusters (COIN) with characteristics of -7- 200535246 (4) SERS activity. For example, we synthesized a silver seed crystal colloid with a diameter of about 12 nanometers, and mixed the silver body with an organic Raman label (for example, 20 // M 8-aza-adenine), and heated by the presence of a reducing agent. This solution produces additional silver from AgN03. The color of the solution changed from yellow to orange, then brown, and finally blue. The color change is quantified by absorbance measurement (Figure 2A). The main absorption peak shifted from 395 nm to red in the first minute, and then remained at about 420 nm. At the same time, a small shoulder peak appears at 500 nm (Figure 2B). After that, the absorbance at the higher length (700 nm) increased to the 62.5 minute time point. During the 12.5 minute time period, the SERS activity reached the maximum level (Figure 2B) because the SERS activity reached the highest level after completing the main peak transition and before the silver aggregate decline began (before the 700 nanometer wave peak reduction). Two phases: particle growth (fusion phase and subsequent particle clustering phase. Both phases are supported by electron microscopy. When the silver seed crystal suspension is heated to 10 (TC in the absence of organic Raman label After 40 minutes, the solution remained pale orange, while most of the silver particles remained less than 10 nanometers. Raman markers were added to the silver seed crystals to dissolve, and the solution was heated to produce orange. At this time, S ERS activity could not be detected It is found that some small silver colloids are transformed into relatively large ones larger than 10 nanometers. After heating, a brownish color is generated, which is accompanied by strong Raman activity. In this case, at this stage, two or more Clusters of primary particles. Scanning electron microscope (SEM) analysis shows that the SERS active particles in this example are aggregates of about 100 nanometers, including primary particles of about 20 to nanometers. Glue After the gold change at 50, the wave is in the theory of Shen.) 硏 Adding liquid to disperse large Changshi group -8- 30 200535246 (5) COIN produces characteristic SERS signal. I compare the SERS-activity of COIN. The data comes from the typical of various reagents. SERS reaction (Figure 3A to 3D). Typical SERS reaction requires the addition of salt to induce the aggregation of nano-particles with strong SERS activity. Figure 3A shows the Raman label (8-aza-adenine) with silver colloid and monovalent salt (+ LiCl ) Typical Raman spectrum when mixed. When the salt (-LiCl) is omitted in the reaction, the SERS signal cannot be detected. On the contrary, a strong Raman signal is measured in the COIN sample without salt (Figure 3B). When salt is used, the Raman signal is greatly reduced, which may be due to the increase in the aggregation and sedimentation of COIN particles. Compared with the typical SERS spectrum, the peaks at 1 100 cm ^ and 1570 cm μ almost completely disappear from the COIN spectrum. Other tests Raman labeling (see the example in Figure 8) also finds spectral differences. For example, COIN particles have negligible effects on experimental Raman labels (1 0 // MN-benzyl adenine, see Figures 9A through B). Raman enhanced activity. The SERS signal is now completely inhibited by 0.3% bovine blood albumin (BSA). In contrast, the signal of COIN does not change significantly when BSA is added, regardless of the presence or absence of salt. Twe en-20®, a Nonionic surfactants, which are generally used in biochemical reactions, obviously inhibit salt-induced aggregation, but cause low-level colloidal aggregation, as shown in individual experiments. It is interesting to find that in the presence of 30% ethanol (with salt) The sers response increased the peak height at 1550 cm-1 compared to the ethanol-free response (Figure 9G). On the other hand, the spectrum and relative peak intensity of the COIN signal are equal to the COIN in water (Figure 3D and Figure 9H). These functional analyses have shown that COIN has chemical and physical properties that are different from the salt-induced colloidal aggregation used in typical SErs reactions. -9- 200535246 (6) In an additional embodiment, a method for manufacturing a composite organic-inorganic nano-cluster is provided. This method can be performed, for example, by reducing metal ions in the presence of a Raman-active organic compound under conditions suitable for forming a metal colloid to obtain clusters of fused or aggregated metal particles. The Raman-active organic compound is adsorbed On the metal particles and / or in the junctions of the metal particles. The nano-cluster of the present invention can be prepared by a physical-chemical method called organic compound assisted metal fusion (OCAMF). Organic compounds can be adsorbed on metal colloids, and aggregates can be caused by changing the zeta potential of the surface of the particles (Figures 7A to B). It is found that the aggregated metal colloids fuse at high temperatures. Organic Raman labels can be incorporated into the agglomerated metal particles to form stable clusters and generate Raman scattering signals with enhanced characteristics. The interaction between the organic Raman-labeled molecule and the metal colloid has multiple advantages. In addition to serving as a signal source, organic molecules promote and stabilize the binding of metal particles, which is beneficial to SMES. On the other hand, the metal particles provide space to hold and stabilize Raman-labeled molecules, especially those in the cluster junction. These composite organic-inorganic nanoclusters (COIN) can be used as indicator (reporter) of molecular probes. This concept is illustrated in Figure 1B, where two types of COINs can be prepared from compounds "A" and "B", and then functionalized using specific affinity probes to connect to the in-depth analysis via the probe To detect analytes "C" and "D". The use of OCAMF-based COIN synthetic chemistry can produce a number of different CO IN characteristics by mixing limited amounts of Raman labels. Therefore, COIN is suitable for multiplex detection. In the simplified case, the Raman spectrum of the sample marked with COIN can be characterized by three parameters: -10- 200535246 (7) (a) The peak position (referred to as L), which depends on the Raman label used (B) the number of peaks (referred to as M), which depends on the number of markers used together in a single COIN, and (c) the peak height (referred to as i), which It depends on the range of relative peak intensity. The total number of possible Raman features (called T) can be calculated from the following formula: {L—ky.k \ where P (i, k) = ik-i + l, which is an intensity multiplier, which can be expressed by Number of different Raman spectra generated for a particular i 値 combined with k (k == 1 to M) markers. In order to illustrate that multiple labels can be mixed to make C Ο IN, we tested three Raman labeling compositions for COIN synthesis (L = 3, M = 3, and i = 2). As shown in Fig. 4 (also refer to Fig. 1), the results of Type I, Type 2 and Type 3 marks are as predicted. These spectral characteristics prove that near-peaks (between 10 cm · 1 between AA and AN) can be visually isolated. Theoretically, by using COAM synthetic chemistry based on COAMF, a variety of organic molecules can be incorporated into COIN as Raman markers, and COIN characteristics of more than one million can be obtained within a Raman shift range of 500 to 2000 cmd. The term organic compound used in the present invention refers to any hydrocarbon molecule containing at least one square group ring and at least one nitrogen atom. Organic compounds may also contain atoms such as 0, S, P, and the like. The Raman-active organic compound used in the present invention refers to an organic molecule that has a unique s E R S characteristic 200535246 (8) in response to laser excitation. Various organic compounds (both Raman and non-Raman activities) can be used as components in nanoclusters. In a specific embodiment, the Raman-active organic compound is a polycyclic aromatic or heteroaromatic compound. Raman-active compounds generally have a molecular weight of less than about 50 channels. In several non-limiting examples, various organic Raman labels (as shown in Table i) are used for COIN synthesis. The compounds tested can be divided into several categories: (a) colorless and non-fluorescent (such as 8-aza-adenine), (b) color dyes (such as methylene blue), (c) fluorescent dyes (such as 9-amine Acridine), and (d) a thiol compound (eg, 6-hydrothiopurine). All compounds are soluble in aqueous solutions below 1 μM. It should be noted that the tensile displacement peaks from c Ο IN do not necessarily conform to S E R S. During the test, more than 40 organic compounds showed positive signals when they were incorporated into COIN (Table 1 and Figure 8). Among them, the fluorescent dye produced the strongest COIN signal.

表1 編號 縮寫 名稱 結構 1 AAD (AA) 8-氮雜-腺嘌呤 fir^ Η 2 BZA (BA) N-苄醯基腺嘌呤 C^° Η N 3 MBI 2-氫硫基·苯并咪Π坐 rvv- -12- 200535246 Ο) 4 APP 4-胺基-吡唑并〔3,4-d〕嘧啶 nh2 Η 5 ZEN 玉米素（Zeatin) ΝΗ/Χ^==</ c6 Η 6 MBL (MB) 亞甲基藍 7 AMA (AN? AM) 9,胺基-吖啶 ΝΗ2 coo 8 EBR 溴乙錠（Ethidine Bromide ) ό 9 BMB 俾斯麥綜（BismarckBrown) Y 10 NBA N-节基-胺基嘿D令 9 c6 Η Ν 11 THN 乙酸硫堇 H2NCCp〇NHj cHsc〇r 12 DAH 3,6-二胺基吖啶 .ixa.Table 1 Number abbreviation name structure 1 AAD (AA) 8-aza-adenine fir ^ Η 2 BZA (BA) N-benzylidene adenine C ^ ° Η N 3 MBI 2-hydrothio · benzimidyl Rvv- -12- 200535246 〇) 4 APP 4-Amino-pyrazolo [3,4-d] pyrimidine nh2 Η 5 ZEN Zeatin ΝΗ / Χ ^ == < / c6 Η 6 MBL ( MB) Methylene blue 7 AMA (AN? AM) 9, amine-acridine NΗ2 coo 8 EBR Ethidine Bromide ό 9 BMB Bismarck Brown Y 10 NBA N-Benyl-Amino Hey D Order 9 c6 Η Ν 11 THN thionine acetate H2NCCCpOHNHj cHsc〇r 12 DAH 3,6-diaminoacridine.ixa.

-13- 200535246 (10) 13 CYP 6-氰基嘌呤 CN ——Ν Η 14 AIC 4-胺基-5-咪.甲醯胺鹽酸鹽 Jl J -HCI o 15 DII 1，3-二亞胺基異D弓陳啉 NH NH 16 R6G 若丹明6G 入 R6G 17 CRV 結晶紫 9 α· A 18 BFU 鹼性品紅 19 ANB 苯胺藍二銨鹽 ◊ο㈣ XX^^rs -03B -^=/ \=>· 20 ACA N-〔（3-(苯胺基亞甲基）-2-氯-1·環己烯-1-基）亞甲基〕苯 胺單鹽酸鹽 ^ ^-NH =N-^ ^ · HCI 21 ATT 六氟磷酸〇- (7-氮雜苯并三 唑小基）-N，N，N，，N，-四甲基脲 鏺 'n(ch3)2 -14- 200535246 (11) 22 AMF 鹽酸9-胺基荛 of nh2 23 BBL 鹼性藍 y〇oac 24 DDA 1，8-二胺基-4,5-二氫-蒽醌 OH 0 OH W nh2 0 nh2 25 PFV 原黃素半硫酸鹽水合物 .ooou .V2h2so4 26 APT 2-胺基-1，1，3·丙烯三腈 h2n cn ncch3 c=c -cn 27 VRA 變胺藍RT鹽 ^ NH N2+ HS04- 28 TAP 4,5,6-三胺基嘧啶硫酸鹽 m. 1 * 29 ABZ 2-胺基-苯并噻唑 30 MEL 三聚氰胺 H入A% 31 PPN 3- (3-D比D定基甲基胺基）丙睛 ch2nhch2ch2cn 32 SSD 銀（I)磺胺噠畊 33 AFL 吖啶黃 CHj ，cox-13- 200535246 (10) 13 CYP 6-cyanopurine CN ——N Η 14 AIC 4-amino-5-imide. Formamidine hydrochloride Jl J -HCI o 15 DII 1,3-diimine Base isoD-bendroline NH NH 16 R6G rhodamine 6G into R6G 17 CRV crystal violet 9 α · A 18 BFU basic fuchsin 19 ANB aniline blue diammonium salt ◊ο㈣ XX ^^ rs -03B-^ = / \ = &·; 20 ACA N-[(3- (aniline methylene) -2-chloro-1 · cyclohexene-1-yl) methylene] aniline monohydrochloride ^ -NH = N- ^ HCI 21 ATT Hexafluorophosphate 0- (7-azabenzotriazole small group) -N, N, N ,, N, -tetramethylurea 鏺 'n (ch3) 2 -14- 200535246 ( 11) 22 AMF 9-Amino hydrochloride of nh2 23 BBL Basic blue yooac 24 DDA 1,8-diamino-4,5-dihydro-anthraquinone OH 0 OH W nh2 0 nh2 25 PFV original yellow Hemisulfate hydrate.ooou .V2h2so4 26 APT 2-amino-1,1,3 · acrylic trinitrile h2n cn ncch3 c = c -cn 27 VRA amine blue RT salt ^ NH N2 + HS04- 28 TAP 4, 5,6-triaminopyrimidine sulfate m. 1 * 29 ABZ 2-amino-benzothiazole 30 MEL melamine H into A% 31 PPN 3- (3-D to D-Aminomethylamino) acryl ch2nhch2ch2cn 32 SSD Silver (I) Aminopyridazin plow 33 AFL acridine yellow CHj, cox

-15- 200535246 (12) 34 AMPT 4-胺基-6-氫硫基吡唑并〔3,4-d 〕嘧啶 N一N 〇Υ〇 35 APU 2-Am-嘌Π令 Η 36 ATH 腺嘌呤硫醇 νη2 Η 37 FAD F-腺嘌呤 ΝΗ2 </ΝχΧ Ν入Ν人F Η 38 MCP 6-氫硫基嘌呤 SH 〇6 Η 39 AMP 4-胺基-6-氫硫基吡唑并〔3,4-d 〕嘧啶 νη2 Η 40 R110 若丹明110 dr 41 ADN 腺嘌呤 Μ Ν 42 AMB 5-胺基-2-氫硫基苯并咪唑 j〇rt SH " Η-15- 200535246 (12) 34 AMPT 4-Amino-6-hydrothiopyrazolo [3,4-d] pyrimidine N-N 〇〇〇35 35 APU 2-Am-purine Η Η 36 ATH adenine Thiol νη2 Η 37 FAD F-Adenine NΗ2 < / Nχχ ΝΝΝFF 38 MCP 6-Hydroxythiopurine SH 〇6 Η 39 AMP 4-Amino-6-hydrothiopyrazolo [3 , 4-d] pyrimidine νη2 Η 40 R110 rhodamine 110 dr 41 ADN adenine M Ν 42 AMB 5-amino-2-hydrothiobenzimidazole j〇rt SH ";

此外，應明瞭此等拉曼活性化合物可包括螢光化合物 或非螢光化合物。例示拉曼活性有機化合物包括（但不限 於）腺嘌呤、4 -胺基-吡唑并（3，4 - d )嘧啶、2 -氟腺嘌呤 、N6-苄醯基腺嘌呤、激動素、二甲基-烯丙基-胺基-腺嘌 呤、玉米素、溴-腺嘌呤、8 -氮雜-腺嘌呤、8 -氮雜鳥嘌呤 -16- 200535246 (13) 、6 -氫硫基嘿D令、4 -胺基-6 -氫硫基吼Π坐幷（3，4 - d )嚼' 0定、 8-氫硫基腺嘌呤、9-胺基-吖啶及其類者。 拉曼活性有機化合物之其他非限制實例包括trit ( 四甲基若丹明異硫醇）、NBD ( 7-硝基苯并-2 _哼+ 3-二哩 )、德克薩斯（T e X a s )紅染料、苯二甲酸、對苯二甲酸 、異苯二甲酸、甲苯堅牢紫、甲苯藍紫、亮甲苯藍、對-胺基苄酸、赤蘚紅、生物素、地谷新配質、5 -竣基-4，，5，-二氯-2’，7’-二甲氧基螢光素、5-殘基_2’，4，，5，，7，-四氯螢光 素、5-羧基螢光素、5-羧基若丹明、6-羧基若丹明、6_殘 基四甲基胺基酞花青、甲亞胺、花青、黃質、琥珀醯螢光 素、胺基吖啶及其類者。此等及其他拉曼活性有機化合物 可得自市售來源（例如 Molecular Probes，Eugene，OR.) ο 當螢光化合物摻入本發明所述之奈米團簇中時，該等 化合物係包括（但不限於）染料、特性螢光蛋白質、鑭系 磷光質及其類者。染料係包括例如若丹明及衍生物，諸如 德克薩斯紅、ROX ( 6-羧基-X-若丹明）、若丹明-NHS及 TAMRA(5/6-羧基四甲基若丹明NHS);螢光素及衍生物 ，諸如5-溴甲基螢光素及FAM(5，-羧基螢光素NHS)、 螢光黃（Lucifer Yellow ) 、：[ A E D AN S、7 - M e2 , N -香豆素- 4-乙酸酯、7-OH-4-CH3-香豆素-3-乙酸酯、7-NH2-4CH3·香 豆素-3-乙酸酯（AMCA )、單溴白蔓、芘三磺酸酯諸如階 式藍及單溴三甲基-胺基白蔓。 該OCAMF化學使各式各樣之拉曼標記摻入金屬膠體 -17- 200535246 (14) 內，以產生數類COIN。簡易之單階化學方法使其可藉著 混合不同比例之數種有機拉曼活性化合物，進行許多具有 不同拉曼特徵之COIN的並行合成。 用於COIN合成之金屬奈米粒子的尺寸可變化，但係 選擇小於所期望形成之C Ο IN尺寸。就某些應用而言，例 如在爐式及回流合成方法中，平均直徑約3至約1 2奈米 之銀粒子用以形成銀C Ο IN，而約1 3至約1 5奈米之金奈 米粒子用以製得金C Ο IN。另一應用中，金合成方法係使 用例如具有約1 0至約8 0奈米之寬幅尺寸分布的銀粒子。 一般期望用以形成奈米團簇之金屬係包括例如銀、金、鉑 、銅、鋁及其類者。此外，可使用多金屬奈米粒子，諸如 例如具有金核心之銀奈米粒子。 一般，就諸如分析物偵測之應用而言’ COIN平均直 徑範圍係約20奈米至約200奈米，較佳COIN平均直徑 係約3 0至約2 0 0奈米範圍，更佳係約4 0至約2 0 0奈米， 更佳係約50至約200奈米，而約50至約150奈米更佳。 本發明特定實施例中，所使用之金屬粒子係爲金屬膠 體。本發明所使用之術語膠體係表示由懸浮於液體（通常 爲水溶液）中之奈米尺寸粒子所組成之複合型流體類別。 在液體中有機分子存在下金屬膠體形成或生長期間，該有 機分子係吸附於懸浮在液體中之初級金屬粒子上及/或介 於初級金屬粒子間之空隙中。用以自金屬膠體形成奈米團 簇的一般金屬係包括例如銀、金、鉑、銅、鋁及其類者。 本發明方法及組成物中所使用之膠體中的金屬粒子之一般 -18- 200535246 (15) 平均尺寸範圍係約3奈米至約1 5奈米。 通常，COIN可如下製備。製備含有適當之金屬陽離 子、還原劑、及至少一種適當之拉受活性有機化合物之水 溶液。溶液組份隨之接受將金屬陽離子還原以形成中性膠 體金屬粒子之條件。因爲金屬膠體之形成係發生於適當之 拉曼活性有機化合物存在時，故該拉曼活性有機化合物即 於膠體形成期間吸附於該金屬上。此類奈米粒子係爲數個 初級金屬粒子之團簇或聚集體，拉曼活性有機化合物吸附 於該金屬粒子表面上且被捕集於該初級金屬粒子間之接合 點中。COIN通常非球形，經常包括溝紋及突起。COIN可 藉薄膜過濾加以單離，而具有不同尺寸之COIN可藉離心 來增加濃度。 本發明進一步實施例中，奈米團簇係包括第二種異於 第一種金屬之金屬，其中該第二種金屬形成覆蓋該奈米團 簇表面之層。製備此種奈米團簇時，COIN係置於含有適 當之第二種金屬陽離子及還原劑之水溶液中。溶液之組份 隨之接受還原該第二種金屬陽離子的條件，以形成覆蓋該 奈米團簇表面之金屬層。特定實施例中，第二種金屬層係 包括金屬諸如例如銀、金、鉑、鋁、銅、鋅、鐵及其類者 。此類奈米團簇可依如同單一金屬C Ο IN之方式加以單離 及/或增濃。 特定實施例中，覆蓋奈米團簇表面之金屬層係稱爲保 護層。保護層可賦予奈米團簇水性安定性。C Ο IN可塗覆 二氧化矽層’以取代金屬保護層或附加於金屬保護層上。 -19- 200535246 (16) 若C O IN已塗覆金屬層，諸如例如金，則二氧化矽層可藉 C Ο IN與例如3 -胺基丙基三甲氧基砂院（A P T M S )之親玻 璃（vitreophilization)而連接於金層。二氧化矽沈積係自 超飽和二氧化矽溶液起始，之後藉著逐滴添加氨及原矽酸 四乙酯（TEOS )而生長二氧化矽層。該塗覆有二氧化矽 之COIN即可使用標準二氧化矽化學加以功能化。 特定其他實施例中，COIN可包括覆蓋金屬表面或二 氧化矽層之有機層。部分實施例中，此等類型之奈米粒子 係藉著使有機化合物吸附或共價連接於該COIN表面而製 備。有機層共價連接於金屬表面可依各種熟習此技術者熟 知之方式達成，諸如例如經由硫醇-金屬鍵結。備擇實施 例中，有機分子可交聯以形成分子網絡。 有機層亦可用以提供膠體安定性及供進一步衍化使用 之官能基。該有機層係視情況交聯以形成固體塗層。例示 有機層係藉著使經辛基胺修飾之聚丙烯酸吸附於COIN上 而製得，該吸附係借助於帶正電胺基。聚合物之羧基隨後 與適當之試劑諸如離胺酸、（1，6 )-二胺基庚烷及其類者 交聯。未反應之羧基可用於進一步衍化。其他官能基亦可 經由經修飾之聚丙烯酸主鏈來導入。 此外，該金屬及有機塗層可依各種組合重疊，以提供 所需之經塗覆COIN性質。例如，COIN可先塗覆金層， 以在施加吸收層、二氧化矽或固體有機塗層之前先密封較 具反應性之銀。即使外層係多孔性，內部金層仍可防止 COIN被不同應用中所使用之試劑所攻擊。另一實例係施 -20- 200535246 (17) 加吸附層於二氧化矽或金層上，以提供附加之膠體安定性 〇 特定另一實施例中，用於C Ο IN之金屬粒子可包括磁 性材料，諸如例如氧化鐵及其類者。磁性C Ο IN可在不離 心下使用一般有效之磁性粒子操作系統來操作。實際上， 磁性可作爲用以分離標有特定生物探針之COIN粒子的機 制。 另一實施例中，提供偵測試樣中之分析物的方法。該 等方法可例如藉著使含有分析物之試樣與具有附著探針之 COIN接觸而進行，其中該探針係鍵結於該分析物，自任 何未錯合之COIN分離任何COIN-分析物錯合物，並偵測 來自該奈米團簇之SERS信號，其中該SERS信號係指示 分析物之存在。 另一實施例中，提供使用一拉曼活性金屬奈米團簇組 識別試樣中之分析物的方法，該組中之各員係具有在該組 中獨特之拉曼特徵。該等方法可例如藉著使可能含有分析 物之試樣與多個奈米團簇接觸；在該試樣與該奈米團簇接 觸時偵測以多重方式偵測SERS信號·，及結合來自該奈米 團簇之SERS信號與該奈米團簇所連接之分析物本體。 其他實施例中，本發明提供區別試樣中生物分析物之 方法，其係藉著包含多種生物分析物之試樣與一平均直徑 約5 0奈米至約2 0奈米之拉曼活性金屬團簇組（該組中之 各員係具有在該組中獨特之拉曼特徵，此特徵係由摻入其 中之至少一種拉曼活性有機化合物產生）及專一性地鍵結 -21 - 200535246 (18) 於已知之生物分析物之探針接觸，條件係適於使探針專一 性地鍵結於存在試樣中之分析物而形成錯合物。分離經鍵 結之團簇，以多重方式偵測所鍵結錯合物中之有機拉曼活 性化合物所發射的拉曼特徵。各個拉曼特徵皆指示試樣中 已知之生物分析物的存在。 COIN可作爲生物分析偵測之標籤，於一實例中，吾 人使用類似標準夾層免疫檢測之檢測流程（圖5A );不 同處係使用其他標籤之夾層免疫檢測中所必需的在專一性 鍵結之後的信號放大步驟在使用COIN作爲分析物標籤時 並不需要（圖5A)。蛋白質間白素- 2(IL-2)係連接於已 預先塗覆抗-IL-2捕集抗體之表面，使得最大平均IL-2分 子密度低於每雷射光束剖面積1分子（0.77分子/12微米2 ’雷射光束內1.3 yoctomole)，塗覆有抗-1L - 2抗體之 C Ο IN係用以偵測被固定之IL - 2分子。如圖5 B所示，發 現平均有28%光譜具有所需之IL-2特徵，顯示所有所施 加之分析物分子有36%偵測率。此偵測率在實驗條件下僅 可能存在每個數據收集區具有平均10個分析物分子，考 慮夾層檢測中可能不完全之鍵結及可能存在非活性COIN 時。 捕集受質係使用對抗IL-2及IL-8之混合抗體製備。 相同地，使用專一性地鍵結於兩分析物之偵測抗體製備兩 組COIN (個別具有AA及BA特徵）。當使用不同比例之 兩種分析物時，在與基於已知之所使用分析物比例預測之 値完全符合之比例下偵測正C Ο IN信號。 -22- 200535246 (19) 用於偵測分析物時，探針可連接於C ΟIN。特定實施 例中，例示探針係爲抗體、抗原、聚核苷酸、寡聚核苷酸 、受體、配體及其類者。術語聚核苷酸於本發明中係廣用 以表示藉磷酸二酯鍵結鍵合之脫氧核糖核苷酸或核糖核苷 酸之序列。術語寡聚核苷酸於本發明係簡便地用來表示作 爲引子或探針之聚核苷酸。通常，可作爲選擇性地融合於 所選擇之核苷酸序列之探針或引子的寡聚核苷酸係至少約 1 〇個核苷酸長，通常至少約1 5個核苷酸長，例如約1 5及 約5 0個核苷酸之間的長度。聚核苷酸探針特別可用於偵 測生物試樣中之互補聚核苷酸，亦可藉著將已知之聚核苷 酸探針與已知之拉曼活性信號（由本發明所述之拉曼活性 有機化合物的組合物構成）配對，而用於DNA定序。 聚核苷酸可爲RNA或DNA，其可爲基因或其部分、 cDNA、合成聚去氧核糖核酸序列或其類者，且可爲單股 或雙股，及DN A/RNA混雜物。在各種實施例中，聚核苷 酸’包括寡聚核苷酸（例如探針或引子）可含有核苷或核 苷酸類似物，或除磷酸二酯鍵結以外之主鏈鍵結。通常， 包含聚核苷酸之核苷酸係天然去氧核糖核苷酸，諸如連接 於2 ’ -去氧核糖之腺嘌呤、胞嘧啶、鳥嘌呤或胸腺嘧啶， 或核糖核苷酸，諸如連接於核糖之腺嘌呤、胞嘧啶、鳥嘌 哈或尿嘧啶。然而，聚核苷酸或寡聚核苷酸亦可含有核苷 酸類似物’包括非天然之合成核苷菱或經修飾之天然核苷 酸。 連接聚核苷酸之核苷酸的共價鍵結通常係爲磷酸二酯 -23- 200535246 (20) 鍵結。然而，該共價鍵結亦可爲任何數種其他鍵結，包括 硫二酯鍵結、硫代磷酸酯鍵結、胜肽類醯胺鍵結或技術界 已知之任何其他可用於連接核苷酸以製得合成聚核苷酸之 鍵結。非天然核苷酸同類物或連接核苷酸或同類物之鍵結 的倂入特別可用於聚核苷酸將暴露於可含有核分解活性之 環境（包括例如組織培養基或投予活體時）之情況，因爲 經修飾之聚核苷酸較不易降解。 本發明所使用之術語選擇性混雜或選擇性混雜化係表 示在相當嚴苛或高度嚴苛條件下混雜化，使得核苷酸序列 與選擇之核苷酸序列締合之優勢高於不相關之核苷酸序列 ，而達到可確識所選擇之核苷酸序列的程度。應認知會發 生某些非專一性之混雜化，但當標的核苷酸序列之混雜化 係充分選擇性，使得可與非專一***互混雜區分時，則可 接受，例如至少高兩倍之選擇性，通常至少高3倍之選擇 性，通常至少約高5倍之選擇性，尤其是至少高約1 〇倍 之選擇性，例如藉由鍵結於標的核酸分子之經標記寡聚核 苷酸相對於除標的分子以外之核酸分子（尤其是除標的核 酸分子以外之實質相同或類似之核酸分子）的量決定。可 進行選擇性混雜之條件可由實驗決定，或可基於例如混雜 用寡聚核苷酸及其欲混雜之序列之相對GC .· AT含量、混 雜用寡聚核苷酸之長度及寡聚核苷酸與其欲混雜之序列之 間的不配（若存在）數目來估計。In addition, it should be understood that such Raman-active compounds may include fluorescent compounds or non-fluorescent compounds. Exemplary Raman-active organic compounds include, but are not limited to, adenine, 4-amino-pyrazolo (3,4-d) pyrimidine, 2-fluoroadenine, N6-benzylidene adenine, kinetin, diamine Methyl-allyl-amino-adenine, zeatin, bromo-adenine, 8-aza-adenine, 8-azaguanine-16- 200535246 (13), 6-hydrothio Let, 4-amino-6-hydrosulfanyl group (3,4-d) chelate, 0-hydrogen adenine, 9-amino-acridine and the like. Other non-limiting examples of Raman-reactive organic compounds include trit (tetramethylrhodamine isothiol), NBD (7-nitrobenzo-2_hum + 3-two miles), Texas (T e X as) red dye, phthalic acid, terephthalic acid, isophthalic acid, toluene fast violet, toluene blue violet, bright toluene blue, p-aminobenzic acid, erythrosine, biotin, digu new compound Quality, 5-junction-4,5, -dichloro-2 ', 7'-dimethoxyfluorescein, 5-residue_2', 4,5,7, -tetrachlorofluorescein Photoin, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-residue tetramethylaminophthalocyanine, methylimine, anthocyanin, xanthophyll, amber fluorene Photon, aminoacridine and the like. These and other Raman-active organic compounds can be obtained from commercially available sources (eg, Molecular Probes, Eugene, OR.) Ο When fluorescent compounds are incorporated into the nanoclusters described in the present invention, these compounds include ( But not limited to) dyes, characteristic fluorescent proteins, lanthanoid phosphors, and the like. Dyes include, for example, rhodamine and derivatives such as Texas Red, ROX (6-carboxy-X-rhodamine), rhodamine-NHS, and TAMRA (5 / 6-carboxytetramethylrhodamine NHS); Luciferin and derivatives, such as 5-bromomethylfluorescein and FAM (5, -carboxyfluorescein NHS), Lucifer Yellow, [AED AN S, 7-M e2 , N-coumarin-4-acetate, 7-OH-4-CH3-coumarin-3-acetate, 7-NH2-4CH3 · coumarin-3-acetate (AMCA), Monobromo white vines, pyrene trisulfonates such as cascade blue and monobromotrimethyl-amino white vines. This OCAMF chemistry incorporates various Raman labels into metal colloids -17- 200535246 (14) to generate several types of COIN. The simple single-stage chemical method makes it possible to perform parallel synthesis of many COINs with different Raman characteristics by mixing several organic Raman active compounds in different proportions. The size of metallic nano-particles used for COIN synthesis can vary, but is chosen to be smaller than the desired size of CO IN. For some applications, for example, in furnace and reflow synthesis methods, silver particles with an average diameter of about 3 to about 12 nanometers are used to form silver C0 IN, and about 13 to about 15 nanometers of gold Nano particles are used to make gold C 0 IN. In another application, the gold synthesis method uses, for example, silver particles having a wide size distribution of about 10 to about 80 nanometers. Metal systems that are generally desired to form nanoclusters include, for example, silver, gold, platinum, copper, aluminum, and the like. In addition, polymetallic nano particles such as, for example, silver nano particles having a gold core can be used. In general, for applications such as analyte detection, the average COIN diameter ranges from about 20 nanometers to about 200 nanometers, preferably the average COIN diameter ranges from about 30 to about 200 nanometers, and more preferably about 40 to about 200 nanometers, more preferably about 50 to about 200 nanometers, and more preferably about 50 to about 150 nanometers. In a specific embodiment of the present invention, the metal particles used are metal colloids. The term gum system used in the present invention refers to a class of composite fluids composed of nano-sized particles suspended in a liquid (usually an aqueous solution). During the formation or growth of the metal colloid in the presence of organic molecules in the liquid, the organic molecules are adsorbed on the primary metal particles suspended in the liquid and / or interstitial spaces between the primary metal particles. Typical metal systems used to form nanoclusters from metal colloids include, for example, silver, gold, platinum, copper, aluminum, and the like. The average size of the metal particles in the colloid used in the method and composition of the present invention is from about 3 nanometers to about 15 nanometers. Generally, COIN can be prepared as follows. An aqueous solution is prepared containing the appropriate metal cation, reducing agent, and at least one suitable tensile active organic compound. The solution components then accept the conditions for reducing metal cations to form neutral colloidal metal particles. Because the formation of a metal colloid occurs when a suitable Raman-active organic compound is present, the Raman-active organic compound is adsorbed on the metal during the formation of the colloid. Such nano particles are clusters or aggregates of several primary metal particles, and Raman-active organic compounds are adsorbed on the surface of the metal particles and trapped in the junctions between the primary metal particles. COIN is usually non-spherical and often includes grooves and protrusions. COIN can be isolated by membrane filtration, and COIN with different sizes can be increased by centrifugation. In a further embodiment of the present invention, the nano-cluster system includes a second metal different from the first metal, wherein the second metal forms a layer covering the surface of the nano-cluster. In preparing such nanoclusters, COIN is placed in an aqueous solution containing a suitable second metal cation and a reducing agent. The components of the solution are then subjected to conditions for reducing the second metal cation to form a metal layer covering the surface of the nanoclusters. In a particular embodiment, the second metal layer system includes metals such as, for example, silver, gold, platinum, aluminum, copper, zinc, iron, and the like. Such nanoclusters can be isolated and / or enriched in the same way as a single metal CO IN. In a specific embodiment, the metal layer covering the surface of the nano-cluster is called a protective layer. The protective layer can impart water stability to the nano-cluster. C O IN can be coated with a silicon dioxide layer 'instead of or in addition to the metal protective layer. -19- 200535246 (16) If the CO IN has been coated with a metal layer, such as, for example, gold, the silicon dioxide layer can be borrowed by C 0 IN and, for example, 3 -aminopropyltrimethoxy sand (APTMS) glass ( vitreophilization). The silicon dioxide deposition system starts from a super-saturated silicon dioxide solution, and then a silicon dioxide layer is grown by adding ammonia and tetraethyl orthosilicate (TEOS) dropwise. The silicon dioxide-coated COIN can be functionalized using standard silicon dioxide chemistry. In certain other embodiments, the COIN may include an organic layer covering a metal surface or a silicon dioxide layer. In some embodiments, these types of nano-particles are prepared by adsorbing or covalently attaching an organic compound to the surface of the COIN. Covalent attachment of the organic layer to the metal surface can be achieved in a variety of ways known to those skilled in the art, such as, for example, via a thiol-metal bond. In alternative embodiments, the organic molecules can be cross-linked to form a molecular network. The organic layer can also be used to provide colloidal stability and functional groups for further derivatization. This organic layer is optionally crosslinked to form a solid coating. Exemplary The organic layer is prepared by adsorbing octylamine-modified polyacrylic acid on COIN, and the adsorption system is based on a positively charged amine group. The carboxyl group of the polymer is then cross-linked with suitable reagents such as lysine, (1,6) -diaminoheptane and the like. Unreacted carboxyl group can be used for further derivatization. Other functional groups can also be introduced via a modified polyacrylic backbone. In addition, the metal and organic coatings can be stacked in various combinations to provide the desired coated COIN properties. For example, COIN can be coated with a gold layer to seal the more reactive silver before applying an absorbent layer, silicon dioxide, or a solid organic coating. Even if the outer layer is porous, the inner gold layer prevents COIN from being attacked by reagents used in different applications. Another example is Shi-20-200535246 (17) An adsorption layer is added on the silicon dioxide or gold layer to provide additional colloidal stability. In a specific embodiment, the metal particles used for C 0 IN may include magnetic properties. Materials such as, for example, iron oxide and the like. The magnetic C Ο IN can be operated without centrifugation using generally effective magnetic particle operating systems. In fact, magnetism can be used as a mechanism to separate COIN particles labeled with specific biological probes. In another embodiment, a method for detecting an analyte in a sample is provided. These methods can be performed, for example, by contacting a sample containing an analyte with a COIN with an attached probe, wherein the probe is bonded to the analyte and any COIN-analyte is separated from any uncombined COIN The complex is detected and a SERS signal is detected from the nanoclusters, where the SERS signal is indicative of the presence of the analyte. In another embodiment, a method for identifying an analyte in a sample using a Raman-active metal nanocluster group is provided, and each member of the group has Raman characteristics that are unique in the group. These methods can, for example, contact multiple nanoclusters by contacting a sample that may contain an analyte; detect SERS signals in multiple ways when the sample is in contact with the nanoclusters; The SERS signal of the nano-cluster and the analyte body to which the nano-cluster is connected. In other embodiments, the present invention provides a method for distinguishing biological analytes in a sample by using a sample containing multiple biological analytes and a Raman active metal having an average diameter of about 50 nm to about 20 nm. Cluster groups (each member in the group has a unique Raman characteristic in the group, which is generated by at least one Raman-active organic compound incorporated therein) and specifically bonds -21-200535246 ( 18) The contact with the probe of a known biological analyte is suitable for the probe to specifically bind to the analyte in the sample to form a complex. The bonded clusters are separated, and the Raman characteristics emitted by the organic Raman-active compounds in the bonded complexes are detected in multiple ways. Each Raman signature indicates the presence of a known biological analyte in the sample. COIN can be used as a label for bioanalytical detection. In one example, we used a detection process similar to the standard sandwich immunoassay (Figure 5A); the difference is that after the specific bonding is required in the sandwich immunoassay using other tags The signal amplification step is not required when using COIN as the analyte tag (Figure 5A). Interleukin-2 (IL-2) is connected to a surface that has been pre-coated with anti-IL-2 capture antibody, so that the maximum average IL-2 molecular density is lower than 1 molecule per laser beam cross-sectional area (0.77 molecules) / 12 micron 2 '1.3 yoctomole in the laser beam), C 0 IN coated with anti-1L-2 antibody is used to detect the immobilized IL-2 molecules. As shown in Figure 5B, an average of 28% of the spectra were found to have the desired IL-2 characteristics, showing a 36% detection rate for all applied analyte molecules. This detection rate is only possible under experimental conditions with an average of 10 analyte molecules per data collection area, considering possible incomplete bonding in sandwich detection and the possibility of inactive COIN. The capture substrate was prepared using a mixed antibody against IL-2 and IL-8. Similarly, two sets of COINs (each with AA and BA characteristics) were prepared using detection antibodies specifically bound to both analytes. When using two analytes in different ratios, detect a positive CO IN signal at a ratio that exactly matches 预测 predicted based on the known ratio of the used analytes. -22- 200535246 (19) When used to detect analytes, the probe can be connected to ΟIN. In specific embodiments, exemplary probes are antibodies, antigens, polynucleotides, oligonucleotides, receptors, ligands, and the like. The term polynucleotide is widely used in the present invention to denote a sequence of deoxyribonucleotides or ribonucleotides bonded by a phosphodiester bond. The term oligonucleotide is simply used in the present invention to denote a polynucleotide as a primer or a probe. Generally, oligonucleotides that can be used as probes or primers that are selectively fused to a selected nucleotide sequence are at least about 10 nucleotides in length, usually at least about 15 nucleotides in length, such as Lengths between about 15 and about 50 nucleotides. Polynucleotide probes are particularly useful for detecting complementary polynucleotides in biological samples, or by combining known polynucleotide probes with known Raman activity signals (the Raman described in the present invention). Compositions of active organic compounds) are used for DNA sequencing. Polynucleotides can be RNA or DNA, they can be genes or parts thereof, cDNA, synthetic DNA sequences, or the like, and they can be single-stranded or double-stranded, and DNA / RNA hybrids. In various embodiments, the polynucleotide ' includes oligonucleotides (e.g., probes or primers) may contain nucleosides or nucleotide analogs, or backbone linkages other than phosphodiester linkages. Generally, a polynucleotide comprising a polynucleotide is a natural deoxyribonucleotide, such as adenine, cytosine, guanine or thymine, or a ribonucleotide, such as a linker, attached to 2'-deoxyribose. In ribose adenine, cytosine, guanine or uracil. However, the polynucleotide or oligonucleotide may also contain nucleotide analogs ' including unnatural synthetic nucleotides or modified natural nucleotides. Polynucleotide-linked covalent bonds are usually phosphodiester -23-200535246 (20) bonds. However, the covalent bond can also be any of a number of other bonds, including thiodiester bonds, phosphorothioate bonds, peptide amidoamine bonds, or any other known nucleoside linkages known in the art The acid is bound to make synthetic polynucleotides. Unnatural nucleotide congeners or linked nucleotides or conjugation incorporation are particularly useful where the polynucleotide will be exposed to an environment that can contain nuclear degrading activity (including, for example, when in tissue culture media or when administered to a living body) This is because modified polynucleotides are less susceptible to degradation. The term selective promiscuous or selective promiscuous as used in the present invention refers to promiscuous under very severe or highly severe conditions, so that the advantage of association of the nucleotide sequence with the selected nucleotide sequence is higher than that of the unrelated Nucleotide sequence to the extent that the selected nucleotide sequence can be identified. It should be recognized that some non-specific hybridization may occur, but it is acceptable when the hybridization of the target nucleotide sequence is sufficiently selective so that it can be distinguished from non-specific interaction hybridization, such as at least two times higher selection Selectivity, usually at least 3 times higher, usually at least about 5 times higher, especially at least about 10 times higher, such as by labeled oligonucleotides bound to the target nucleic acid molecule Relative to the amount of nucleic acid molecules other than the target molecule (especially nucleic acid molecules that are substantially the same or similar except the target nucleic acid molecule) is determined. The conditions under which selective hybridization can be performed can be determined experimentally, or can be based on, for example, the relative GC of the oligonucleotide for hybridization and the sequence to be hybridized. The AT content, the length of the oligonucleotide for hybridization, and the oligonucleoside Estimate the number of mismatches (if any) between the acid and the sequence to be promiscuous.

愈來愈嚴苛之條件的實例如下：在約室溫下之2 X SSC/0.1%SDS (混雜條件）；在約室溫下之 〇.2 X -24 - 200535246 (21) SSC/0.1%SDS (低嚴苛條件）；在約 42 t下之0.2 X SSC/0.1%SDS (中度嚴苛條件）；及在約68°C下之0.1 X SSC/0.1%SDS (高度嚴苛條件）。洗滌可僅使用此等條件 中之一進行，例如高度嚴苛條件，或可依前述順序使用各 個條件，例如各1 0至1 5分鐘，重複任一或所有所列之步 驟。然而，如前文所述，最佳條件可視所涉及之特定混雜 反應而改變，且可以實驗決定。 部分實施例中，有機層可包括抗體探針。本發明所使 用之術語抗體係使用其最廣義意義，包括單株及多株抗體 ’及該等抗體之抗原鍵結片段。可使用於本發明方法之抗 體或其抗原鍵結片段係例如具有針對於分析物之抗原決定 部位的專一性鍵結活性之特性。 於本發明特定態樣中，抗體係與奈米團簇結合。該抗 體係例如包括天然抗體及非天然抗體，包括例如單鏈抗體 、嵌合、雙官能性且人化抗體，及其抗原結合性片段。該 等非天然抗體可使用固相胜肽合成來建構，可重組製得或 可例如藉著篩選由可變重鏈及可變輕鏈所組成之組合庫而 得到。此等及其他製造例如嵌合、人化、CDR-接枝、單鏈 且雙官能性抗體之方法係熟習此技術者所熟知。 術語專一性鍵結或專一性鍵結活性在使用於抗體時係 表示抗體與特定抗原決定部位之相互作用具有至少約 lxl(T6之解離常數，通常至少約10xl0〃，通常至少約 1χ10_8，尤其是至少約1χ10·9或ιχ1(Γΐ()或較低。此情況 下’抗體保持針對抗原之抗原決定部分之專一性鍵結活性 - 25- 200535246 (22) 的Fab、F ( ab，）2、Fd及Fv片段係包括於抗體定義內。 本發明內容中，術語配位體係表示受體之天然專一性 鍵結配合物、受體之合成專一性鍵結配合物或天然或合成 配位體之適當衍生物。如同熟習此技術者所知，分子（或 巨分子複合物）可同時爲受體及配位體。通常，具有較小 分子量之鍵結配合物係稱爲配位體，而具有較大分子量之 鍵結配合物係稱爲受體。 另一實施例中，提供測定試樣中之分析物的方法。該 等方法可例如藉著含分析物之試樣與包括探針之奈米團簇 接觸而進行，其中該探針係鍵結於分析物；並偵測由奈米 團簇所發射之SERS信號，其中該信號係指示分析物之存 在。該試樣更常含有一群生物分析物，試樣與一組本發明 所述之COIN接觸，其中該組中之各員係具有專一性鍵結 於已知生物分析物之探針，而該組員內收納有不同組合之 拉曼活性有機化合物，以提供獨特之拉曼記號，可立即得 出與該探針所專一性鍵結之已知分析物的關連。 分析物係表示任何分子或化合物。分析物可爲固體、 液體、3¾體或蒸汽相。氣體或蒸汽相分析物係表不分子或 化合物係存在於例如液體之頂部空間、環境空氣中、呼吸 試樣、氣體或任何前述者之污染物。應認知氣體或蒸汽相 之物理狀態可因壓力、溫度及藉例如鹽之存在或添加來影 響液體之表面張力而改變。 如前文所述，本發明方法在特定態樣中係偵測分析物 對探針之鍵結。該分析物可包含特定鍵結對（sbp )中之 -26- 200535246 (23) 一員且可爲配位體，其係單價（單抗原決定部位）或多價 (多抗原決定部位），通常爲抗原或半抗原，且係爲單一 種化合物或共用至少一個共用抗原決定部位或決定素部位 之多種化合物。該分析可爲細胞之一部分，諸如具有血液 基抗原諸如A、B、D等或HLA抗原之細菌或細胞，或微 生物例如細菌、真菌、原生動物或病毒。本發明特定態樣 中，該分析物係帶電。 特定鍵結對中之一員（sbp員）係爲兩不同分子中之 一，具有位於表面上或孔穴中專一性地鍵結且因而定義爲 與另一分子之特定空間及極性有機化互補之區域。專一性 鍵結對之成員稱爲配位體及受體（抗配位體）或分析及探 針。因此，探針係爲專一性鍵結分析物之分子。此等通常 係爲免疫對諸如抗原-抗體之成員，唯其他專一性鍵結對 諸如生物素-卵蛋白、激素-激素受體、核酸雙重物、IgG-蛋白質A、聚核苷酸對諸如DNA-DNA、DNA-RNA及其類 者非免疫對，但仍包括於本發明及sbp員之定義中。 專一性鍵結係爲兩不同分子中之一對另一者相較於其 他分子之實質較低識別性的專一性識別。通常，該等分子 在其表面或孔穴中具有產生介於該兩分子間之專一性識別 的區域。專一性鍵結之實例係爲抗體-抗原相互作用、酶-受質相互作用、聚核苷酸混雜相互作用等。非專一性鍵結 係爲分子間之非共價鍵結，其與專一性表面結構相對無關 。非專一性鍵結可得自數項因素，包括分子間之疏水性相 互作用。 -27- 200535246 (24) 本發明奈米團簇可用以偵測特定標的分析物例如核酸 、寡聚核苷酸、蛋白質、酶、抗體或抗原之存在。該奈米 團簇亦可用以篩檢鍵結於特定標的之生物活性劑諸如例如 藥物候選物或偵測如污染物之作用劑。如前文所討論，可 設計探針部分之任何分析物諸如胜肽、蛋白質、寡聚核苷 酸或適當體（a p t a m e r )皆可與所揭示之奈米團簇結合使 用。 配位體分析物包括聚（胺基酸），諸如例如聚胜肽及 蛋白質、多醣、激素、核酸及其組合物。該等組合物係包 括細菌、病毒、朊病毒、細菌、染色體、基因、粒線體、 核、細胞膜其類者之成份。其他可能之分析物包括藥物、 代謝物、殺蟲劑、污染物及其類者。所硏究之藥物中特別 包括生物鹼。生物鹼中特別有嗎啡生物鹼，其包括嗎啡、 可待因（codeine )、***（heroin )、右美沙芬（ dextromethorphan )、及衍生物及代謝物；古柯鹼生物鹼 ，其包括古柯鹼及苄基可卡終鹼、其衍生物及代謝物；麥 角生物鹼，包括麥角酸之二乙基醯胺；類固醇生物鹼；咪 唑基生物鹼；喹唑啉生物鹼；異喹啉生物鹼；喹啉生物鹼 ，包括奎寧及奎尼丁；雙萜生物鹼、其衍生物及代謝物。 術語分析物係進一步包括聚核苷酸分析物，諸如下文 所定義之聚核苷酸。此等包括例如111-1^八、1-1^八、1-RNA、DNA、DNA-RNA雙重物。術語分析物亦包括係爲 聚核苷酸鍵結劑之受體，諸如例如胜肽核酸（PNA )、限 制酶、活化劑、阻遏劑、核酸酶、聚合酶、組蛋白、修補 -28- 200535246 (25) 酶、化學療劑及其類者。 分析物可爲直接於試樣諸如來自宿主之體液中發現的 分子。該試樣可直接檢測，或可預先處理以使分析物更立 即地偵測。此外，所硏究之分析物可藉著偵測所硏究分析 物之探針活性劑諸如與所硏究分析物互補之專一性鍵結對 成員而決定’僅在所硏究分析物存在於試樣中時可測得其 存在。因此’分析物探針活性劑變成在檢測中所偵測之分 析物。體液可爲例如尿液、血液、血漿、血淸、唾液、精 液、糞便、痰、腦脊髓液、淚液、黏液及其類者。 通常’探針可經由探針吸附於COIN表面上而連接於 COIN。或COIN可經由生物素-卵蛋白鍵與探針偶合。例 如，卵蛋白或鏈親和素（或其類似物）可吸附於C Ο IN表 面上’經生物修飾之探針與經卵蛋白或鏈親和素修飾之表 面接觸’形成生物素-卵蛋白（或生物素-鏈親和素）鍵。 卵蛋白或鏈親和素可視情況與另一蛋白質（諸如BS A )結 合吸附且視情況交聯。此外，就具有包括羧酸或胺官能基 之官能層之COIN而言，具有對應之胺或羧酸官能基之探 針可經由水溶性碳化二醯亞胺偶合劑諸如EDC ( 1-乙基- 3-(3 -二甲基胺基丙基）碳化二醯亞胺）來連接，該偶合劑 係將殘酸官能基與胺基偶合。 連接於各種官能基之核苷酸可購得（例如，購自 Molecular Probes, Eugene, OR； Quiagen ( Operon ),Examples of increasingly severe conditions are as follows: 2 X SSC / 0.1% SDS (hybrid condition) at about room temperature; 0.2 X -24-200535246 (21) SSC / 0.1% at about room temperature SDS (low severe conditions); 0.2 X SSC / 0.1% SDS (moderately severe conditions) at approximately 42 t; and 0.1 X SSC / 0.1% SDS (highly severe conditions) at approximately 68 ° C . Washing can be performed using only one of these conditions, such as highly stringent conditions, or each condition can be used in the foregoing order, such as 10 to 15 minutes each, repeating any or all of the listed steps. However, as mentioned earlier, the optimal conditions can vary depending on the particular promiscuous reaction involved and can be determined experimentally. In some embodiments, the organic layer may include an antibody probe. The term anti-system used in the present invention is used in its broadest sense and includes single and multiple antibody antibodies' and antigen-binding fragments of these antibodies. An antibody or an antigen-binding fragment thereof that can be used in the method of the present invention has, for example, a property that has specific binding activity to an epitope of an analyte. In a particular aspect of the invention, the anti-system is combined with nanoclusters. The antibody system includes, for example, natural antibodies and non-natural antibodies, including, for example, single-chain antibodies, chimeric, bifunctional, and humanized antibodies, and antigen-binding fragments thereof. Such non-natural antibodies can be constructed using solid-phase peptide synthesis, can be produced recombinantly, or can be obtained, for example, by screening a combinatorial library consisting of variable heavy chains and variable light chains. These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art. The term specific binding or specific binding activity when used in an antibody means that the interaction of the antibody with a specific epitope has a dissociation constant of at least about 1xl (T6, usually at least about 10x10〃, usually at least about 1x10_8, especially At least about 1 × 10 · 9 or ιχ1 (Γΐ () or lower. In this case, the 'antibody maintains specific binding activity against the epitope of the antigen-Fab, F (ab,) 25-200535246 (22) 2, Fd and Fv fragments are included in the definition of antibody. In the context of the present invention, the term coordination system means a natural specific binding complex of a receptor, a synthetic specific binding complex of a receptor, or a natural or synthetic ligand. Appropriate derivatives. As is known to those skilled in the art, molecules (or macromolecular complexes) can be both receptors and ligands. Generally, a bond complex with a smaller molecular weight is called a ligand and has Larger molecular weight bond complexes are referred to as acceptors. In another embodiment, methods for determining analytes in a sample are provided. These methods can be performed, for example, by analyte-containing samples and probes Rice clusters Touch it, where the probe is bound to the analyte; and detect the SERS signal emitted by the nanoclusters, where the signal is indicative of the presence of the analyte. The sample more often contains a group of biological analytes. In contact with a group of COIN according to the present invention, each member of the group has a probe that specifically binds to a known biological analyte, and the group member contains different combinations of Raman-active organic compounds, In order to provide a unique Raman signature, the correlation with a known analyte that is specifically bonded to the probe can be immediately obtained. Analyte means any molecule or compound. Analyte can be solid, liquid, 3¾ body or vapor A gas or vapor phase analyte is a molecule or compound that exists in, for example, the headspace of a liquid, ambient air, a breath sample, a gas, or any of the foregoing contaminants. It should be recognized that the physical state of the gas or vapor phase may be Changes due to pressure, temperature, and the surface tension of a liquid by the presence or addition of salt, for example. As mentioned earlier, the method of the present invention detects analyte-to-probe in a particular aspect. Bonding. The analyte may include a member of -26- 200535246 (23) in a specific bonding pair (sbp) and may be a ligand, which is monovalent (single epitope) or multivalent (multiple epitope), It is usually an antigen or hapten and is a single compound or multiple compounds that share at least one common epitope or determinin site. The analysis can be part of a cell, such as a blood-based antigen such as A, B, D, etc. or Bacteria or cells of the HLA antigen, or microorganisms such as bacteria, fungi, protozoa, or viruses. In a particular aspect of the invention, the analyte is charged. One of the specific binding pairs (sbp member) is one of two different molecules Has a region that is specifically bonded on the surface or in a cavity and is therefore defined as a region that is complementary to the specific space and polarity of another molecule. Specific binding members are called ligands and receptors (antiligands) or assays and probes. Therefore, probes are molecules that specifically bind to the analyte. These are usually members of immune pairs such as antigen-antibodies, but other specific binding pairs such as biotin-ovin, hormone-hormone receptors, nucleic acid duplexes, IgG-protein A, polynucleotide pairs such as DNA- DNA, DNA-RNA, and the like are not immune pairs, but are still included in the definition of the present invention and sbp members. Specific bonding is the specific identification of one of two different molecules that is substantially less recognizable of the other than the other molecules. Generally, these molecules have regions on their surface or in the pores that give rise to a specific identification between the two molecules. Examples of specific bonding are antibody-antigen interactions, enzyme-substance interactions, polynucleotide hybrid interactions, and the like. Non-specific bonding is a non-covalent bond between molecules, which is relatively unrelated to the specific surface structure. Non-specific bonding can be derived from several factors, including hydrophobic interactions between molecules. -27- 200535246 (24) The nanoclusters of the present invention can be used to detect the presence of specific target analytes such as nucleic acids, oligonucleotides, proteins, enzymes, antibodies or antigens. The nanoclusters can also be used to screen bioactive agents bound to specific targets such as, for example, drug candidates or to detect agents such as contaminants. As discussed previously, any analyte in the probe portion, such as peptides, proteins, oligonucleotides, or appropriate bodies (ap t a m e r) can be used in conjunction with the disclosed nanoclusters. Ligand analytes include poly (amino acids) such as, for example, polypeptides and proteins, polysaccharides, hormones, nucleic acids, and combinations thereof. These compositions include components of bacteria, viruses, prions, bacteria, chromosomes, genes, mitochondria, nuclei, cell membranes and the like. Other possible analytes include drugs, metabolites, pesticides, pollutants and the like. Among the drugs studied are alkaloids. Among the alkaloids are morphine alkaloids, which include morphine, codeine, heroin, dextromethorphan, derivatives and metabolites; ***e alkaloids, which include ***e And benzyl coca terminal base, its derivatives and metabolites; ergot alkaloids, including diethylphosphonium lysergic acid; steroid alkaloids; imidazolyl alkaloids; quinazoline alkaloids; isoquinoline organism Alkaloids; quinoline alkaloids, including quinine and quinidine; diterpene alkaloids, their derivatives and metabolites. The term analyte system further includes a polynucleotide analyte, such as a polynucleotide as defined below. These include, for example, 111-1 ^ A, 1-1 ^ A, 1-RNA, DNA, DNA-RNA duplex. The term analyte also includes receptors that are polynucleotide binding agents, such as, for example, peptide nucleic acid (PNA), restriction enzymes, activators, repressors, nucleases, polymerases, histones, repair-28- 200535246 (25) Enzymes, chemotherapeutics and the like. Analytes can be molecules found directly in a sample, such as a body fluid from a host. The sample can be detected directly, or it can be pre-processed for more immediate detection of the analyte. In addition, the analyte under investigation can be determined by detecting the probe active agent of the analyte under investigation, such as a specifically bonded pair member complementary to the analyte under investigation, 'only if the analyte under investigation exists in the test Its presence can be measured in the sample. Thus the 'analyte probe active agent becomes the analyte detected in the assay. The bodily fluid may be, for example, urine, blood, plasma, blood clots, saliva, semen, stool, sputum, cerebrospinal fluid, tear fluid, mucus, and the like. In general, the 'probe' can be attached to the COIN via the probe adsorbed on the surface of the COIN. Or COIN can be coupled to the probe via a biotin-ovin bond. For example, egg protein or streptavidin (or an analogue thereof) can be adsorbed on the surface of C0 IN 'a biologically modified probe in contact with an egg protein or streptavidin-modified surface' to form a biotin-ovin (or Biotin-streptavidin) bond. Ovalin or streptavidin may optionally be bound to another protein (such as BS A) and cross-linked as appropriate. Further, in the case of a COIN having a functional layer including a carboxylic acid or amine functional group, a probe having a corresponding amine or carboxylic acid functional group may be passed through a water-soluble carbodiimide coupling agent such as EDC (1-ethyl- 3- (3-dimethylaminopropyl) carbodiimide), the coupling agent couples the residual acid functional group with the amine group. Nucleotides linked to various functional groups are commercially available (eg, from Molecular Probes, Eugene, OR; Quiagen (Operon),

Valencia, CA;及 IDT (Integrated DNA Technologies)， Coral vi 1 le，IA )且倂入寡聚核苷酸或聚核苷酸內。經生物 -29- 200535246 (26) 素修飾之核蘇菱係市售（例如購自P i e r c e B i o t e c h η ο 1 o g y， Rockford，IL 或 Pano m ics，Inc. Redwood City，CA)，且 經修飾之核苷酸可在習用放大技術期間倂入核酸內。寡聚 核苷酸可使用市售寡聚核苷酸合成儀（例如 Applied Biosystems，Foster City，CA)製備。此外，經修飾之核苷 酸可使用已知反應合成，諸如例如N e 1 s ο η，P .，S h e r m a η · Gold，R，and Leon，R，’’A New and Versatile Reagent for Incorporating Multiple Primary Aliphatic Amines into Synthetic Oligonucleotides, MNucleic Acids Res., 17:7179-7186(1989)及 Connolly，B·，Rider，P. "Chemical Synthesis of Oligonucleotides Containing a Free Sulfhydry 1 Group and Subsequent Attachment of Thiol Specific Probes， ”Nucleic Acids Res·，13:4485-4502 ( 1985)所揭示。或可 購得含有各種反應性基團諸如生物素、羥基、氫硫基、胺 基或羧基之核苷酸前驅物。在寡聚核苷酸合成之後， COIN可使用標準化學連接。具有任何所需序列之寡聚核 苷酸（具有或不具有供COIN連接使用之反應性基團）皆 亦可購自各種來源（例如 Midland Certified Reagents， Midland, TX) 〇 探針諸如多醣亦可經由 Aslam，M. and Dent，Α.， Bioconjugation: Protein Coupling Techniques for theValencia, CA; and IDT (Integrated DNA Technologies), Coral vi 1 le, IA) and incorporated into the oligonucleotide or polynucleotide. Bio-29- 200535246 (26) primed nuclear thulium is commercially available (for example, from Pierce Biotech η ο 1 ogy, Rockford, IL or Panomics, Inc. Redwood City, CA) and modified Nucleotides can be incorporated into nucleic acids during conventional amplification techniques. Oligonucleotides can be prepared using a commercially available oligonucleotide synthesizer (e.g., Applied Biosystems, Foster City, CA). In addition, modified nucleotides can be synthesized using known reactions, such as, for example, Ne 1 s ο η, P., Sherma η · Gold, R, and Leon, R, `` A New and Versatile Reagent for Incorporating Multiple Primary Aliphatic Amines into Synthetic Oligonucleotides, MNucleic Acids Res., 17: 7179-7186 (1989) and Connolly, B., Rider, P. " Chemical Synthesis of Oligonucleotides Containing a Free Sulfhydry 1 Group and Subsequent Attachment of Thiol Specific Probes, "Nucleic Acids Res., 13: 4485-4502 (1985). Or, nucleotide precursors containing various reactive groups such as biotin, hydroxyl, hydrogenthio, amine, or carboxyl groups are commercially available. After polynucleotide synthesis, COINs can be joined using standard chemistry. Oligonucleotides with or without any desired sequence (with or without reactive groups for COIN attachment) can also be purchased from various sources (eg Midland Certified Reagents, Midland, TX). Probes such as polysaccharides can also be obtained via Aslam, M. and Dent, A., Bioconjugation: Protein Coupling Techniques for the

Biomedical Sciences，Grove’s Dictionaries，Inc., 229,254 (1 9 9 8 )所揭示之方法連接於C O IN。該等方法包括（但 不限於）過碘酸鹽氧化偶合反應及雙-琥珀醯亞胺酯偶合 -30- 200535246 (27) 反應。 以下段落包括有關COIN探針（連接有探針之複合有 機-無機奈米團簇（COIN ))之例示應用的其他細節。應 明瞭採用C Ο IN探針之應用的多個附加特例可使用本發明 教示來加以確定。熟習此技術者應認知聚胜肽與其標的分 子間之許多相互作用可使用經COIN標記之聚胜肽來偵測 。在一組例示應用中’經COIN標記之抗體（即鍵結於 COIN之抗體）係用以偵測標記有COIN之抗體與溶液中 或固體擔體上之抗原的相互作用。應明瞭該等免疫檢定可 使用已知方法進行，諸如例如 ELISA檢定、西方（ Western )印漬法或蛋白質陣列，採用經C0IN標記之抗體 或經COIN標記之二級抗體取代標記有酶或放射性化合物 的初級或二級抗體。 另一組例示方法係使用COIN來偵測標的核酸。該方 法可用於例如偵測臨床試樣內之感染劑、偵測自基因DNA 或RNA或信息RN A衍生之放大產物或偵測無性繁殖系內 之基因（cDNA )嵌入物。針對標的聚核苷酸之偵測的特 定方法中，寡聚核苷酸係使用技術界已知方法合成。寡聚 核苷酸探針隨之用以將C0IN粒子官能化，以產生標記有 COIN之寡聚核苷酸探針。該標有C0IN之寡聚核苷酸探 針係用於混雜化反應中，以偵測標有COIN之寡聚核苷酸 探針對標記聚核苷酸的專一性鍵結。例如，該標有COIN 之寡聚核苷酸探針可用於北方（Northern)印漬或南方（ Southern)印漬反應。或標有COIN之寡聚核苷酸探針可 -31 - 200535246 (28) 施加於包括與固體擔體締合之標的聚核苷酸之反 中，以捕集標有COIN之寡聚核苷酸探針。所捕 COIN之寡聚核苷酸探針隨之可使用拉曼光譜偵 自或不自固體擔體釋出。位於經捕集標有COIN 苷酸探針上的專一性拉曼標記之偵測確認寡聚核 的核苷酸序列，又提供有關標的聚核苷酸之核苷 資料。 另一例示組專一性應用中，標有COIN之核 以測定標的聚核苷酸中發生單一鹽基變化的核苷 應用包括測定”熱區”點突變並識別位在單一核苷 (SNP )部位上的鹽基。例如，製備寡聚核苷酸 於多形部位旁立即混雜化。引子-包括單一鹽基 之標的聚核苷酸，及聚合酶係包括於延伸反應混 該反應混合物係包括四種鏈終止三磷酸酯，各連 之COIN標記。隨之進行延伸反應，且若爲同源 四種鏈終止核苷酸中僅有一種添加於引子末端， 有COIN之延伸引子。位於延伸引子上之COIN 使用拉曼光譜偵測。標記之識別確認添加於單一 部位上之核苷酸，以確認標的聚核苷酸中發生單 化的核苷酸。 在本發明方法中，試樣包括各式各樣之分析 使用本發明所述之奈米團簇分析。例如，試樣可 樣，包括大氣空氣、環境空氣、水、泥漿、土壤 。此外，試樣可爲生物試樣，包括例如患者呼氣 應混合物 集之標有 測，首先 之寡聚核 苷酸探針 酸序列的 苷酸係用 酸。此等 酸多形性 引子，以 變化部位 合物中。 接有獨特 SNP，貝丨J 以生成標 標記隨後 鹽基變化 一鹽基變 物，其可 爲5哀境g式 及其類者 、唾液、 -32- 200535246 (29) 血液、尿液、糞便、各種組織及其類者。 採用本發明所述奈米團簾之本發明方法的工業應用包 括環境毒物及治療、生物藥劑、材料品管、偵測食品及農 產品之病原、麻醉劑偵測、汽車油或輻射物流體偵測、呼 氣酒精分析器、危險物辨識、***偵測、暫時性發射辨識 、醫藥診斷劑、魚新鮮度、在生物醫藥用途及醫藥診斷用 途中細菌及微生物在體外及體內之偵測及分級、偵測重工 業製造、環境空氣偵測、工作人員保護、發射控制、產物 品質測試、洩漏偵測及辨識、油/氣石化應用、可燃氣體 偵測、H2S偵測、危險洩漏偵測及辨識、緊急反應及法律 實施應用、非法物質偵測及辨識、縱火硏究、密閉空間監 視、公共設施及能量應用、發射偵測、變壓器失效偵測、 食品/飮料/農業應用、新鮮度偵測、水果成熟控制、發酵 過程偵測及控制應用、調味組成物及辨識、產物品質及辨 識、冷凍劑及煙薰劑偵測、化粧品/香水/香料調配物、產 物品質測試、個人辨識、化學/塑料/醫藥應用、洩漏偵測 、溶劑回收效力、周界偵測、產物品質測試、危險廢棄部 位應用、暫時性發射偵測及辨識、洩漏偵測及辨識、周界 偵測、輸送、危險物偵測、補給燃料操作、運輸容器檢查 、柴油/汽油/航空燃料辨識、建築/住宅天然氣偵測、甲醒 偵測、煙霧偵測、火焰偵測、自動換氣控制應用（烹煮、 抽煙等）、空氣引入偵測、醫院/醫藥麻醉及殺菌氣體偵 測、感染性疾病偵測及呼吸應用、體液分析、醫藥應用、 藥物發現、遙控手術及其類者。 -33- 200535246 (30) 另一'種以感測器爲主之在引擎流體中之流體偵測裝胃 的應用係爲油/抗凍劑偵測器、用於空氣/燃料最佳化之引 擎診斷器、柴油燃料品質、揮發性有機碳測量（VOC )、 精煉廠中之暫時性氣體、食品品質、口臭、土壤及水污染 、空氣品質偵測、燃燒安全性、化學武器辨識、危險物質 工作組之使用、***偵測、呼吸分析器、氧化乙儲或麻醉 劑偵測器。 另一實施例中，提供偵測試樣中之分析物的系統。該 等系統包括包含多於一個奈米團簇之陣列；含有至少一分 析物之試樣；拉曼光譜儀；及包括用以分析試樣之演算法 之電腦。 可使用各式各樣之分析技術來分析本發明所述之 C0IN粒子。該等技術包括例如核磁共振光譜（NMR)、 光子相關光譜（PCS) 、IR、表面電漿共振（SPR) 、XPS 、掃描探針顯微鏡（SPM ) 、SEM、TEM、原子吸收光譜 、元素分析、UV-vis、螢光光譜及其類者。 進行本發明時，拉曼光譜儀可爲設計以藉拉曼光譜偵 測且定量本發明奈米團簇之偵測單元的一部分。使用拉曼 光譜偵測標有拉曼之分析物（例如核苷酸）的方法係技術 界已知（參照例如美國專利第5,3〇6,4 03號；第6,002,47 1 號；第 6，1 74,677號）。已揭示表面增強拉曼光譜法（ SERS )、表面增強共振拉曼光譜法（SERRS )及相干反斯 托克斯拉曼光譜（CARS )的改變。 拉曼偵測單元之非限制實例係揭示於美國專利第 -34- 200535246 (31) 6,002,47 1號中。激發光束係藉5 3 2奈米波長之倍頻 Nd:YAG雷射或365奈米波長倍頻Ti:藍寶石雷射生成。可 使用脈衝雷射光束或連續雷射光束。激發光束通經共焦光 學儀器及顯微鏡物鏡，聚焦於流動通道及/或流通槽件。 來自經標記奈米團簇之拉曼發射光集中於顯微鏡物鏡及共 焦光學儀器，且耦合於用於光譜離解之單色發光器。共焦 光學儀器包括二色濾器、障濾器、共焦針孔、透鏡及用以 降低背景信號之面鏡的組合。可使用標準全場型光學儀器 及共焦光學儀器。拉曼發射信號係以拉曼偵測器偵測，包 括以計數並將信號數位化之電腦爲界面之突崩光電二極體 〇 拉曼偵測單元之另一實例係揭示於美國專利第 5，306,403號中，包括具有砷化鎵光電倍增管（RC A Model C3 1 03 4 或 B ur 1 e I n d u s t r i e s Μ 〇 d e 1 C 3 1 0 3 4 0 2 )而在單光子 計數模式下操作之雙柵型光譜儀。激發光源包括來自 SpectraPhysics，Model 166之514.5奈米線氬離子雷射及 氪離子雷射之647.1奈米線（Innova 70，Coherent)。 備擇激發光源包括3 3 7奈米之氮雷射（Laser Science Inc.)及325奈米之氦-鍋雷射（Liconox)(美國專利第 6，1 74,677號）、發光二極體、Nd:YLF雷射及各種離子雷 射及/或染料雷射。激發光束可使用帶通型濾器（C 0 r i 0 n ) 光譜純化，且可使用6X物鏡（Newport，Model L6X )聚 焦於流動通道及/或流通槽件。該物鏡可用藉著使用全息 光束***器（Kaiser Optical Systems，Inc.，Model HB 647- -35- 200535246 (32) 2 6N 1 8 )激發奈米團簇之拉曼活性有機化合物且收集拉曼 信號’產生激發光束及發射拉曼信號的直角幾何圖形。全 息凹口波濾波器（K a i s e r Ο p t i c a 1 S y s t e m s，I n c ·)可用以減 少Rayleigh散射輻射。備擇拉曼偵測器包括裝置有紅色增 強鼠何親合裝置（R E -1 C C D )偵測系統（p r丨n c e t 〇 n Instruments)之ISA HR-3 20光譜圖。可使用其他類型之 偵測器，諸如富利葉轉換光譜圖（基於Michael son干涉儀 )、帶電注射裝置、光電二極體陣列、InGaAs偵測器、 電子倍增CCD、強化CCD及/或光電晶體陣列。 技術界已知之拉曼光譜法或相關技術的任何適當形式 或構型可用於偵測本發明奈米團簇，包括但不限於正常拉 曼散射、共振拉曼散射、表面增強拉曼散射、表面增強共 振拉曼散射、相干反斯托克斯拉曼光譜法（CARS )、激 勵拉曼散射、逆拉曼光譜法、激勵增益拉曼光譜法、超拉 曼散射、分子光學雷射檢測器（Μ Ο L E )或拉曼微探針或 拉曼顯微鏡或共焦拉曼顯微光譜法、三維或掃描拉曼、拉 曼飽和光譜法、時間離析共振拉曼、拉曼去耦光譜法或 UV-拉曼顯微鏡。 本發明特定態樣中，用以偵測本發明奈米團簇之系統 係包括干擾處理系統。例示干擾處理系統可收納包括通信 用匯流排及用以處理干擾之處理器的電腦。該干擾處理及 控制系統可另外包括技術界已知之任何周邊裝置，諸如記 憶體、顯示器、鍵盤及/或其他裝置。 特定實例中，偵測單元可操作耦合於干擾處理系統。 -36- 200535246 (33) 來自偵測單元之數據可藉處理器及儲存於記憶體中之數據 處理。針對各種拉曼標記之發射曲線的數據亦可儲存於記 憶體中。該處理器可比較來自流動通道及/或流通槽件中 複合型有機-無機奈米團簇的發射光譜，以辨識拉曼活性 有機化合物。該處理器可分析來自偵測單元之數據，以決 定例如由本發明奈米團簇探針所鍵結之聚核苷酸的序列。 干擾處理系統亦可進行標準程序，諸如扣除背景信號。The method disclosed in Biomedical Sciences, Grove's Dictionaries, Inc., 229,254 (1 9 9 8) is connected to CO IN. These methods include, but are not limited to, the periodate oxidation coupling reaction and the bis-succinimide coupling -30-200535246 (27) reaction. The following paragraphs include additional details regarding exemplary applications of COIN probes (composite organic-inorganic nanoclusters (COIN) with probes attached). It should be noted that a number of additional special cases of applications using CO IN probes can be determined using the teachings of the present invention. Those skilled in the art should recognize that many interactions between polypeptides and their target molecules can be detected using COIN-labeled polypeptides. In a set of exemplary applications, ' COIN-labeled antibodies (i.e., antibodies that are bound to COIN) are used to detect the interaction of a COIN-labeled antibody with an antigen in solution or on a solid support. It should be understood that such immunoassays can be performed using known methods, such as, for example, ELISA assays, Western blotting, or protein arrays, using COIN-labeled antibodies or COIN-labeled secondary antibodies instead of enzymes or radioactive compounds. Primary or secondary antibodies. Another set of exemplary methods uses COIN to detect the target nucleic acid. This method can be used, for example, to detect infectious agents in clinical samples, to detect amplified products derived from genetic DNA or RNA or information RN A, or to detect gene (cDNA) inserts in clonal propagation lines. In a specific method for detecting the target polynucleotide, the oligonucleotide is synthesized using a method known in the technical field. Oligonucleotide probes were then used to functionalize the COIN particles to produce COIN-labeled oligonucleotide probes. The COIN-labeled oligonucleotide probe is used in the hybridization reaction to detect the specific binding of the COIN-labeled oligonucleotide probe to the labeled polynucleotide. For example, the COIN-labeled oligonucleotide probe can be used in Northern or Southern blotting reactions. Or COIN-labeled oligonucleotide probes can be applied to the reverse of the target polynucleotides associated with the solid support to capture the oligonucleosides labeled with COIN. Acid probe. The captured COIN oligonucleotide probes can then be detected using Raman spectroscopy or not released from a solid support. Detection of specific Raman-labeled nucleotides captured on the COIN-labeled probes confirms the nucleotide sequence of the oligo, and provides nucleoside information about the target polynucleotide. In another group-specific application, nucleosides marked with COIN are used to determine a single base change in the target polynucleotide. Applications include measuring "hot spot" point mutations and identifying single nucleoside (SNP) sites. On the base. For example, oligonucleotides are prepared and immediately hybridized next to the polymorphic site. Primers-Includes a single base of the target polynucleotide, and a polymerase system is included in the extension reaction mix. The reaction mixture includes four chain-terminated triphosphates, each with a COIN label. Then an extension reaction is performed, and if it is homologous, only one of the four chain termination nucleotides is added to the end of the primer, and there is an extension primer of COIN. The COIN on the extension primer is detected using Raman spectroscopy. The identification of the label confirms the nucleotides added to a single site to confirm the singulated nucleotides in the target polynucleotide. In the method of the present invention, the sample includes a variety of analyses using the nanocluster analysis according to the present invention. For example, samples can be sampled, including atmospheric air, ambient air, water, mud, soil. In addition, the sample may be a biological sample, including, for example, a patient's exhalation mixture set labeled with a measurement, first an oligonucleotide probe, an acid sequence of an acid sequence of an acid. These acid polymorphic primers are used to change position in the compound. With a unique SNP, the label is used to generate a label followed by a salt-based change, which can be a type 5 and its type, saliva, -32- 200535246 (29) blood, urine, feces , Various organizations and theirs. Industrial applications of the method of the invention using the nano-ball curtains of the present invention include environmental toxicants and treatments, biological agents, material quality control, detection of pathogens in food and agricultural products, detection of anesthetic agents, detection of automobile oil or radioactive fluids, Breath alcohol analyzer, dangerous substance identification, explosion detection, temporary emission identification, medical diagnostics, fish freshness, detection and classification of bacteria and microorganisms in vitro and in vivo in biomedical and medical diagnostic applications, detection, detection Heavy industry manufacturing, ambient air detection, worker protection, launch control, product quality testing, leak detection and identification, oil / gas and petrochemical applications, combustible gas detection, H2S detection, hazardous leak detection and identification, emergency response And law enforcement applications, illegal substance detection and identification, arson research, confined space surveillance, public facilities and energy applications, launch detection, transformer failure detection, food / feeding / agricultural applications, freshness detection, fruit maturity control , Fermentation process detection and control applications, flavoring composition and identification, product quality and identification, refrigerants and fumigation Agent detection, cosmetics / perfume / fragrance formulations, product quality testing, personal identification, chemical / plastic / pharmaceutical applications, leak detection, solvent recovery effectiveness, perimeter detection, product quality testing, hazardous waste application, temporary Launch detection and identification, leak detection and identification, perimeter detection, transportation, hazardous material detection, refueling operations, inspection of transportation containers, diesel / gasoline / aviation fuel identification, building / residential natural gas detection, armor detection Testing, smoke detection, flame detection, automatic ventilation control applications (cooking, smoking, etc.), air introduction detection, hospital / medicine anesthesia and sterilization gas detection, infectious disease detection and respiratory applications, body fluid analysis, Medical applications, drug discovery, remote surgery and the like. -33- 200535246 (30) Another type of sensor-based fluid detection in engine fluids is the application of oil / antifreeze detectors for air / fuel optimization. Engine diagnostics, diesel fuel quality, volatile organic carbon measurement (VOC), temporary gases in refineries, food quality, bad breath, soil and water pollution, air quality detection, combustion safety, chemical weapon identification, hazardous substances Workgroup use, explosion detection, breath analyzer, ethylene oxide storage or anesthetic detector. In another embodiment, a system for detecting an analyte in a sample is provided. The systems include an array containing more than one nano-cluster; a sample containing at least one analyte; a Raman spectrometer; and a computer including an algorithm for analyzing the sample. Various analysis techniques can be used to analyze COIN particles according to the present invention. These technologies include, for example, nuclear magnetic resonance spectroscopy (NMR), photon correlation spectroscopy (PCS), IR, surface plasmon resonance (SPR), XPS, scanning probe microscope (SPM), SEM, TEM, atomic absorption spectroscopy, elemental analysis, UV-vis, fluorescence spectrum, and the like. In carrying out the present invention, the Raman spectrometer may be part of a detection unit designed to detect and quantify the nanoclusters of the present invention by Raman spectroscopy. Methods for detecting Raman-labeled analytes (such as nucleotides) using Raman spectroscopy are known in the art (see, for example, U.S. Patent No. 5,306,4 03; 6,002,47 1; No. 6, 1 74,677). Changes in surface enhanced Raman spectroscopy (SERS), surface enhanced resonance Raman spectroscopy (SERRS), and coherent anti-Stokes Raman spectroscopy (CARS) have been revealed. A non-limiting example of a Raman detection unit is disclosed in U.S. Patent No. -34-200535246 (31) 6,002,47 1. The excitation beam is generated by a 5 3 2 nm wavelength doubling Nd: YAG laser or a 365 nm wavelength doubling Ti: sapphire laser. Either a pulsed laser beam or a continuous laser beam can be used. The excitation beam passes through the confocal optical instrument and the microscope objective lens, and is focused on the flow channel and / or the flow channel member. The Raman emission from the labeled nanoclusters is concentrated on the microscope objective and confocal optics, and is coupled to a monochromatic emitter for spectral dissociation. Confocal Optical instruments include a combination of a dichroic filter, a barrier filter, a confocal pinhole, a lens, and a mirror to reduce the background signal. Standard full-field optics and confocal optics can be used. The Raman emission signal is detected by a Raman detector, including a burst photodiode with a computer that counts and digitizes the signal. Another example of a Raman detection unit is disclosed in US Patent No. No. 306,403 includes gallium arsenide photomultiplier tubes (RC A Model C3 1 03 4 or Bur 1 e I ndustries Μ ode 1 C 3 1 0 3 4 0 2) and operates in single photon counting mode. Double grid type spectrometer. Excitation light sources include 514.5 nanometer argon ion lasers from SpectraPhysics, Model 166 and 647.1 nanometer ray lasers (Innova 70, Coherent). Alternative excitation light sources include a 3 3 7 nm nitrogen laser (Laser Science Inc.) and a 325 nm helium-pot laser (Liconox) (US Pat. No. 6,174,677), a light emitting diode, Nd : YLF laser and various ion lasers and / or dye lasers. The excitation beam can be spectrally purified using a band-pass filter (C 0 r i 0 n), and a 6X objective lens (Newport, Model L6X) can be used to focus on the flow channel and / or flow cell. The objective lens can use a holographic beam splitter (Kaiser Optical Systems, Inc., Model HB 647- -35- 200535246 (32) 2 6N 1 8) to excite Raman-active organic compounds of nanoclusters and collect Raman signals. 'Right-angle geometry that generates an excitation beam and emits a Raman signal. A full-indentation notch wave filter (K a s e r ο p t i c a 1 S y s t e m s, I n c ·) can be used to reduce Rayleigh scattered radiation. The optional Raman detector includes an ISA HR-3 20 spectrum of a red enhanced mouse affinity device (R E -1 C CD) detection system (p r n c e t 〇 n Instruments). Other types of detectors can be used, such as Fourier transform spectrogram (based on Michael son interferometer), charged injection device, photodiode array, InGaAs detector, electron multiplying CCD, enhanced CCD and / or optoelectronic crystal Array. Any suitable form or configuration of Raman spectroscopy or related technologies known in the technical field can be used to detect the nanoclusters of the present invention, including but not limited to normal Raman scattering, resonance Raman scattering, surface enhanced Raman scattering, surface Enhanced resonance Raman scattering, coherent anti-Stokes Raman spectroscopy (CARS), excited Raman scattering, inverse Raman spectroscopy, excitation gain Raman spectroscopy, super Raman scattering, molecular optical laser detector ( Μ Ο LE) or Raman microprobe or Raman microscope or confocal Raman microspectroscopy, three-dimensional or scanning Raman, Raman saturation spectroscopy, time-resolved resonance Raman, Raman decoupling spectroscopy, or UV -Raman microscope. In a specific aspect of the present invention, the system for detecting the nano clusters of the present invention includes an interference processing system. An exemplary interference processing system may contain a computer including a communication bus and a processor for processing interference. The interference processing and control system may additionally include any peripheral devices known in the art, such as a memory, a display, a keyboard, and / or other devices. In a specific example, the detection unit is operatively coupled to the interference processing system. -36- 200535246 (33) The data from the detection unit can be processed by the processor and the data stored in the memory. Data for emission curves for various Raman markers can also be stored in memory. The processor can compare emission spectra from composite organic-inorganic nanoclusters in flow channels and / or flow cells to identify Raman-active organic compounds. The processor can analyze the data from the detection unit to determine, for example, the sequence of a polynucleotide bound by the nanocluster probe of the present invention. The interference processing system can also perform standard procedures such as subtracting background signals.

雖然特定本發明方法可在程式化處理器控制下進行， 但在本發明備擇實施例中，該等方法可藉任何可程序化或 硬編碼邏輯（諸如現場可程式化閘極陣列（F P G A ) 、T 丁 L 邏輯或應用專一性積體電路（A S I C ))完全或部分執行。 此外，所揭示之方法可藉可程式化通用型電腦組件及/或 定製之硬體組件之任何組合來進行。 在數據收集操作之後，一般係將數據記錄於數據分析 操作。爲幫助分析操作，該偵測單元所得之數據一般係使 用釋位電腦來分析。該電腦一般係經適當地程式化，以接 收並儲存來自偵測單元之數據，且分析並記錄所收集之數 據。 特定之本發明實施例中，可使用定製之軟體組合來分 析得自偵測單元之數據。本發明備擇實施例中，數據分析 可使用干擾處理系統及公開販售之軟體組合來進行。 本發明另一實施例中，提供包含多個埋置且一起保持 於聚合物珠粒內之COIN的微球。該等微球可產生較個別 COIN強且較固定之SERS信號。大型微球之聚合物塗層 -37- 200535246 (34) 亦可提供連接生物分子（諸如探針）之表面積。該結構特 色係爲a )由聚合之有機化合物所形成之結構框架；b )多 個埋置於各微型粒子中之COIN; c)具有適於連接所需之 官能基（諸如鍵合基、探針及其類者）之官能基的表面（ 圖1 2 )。以下列出數種製造本實施例微球之方法。 包含法（圖1 3 ):此項硏究採用公認之用以製備均勻 膠乳微球的乳化聚合技術，不同處係C Ο IN係在起始聚合 之前先導入該微胞內。如圖13之流程圖所示，本發明方 法之此態樣包括下列步驟：1 )先藉著水與界面活性劑（ 例如辛醇）均質化來製備具有所需尺寸之微胞。2 ) COIN 粒子與疏水劑（例如SDS ) —起導入。後者有助於COIN 輸送至微胞內部。3 )微胞以安定劑（例如酪蛋白）保護 防止聚集。4)導入單體（例如苯乙烯或甲基丙烯酸甲酯 )。5 )最後，使用游離之自由基起始劑（例如過氧化物 或過硫酸鹽）來起始聚合，以製得埋置COIN之膠乳珠粒 〇 此外，已埋置於固體有機聚合物珠粒內之COIN可用 以形成微球。該珠粒之聚合物可防止微胞中及最終產物（ 微球）中之COIN之間直接接觸。此外，各珠粒中COIN 之數量可藉著改變珠粒空隙中聚合物厚度而加以調整。珠 粒之聚合物材料不需生成信號，聚合物之功能係結構。 該微球尺寸最高達微米，各以具有包含許多藉珠粒之 結構聚合物保持在一起之個別COIN粒子的結構之功能性 單元形式操作。因此，在單一微球內有數個埋置於結構聚 -38- 200535246 (35) 合物中之COIN，該結構聚合物係爲珠粒之主要內部及外 部結構材料。結構聚合物亦作爲表面連接鍵合劑、衍生物 或用以官能化以連接探針。因爲各個COIN包含初級金屬 粒子之團簇，金屬粒子上吸附有至少一個拉曼活性有機化 合物，珠粒之聚合物大部分不與拉曼活性有機化合物（其 在膠體形成期間吸附於COIN結構之初級金屬粒子接合點 中時被捕集）接觸，因此不減弱其拉曼活性。該等位於 COIN周邊上可與微球之結構聚合物接觸的拉曼活性有機 分子作爲拉曼活性分子之效果較低。 浸入法（圖1 4 ):先製得微球並使之接觸個別合成之 COIN。在特定條件（諸如在有機溶劑中）下，珠粒之微 孔放大到足以使C Ο IN擴散入內。在液相變成水相之後， 珠粒之探針關閉，將COIN埋置於聚合物珠粒內。例如1 )苯乙烯單體與二乙烯基苯乙烯與丙烯酸共聚，經由乳化 聚合形成均勻尺寸之珠粒。2 )該珠粒以有機溶劑（諸如 氯仿/丁醇）潤脹，將一組特定比例之COIN導入，使得 COIN擴散至潤脹之珠粒內。3 )該珠粒隨之置入非溶劑中 以使珠粒收縮，使得C Ο IN捕集於內部而形成安定、均勻 之封包有COIN的珠粒。 內建法（圖1 5 ):此方法中，先製得微球珠粒，使之 於有機溶劑中與拉曼標記及銀粒子接觸。在此條件下，珠 粒之微孔放大到足以使標記及銀粒子擴散至內部。COIN 團簇係於銀膠體於有機拉曼標記存在下彼此遭遇之時於微 球珠粒內部形成。可使用熱及光來加速銀粒子之聚集及熔 -39- 200535246 (36) 合。最後，液相變成氣相’封住C0IN。例如1 ) 單體係與二乙烯基苯乙烯與丙烯酸共聚而經由乳化 成均勻尺寸之珠粒。2 )該等珠粒隨後以有機溶劑 仿/ 丁醇潤脹，導入一組在特定比例之拉曼活性分子 如8-氮雜-腺嘌呤及N-苄醯基腺嘌呤），使得分子擄 所潤脹之珠粒內。在相同溶劑中之Ag膠體懸浮液隨 珠粒混合以形成封包Ag粒子之珠粒。3 )溶劑換成使 收縮者，以將拉曼標記及Ag粒子捕集於內部。控制 序，使得Ag粒子彼此接觸，拉曼分子位於接合點中 成位於珠粒內部之COIN。當使用中間尺寸之銀膠體 如60奈米膠體時，個別添加拉曼標記（在添加銀之 之後），以於珠粒內部誘發膠體聚集（形成COIN ) 用1至1〇奈米膠體時，可一起添加標記。隨之使用 熱誘發珠粒內部之活性COIN形成。 外加法（圖16):此方法中，先使用固體核心 COIN連接之擔體。該核心可爲金屬（金及銀）、無 氧化鋁、赤鐵礦及二氧化矽）或有機（聚苯乙烯、膠 粒子。COIN對核心粒子之連接可藉靜電吸引、凡得 及/或共價鍵結來誘發。連接之後，組合體塗覆以聚 ，以使結構安定化，同時提供具有官能基之表面。可 前述方法建立多層COIN層。COIN珠粒之尺寸可藉 尺寸及C ΟIN層數量來控制。例如1 ) 〇 . 5微米帶正電 粒子與帶負電之COIN混合，2 )膠乳-COIN複合物塗 可交聯之聚合物，諸如聚-丙烯酸。3 )聚合物塗層以 :乙烯 :合形 如氯 (例 丨散至 :之與 .珠粒 該程 ，形 諸如 前或 0使 光或 作爲 機（ 乳） 瓦力 合物 基於 核心 膠乳 覆以 鍵合 -40- 200535246 (37) 分子諸如離胺酸交聯’以形成不可溶之外殼。殘留（未反 應）羧基作用供第二層COIN連接或探針連接使用之官能 基。附加之官能基亦可經由共聚或於交聯過程中導入。 【實施方式】 實施例1 一般重點 化學試劑：生物試劑包括抗-IL-2及抗-IL-8抗體係購 自 BD Biosciences Inc。捕集抗體係爲自小鼠產生之單株 抗體，偵測抗體係爲自小鼠產生且與生物素軛合之多株抗 體。液體鹽溶液及緩衝劑係購自 Ambion, Inc. ( Austin， TX，USA)，其包括 5 M NaCl，1 OX PBS ( 1 x PBS 1 37 mM NaCl，2.7 mM KC1，8 mM Na2HP〇4,及 2 mM KH2P04，pH 7.4 )。除非另有陳述，否則所有其他化學物品係購自 Sigma Aldrich Chemical Company ( St. Louis, MO? USA) 之最高品質。實驗所使用之去離子水具有18.2 x 106歐 姆-厘米之電阻，以水純化單元（Nanopure Infinity， Barnstead，USA)得到。 銀種晶粒子合成：硝酸銀（AgN03 )及檸檬酸鈉（檸 檬酸Na3)之儲液經0.1微米聚醯胺膜濾器（Schleicher and Schuell, NH，USA)過濾兩次，其於使用之前充分淋 洗。新製得硼水合鈉溶液（5 0 mM )，且在製備之後的2 小時內使用。藉著於劇烈攪拌下迅速將5 0毫升溶液A ( 含有8.0 0 m Μ檸檬酸鈉、〇 . 6 0 m Μ硼水合鈉及2 · 0 0 m Μ氫 -41 - 200535246 (38) 氧化鈉）添加於50毫升溶液B (含有4.00 mM硝酸鈉） 而製備銀種晶粒子。將溶液B添加於溶液A導致具有更高 之聚合度分散性的懸浮液。銀種晶懸浮液儲存黑暗中’且 在製備後的一週內使用。使用之前，以光子相關光譜法（ PCS，Zetasizer 3 000 HS，Malvern)分析該懸浮液，以確定 強度-平均直徑（z-平均値）係介於10至12奈米之間’而 聚合度分散性指數低於0.25。 金種晶合成：使用家用微波爐（1350瓦，Panasonic )製備金奈米粒子。一般，在玻璃瓶（1〇〇毫升）中之40 毫升含有0.5 mM HAuC14及2.0 mM檸檬酸鈉之水溶液於 微波爐中使用最大功率加熱至沸騰，之後使用低功率設定 値，保持溶液溫和沸騰5分鐘。將2.0克PTFE沸石（6 毫米，Saint-Gobain A1069103，經由 VWR)添加於溶液 中，以促進溫和且有效之沸騰。形成之溶液具有薔薇紅色 。藉PCS測量顯示該金溶液具有一般z-平均値13奈米， 聚合度分散性指數<0.04。 COIN合成：可使用備擇方法。 回流方法：以銀種晶製備COIN粒子時，一般在導入 拉曼標記之前於回流系統中將5 0毫升銀種晶懸浮液（等 於2·0 mM Ag+ )加熱至沸騰。之後逐滴或分小批量（50 至100微米）地添加硝酸銀儲液（0.50 Μ )，以誘發銀種 晶粒子之生長及聚集。總共可添加2.5 mM硝酸銀。該溶 液保持沸騰，直至懸浮液變成極混濁之暗棕色。此時，藉 著將膠體溶液移入玻璃瓶內而迅速降低溫度，之後將其儲 -42 - 200535246 (39) 存於室溫。最佳加熱時間係視拉曼標記之性質及硝酸銀添 加量而定。已發現在停止加熱之前藉PCS或UV-Vis光譜 儀確認粒子達到所需尺寸範圍（例如平均80至1 〇〇奈米 )係有幫助。一般，暗棕色係表示團簇形成且伴有拉曼活 性。 製備具有金種晶之COIN粒子時，金種晶先於拉曼標 記（例如 2 0 // Μ 8 -氮雜-腺嘌呤）存在下自0.2 5 mM HAhC14製備。該金種晶溶液力[]熱至沸騰之後，個別地添 加硝酸銀及檸檬酸鈉儲液（〇 . 5 0 Μ )，使得最終金懸浮液 含有1·0 mM AgN03及1.0 mM檸檬酸鈉。氯化銀沉澱物 可在硝酸銀添加後立即形成，但在加熱時迅速消失。沸騰 之後’橙棕色發展出來且安定下來，添加另外一份（50至 1〇〇微升）硝酸銀及檸檬酸鈉儲液（各0.50 M)，以誘發 綠色之發展，其指示團簇形成且伴生拉曼活性。 該方法產生具有不同顏色之COIN，主要因爲在團簇 形成之前的初級粒子尺寸差異。 烘烤法：COIN亦可簡便地使用對流爐製備。銀種晶 懸浮液與檸檬酸鈉及硝酸銀溶液於2〇毫升玻璃管瓶中混 合。混合物之最終體積一般爲1 0毫升，其含有銀粒子（ 等於〇·5 mM銀離子）、1.0 mM硝酸銀及2.0 mM檸檬酸 鈉（包括來自種晶懸浮液之部分）。玻璃管瓶於設定在95 °C之爐中培育60分鐘，之後儲存於室溫。同時測試標記 濃度範圍。測試混濁之帶棕色批物之拉曼活性及膠體安定 性。丟棄具有明顯沉降（發生於標記濃度太高時）之批物 -43- 200535246 (40) 。未顯示充分濁度之批物可長時間（最長達3日）保持於 室溫，以使之形成團簇物。許多情況下’懸浮液因聚集而 隨時間變得更混濁，在2 4小時內顯示出強拉曼活性。可 使用安定劑諸如牛血淸蛋白（BSA )來終止聚集並安定化 該COIN粒子。 使用類似硏究來製備具有金核心之C Ο IN。簡言之，3 毫升於拉曼標記存在下製備之金懸浮液（〇·5〇 mM Au3+) 於2 0毫升玻璃管瓶中與7毫升檸檬酸銀溶液（混合前含 有5.0 mM硝酸銀及5.0 mM檸檬酸鈉）混合。管瓶置入對 流爐中並加熱至9 5 °C歷經1小時。可同時使用不同濃度之 經標記金種晶，以製得具有充分拉曼活性之批物。應注意 COIN試樣之尺寸及拉曼活性可不均勻。吾人一般使用離 心（200至2,000 xg歷經5至10分鐘）或過濾（200 kDa ，1000 kDa 或 0.2 微米濾器，Pall Life Sciences，經由 VWR)以增濃在50至100奈米範圍內之粒子。該COIN 粒子可在增濃之前塗覆以例如BSA或抗體。所製備之部 分批量COIN (在合成未進一步處理）於室溫下保持安定 超過3個月，而不會有明顯之物性及化性改變。 冷方法：100毫升銀粒子（1 mM銀原子）與1毫升拉 曼標記溶液（一般1 mM )混合。之後，添加5至1 0毫升 0.5 M LiCl溶液以誘發銀聚集。一旦懸浮液在目測下變暗 (因聚集所致），則添加0.5% BSA以抑制該聚集過程。 之後，懸浮液於4500g下離心15分鐘。移除上淸液（大 部分爲單一粒子）之後，片粒再懸浮於1 mM檸檬酸鈉溶 -44- 200535246 (41) 液中。洗滌程序共重複三次。在最後一次洗滌之後，再懸 浮之片粒經〇 · 2微米膜濾器過濾，以移除大型聚集物。收 集濾液爲 COIN懸浮液。COIN之濃度藉著與} mM供 SERS使用之銀膠體比較在400奈米下的吸光度，以i mM 檸檬酸鈉調至1 · 〇或1 · 5 m Μ。 應注意C ΟIΝ試樣之尺寸及拉曼活性可不均勻。吾人 一般使用離心（200至2,0 00 X g歷經5至1〇分鐘）或過 濾（200 kDa，1000 kDa 或 0.2 微米濾器，Pall LifeAlthough specific methods of the invention may be performed under the control of a programmable processor, in alternative embodiments of the invention, the methods may be implemented by any programmable or hard-coded logic such as a field programmable gate array (FPGA) Logic or application specific integrated circuit (ASIC)) is fully or partially implemented. In addition, the disclosed method can be performed by any combination of programmable general purpose computer components and / or custom hardware components. After the data collection operation, the data is generally recorded in a data analysis operation. To assist the analysis operation, the data obtained by the detection unit is generally analyzed using a release computer. The computer is generally properly programmed to receive and store data from the detection unit, and analyze and record the collected data. In a specific embodiment of the present invention, a customized software combination may be used to analyze the data obtained from the detection unit. In an alternative embodiment of the present invention, data analysis may be performed using a combination of an interference processing system and a publicly available software. In another embodiment of the present invention, microspheres comprising a plurality of COINs embedded and held together in polymer beads are provided. These microspheres can produce stronger and more fixed SERS signals than individual COINs. Polymer Coatings for Large Microspheres -37- 200535246 (34) It also provides surface area for attaching biomolecules such as probes. The structural features are a) a structural framework formed by polymerized organic compounds; b) multiple COINs embedded in each microparticle; c) having functional groups suitable for connection (such as bonding groups, probes, etc.) Needle and the like) functional surface (Figure 12). Several methods for manufacturing the microspheres of this embodiment are listed below. Inclusion method (Figure 1 3): This study uses the accepted emulsion polymerization technology for preparing homogeneous latex microspheres. The different locations are the C 0 IN systems that are introduced into the cells before starting polymerization. As shown in the flow chart of FIG. 13, this aspect of the method of the present invention includes the following steps: 1) First, prepare cells with a desired size by homogenizing water with a surfactant (such as octanol). 2) COIN particles are introduced together with a hydrophobic agent (such as SDS). The latter facilitates the delivery of COIN into the microcell. 3) Microcells are protected from stabilizing agents (such as casein) from aggregation. 4) Introduce a monomer (such as styrene or methyl methacrylate). 5) Finally, use a free radical initiator (such as peroxide or persulfate) to initiate polymerization to obtain latex beads with embedded COIN. In addition, solid organic polymer beads have been embedded The COIN inside can be used to form microspheres. The polymer of the beads prevents direct contact between COINs in the cells and in the final product (microspheres). In addition, the amount of COIN in each bead can be adjusted by changing the polymer thickness in the bead voids. The polymer material of the beads does not need to generate signals, and the functional structure of the polymer. The microspheres are up to a micron in size, each operating in the form of a functional unit having a structure of individual COIN particles containing a number of structural polymers held together by beads. Therefore, there are several COINs embedded in a structural poly-38-200535246 (35) compound in a single microsphere. This structural polymer is the main internal and external structural material of the beads. Structural polymers also function as surface-attachment bonding agents, derivatives, or to be functionalized to connect probes. Because each COIN contains clusters of primary metal particles, at least one Raman-active organic compound is adsorbed on the metal particles, and most of the polymer of the beads does not interact with Raman-active organic compounds (which are adsorbed on the primary of the COIN structure during colloid formation) Metal particles are trapped in the junction), so their Raman activity is not reduced. These Raman-active organic molecules, which can be in contact with the microsphere's structural polymer on the periphery of the COIN, are less effective as Raman-active molecules. Immersion method (Figure 1 4): Microspheres are first prepared and brought into contact with individually synthesized COIN. Under certain conditions (such as in organic solvents), the micropores of the beads are enlarged enough to allow CO IN to diffuse into it. After the liquid phase becomes the aqueous phase, the probe of the beads is closed, and COIN is buried in the polymer beads. For example, 1) styrene monomer is copolymerized with divinylstyrene and acrylic acid, and beads of uniform size are formed through emulsion polymerization. 2) The beads are swelled with an organic solvent (such as chloroform / butanol), and a specific set of COIN is introduced so that the COIN diffuses into the swollen beads. 3) The beads are then placed in a non-solvent to shrink the beads, so that CO IN is trapped inside to form a stable, uniform bead with COIN. Built-in method (Figure 15): In this method, microsphere beads are first prepared and brought into contact with Raman labels and silver particles in an organic solvent. Under these conditions, the micropores of the beads are enlarged enough to allow the label and silver particles to diffuse inside. COIN clusters are formed inside the microsphere beads when silver colloids encounter each other in the presence of organic Raman labels. Heat and light can be used to accelerate the aggregation and fusion of silver particles. Finally, the liquid phase changes to the gas phase 'to seal COIN. For example, 1) a single system is copolymerized with divinylstyrene and acrylic acid to emulsify to form beads of uniform size. 2) The beads are then swelled with organic solvent imitation / butanol, and a set of Raman-active molecules such as 8-aza-adenine and N-benzyl adenine are introduced in a specific ratio, so that the molecular structure Inside the swelling beads. A colloidal suspension of Ag in the same solvent is mixed with the beads to form beads that encapsulate the Ag particles. 3) The solvent is changed to a shrinker to trap Raman marks and Ag particles inside. Control the sequence so that the Ag particles are in contact with each other, and the Raman molecules are located in the junction to form the COIN inside the beads. When using silver colloids of intermediate size, such as 60 nm colloids, individually add Raman labels (after adding silver) to induce colloidal aggregation (formation of COIN) within the beads. When using 1 to 10 nm colloids Add tags together. The subsequent use of heat induces the formation of active COIN inside the beads. Additive method (Figure 16): In this method, a solid core COIN connected carrier is used first. The core can be metal (gold and silver), alumina-free, hematite and silicon dioxide) or organic (polystyrene, colloidal particles. The connection of COIN to the core particles can be attracted by static electricity, where available and / or shared) Induced by valence bonding. After the connection, the assembly is coated with poly to stabilize the structure while providing a surface with functional groups. Multi-layer COIN layers can be established by the methods described above. The size of the COIN beads can be measured by the size and the C OIN layer The quantity is controlled. For example, 1) 0.5 micron positively charged particles are mixed with negatively charged COIN, and 2) the latex-COIN composite is coated with a cross-linkable polymer, such as poly-acrylic acid. 3) The polymer coating is: ethylene: shaped like chlorine (eg, scattered to: and its. Beads this way, shaped like the front or 0 to make light or as an organic (emulsion) tile based on the core latex coated with Bonding -40- 200535246 (37) Molecules such as lysine are cross-linked to form an insoluble shell. Residual (unreacted) carboxyl groups act as functional groups for second-layer COIN connection or probe connection. Additional functional groups It can also be introduced through copolymerization or during the cross-linking process. [Embodiment] Example 1 General key chemical reagents: Biological reagents include anti-IL-2 and anti-IL-8 antibody systems purchased from BD Biosciences Inc. Capture antibody system It is a single antibody produced from mice, and the detection system is a multi-body antibody produced from mice and conjugated with biotin. Liquid salt solution and buffer were purchased from Ambion, Inc. (Austin, TX, USA) It includes 5 M NaCl, 1 OX PBS (1 x PBS 1 37 mM NaCl, 2.7 mM KC1, 8 mM Na2HP〇4, and 2 mM KH2P04, pH 7.4). Unless otherwise stated, all other chemicals were purchased Highest quality from Sigma Aldrich Chemical Company (St. Louis, MO? USA) The deionized water used in the experiment has a resistance of 18.2 x 106 ohm-cm and is obtained by a water purification unit (Nanopure Infinity, Barnstead, USA). Synthesis of silver seed particles: silver nitrate (AgN03) and sodium citrate (Na3 citrate) The stock solution was filtered twice through a 0.1 micron polyamide membrane filter (Schleicher and Schuell, NH, USA), which was thoroughly rinsed before use. A new sodium boron hydrate solution (50 mM) was prepared, and after the preparation, Use within 2 hours. By vigorously stirring 50 ml of solution A (containing 8. 0 0 Μ sodium citrate, 0.6 0 Μ sodium boron hydrate and 2. 0 0 Μ hydrogen-41-200535246 ( 38) Sodium oxide) was added to 50 ml of solution B (containing 4.00 mM sodium nitrate) to prepare silver seed crystal grains. Adding solution B to solution A resulted in a suspension with a higher degree of polymer dispersion. Silver seed crystal suspension Store in the dark 'and use within one week after preparation. Prior to use, the suspension was analyzed by photon correlation spectroscopy (PCS, Zetasizer 3 000 HS, Malvern) to determine the intensity-average diameter (z-average radon) system Between 10 and 12 nm 'and A polydispersity index of less than 0.25 degree gold seed crystal synthesized: using a domestic microwave oven (1350 Watts, Panasonic) Preparation of gold nanoparticles. Generally, 40 ml of a glass bottle (100 ml) containing 0.5 mM HAuC14 and 2.0 mM sodium citrate in a microwave oven is heated to boiling with the maximum power, and then a low power setting is used, and the solution is gently boiled for 5 minutes . 2.0 grams of PTFE zeolite (6 mm, Saint-Gobain A1069103, via VWR) was added to the solution to promote gentle and effective boiling. The resulting solution has a rose red color. The PCS measurement showed that the gold solution had a general z-average of 奈 13 nm, and a polymerization degree dispersion index < 0.04. COIN Synthesis: Alternative methods can be used. Reflow method: When preparing COIN particles with silver seed crystals, generally 50 ml of silver seed crystal suspension (equivalent to 2.0 mM Ag +) is heated to boiling in a reflux system before introduction of Raman labeling. Then add the silver nitrate stock solution (0.50 M) dropwise or in small batches (50 to 100 microns) to induce the growth and aggregation of silver seed particles. A total of 2.5 mM silver nitrate can be added. The solution remained boiling until the suspension became extremely cloudy and dark brown. At this time, quickly reduce the temperature by moving the colloidal solution into a glass bottle, and then store it at -42-200535246 (39) at room temperature. The optimal heating time depends on the nature of the Raman mark and the amount of silver nitrate added. It has been found to be helpful to confirm that the particles have reached the desired size range (for example, on average 80 to 1000 nm) by PCS or UV-Vis spectrometer before stopping heating. In general, dark brown lines indicate cluster formation with Raman activity. When preparing COIN particles with gold seed crystals, gold seed crystals are prepared from 0.2 5 mM HAhC14 in the presence of a Raman tag (eg, 20 / M 8-aza-adenine). After the gold seed solution was heated to boiling, silver silver nitrate and sodium citrate stock solution (0.50 M) were individually added so that the final gold suspension contained 1.0 mM AgN03 and 1.0 mM sodium citrate. Silver chloride precipitates formed immediately after the addition of silver nitrate, but quickly disappeared upon heating. After boiling, 'orange-brown developed and settled, adding another (50 to 100 microliters) of silver nitrate and sodium citrate stock solution (0.50 M each) to induce green development, indicating cluster formation and accompanying Raman activity. This method produces COINs with different colors, mainly due to the difference in primary particle size before cluster formation. Baking method: COIN can also be prepared simply by using a convection oven. The silver seed crystal suspension was mixed with sodium citrate and silver nitrate solution in a 20 ml glass vial. The final volume of the mixture is generally 10 ml, which contains silver particles (equivalent to 0.5 mM silver ions), 1.0 mM silver nitrate, and 2.0 mM sodium citrate (including the portion from the seed crystal suspension). The glass vials were incubated in an oven set at 95 ° C for 60 minutes, and then stored at room temperature. The marker concentration range is also tested. The turbid brownish batch was tested for Raman activity and colloidal stability. Discard batches with significant settling (occurring when the labeled concentration is too high) -43- 200535246 (40). Batches that do not show sufficient turbidity can be kept at room temperature for a long time (up to 3 days) to allow them to form clusters. In many cases, the 'suspensions became more cloudy with time due to aggregation, and showed strong Raman activity within 24 hours. A stabilizing agent such as bovine hemoglobin (BSA) can be used to stop aggregation and stabilize the COIN particles. A similar study was used to prepare CO IN with a gold core. Briefly, 3 ml of a gold suspension (0.50 mM Au3 +) prepared in the presence of Raman labeling was placed in a 20 ml glass vial with 7 ml of silver citrate solution (containing 5.0 mM silver nitrate and 5.0 mM before mixing Sodium citrate). The vial was placed in a convection oven and heated to 95 ° C for 1 hour. Labeled gold seed crystals of different concentrations can be used simultaneously to produce batches with sufficient Raman activity. It should be noted that the size and Raman activity of the COIN sample may not be uniform. We generally use centrifugal (200 to 2,000 xg for 5 to 10 minutes) or filtration (200 kDa, 1000 kDa or 0.2 micron filter, Pall Life Sciences, via VWR) to thicken particles in the range of 50 to 100 nanometers. The COIN particles can be coated with, for example, BSA or antibodies before being enriched. Part of the prepared batch of COIN (synthesized without further processing) remained stable at room temperature for more than 3 months without significant physical and chemical changes. Cold method: 100 ml of silver particles (1 mM silver atoms) are mixed with 1 ml of Raman labeling solution (typically 1 mM). After that, 5 to 10 ml of a 0.5 M LiCl solution was added to induce silver aggregation. Once the suspension darkened visually (due to aggregation), 0.5% BSA was added to suppress the aggregation process. After that, the suspension was centrifuged at 4500 g for 15 minutes. After removing the supernatant liquid (mostly single particles), the tablets were resuspended in 1 mM sodium citrate-44-200535246 (41) solution. The washing procedure was repeated three times in total. After the last wash, the resuspended pellets were filtered through a 0.2 micron membrane filter to remove large aggregates. The collected filtrate was a COIN suspension. The concentration of COIN was adjusted to 1 · 0 or 1 · 5 mM with i mM sodium citrate by comparing the absorbance at 400 nm with} mM silver colloid for SERS. It should be noted that the size and Raman activity of the CONO sample may not be uniform. We generally use centrifugation (200 to 2,000 X g for 5 to 10 minutes) or filtration (200 kDa, 1000 kDa or 0.2 micron filter, Pall Life

Sciences’經由VWR)以增濃在50至loo奈米範圍內之 粒子。該COIN粒子可在增濃之前塗覆以保護劑（例如 BSA、抗體）。所製備之部分批量COIN (在合成未進一 步處理）於室溫下保持安定超過3個月，而不會有明顯之 物性及化性改變。 粒徑測量：銀及金種晶粒子及COIN之尺寸係使用光 子相關光譜法（PCS，Zetasizer3 3 000 HS 或 Nano-ZS， Malvern )測定。所有測量皆於25 °C使用 He-Ne雷射於 63 3奈米下進行。試樣視需要以DI水稀釋。TEM分析： 透射式電子顯微鏡（TEM )分析係使用塗覆碳之銅柵來製 備試樣。試樣懸浮液係使用完全玻璃之噴霧器（Ted Pella )噴灑於栅上。或試樣懸浮液滴（20微升）沈積於該柵上 。五分鐘後，以一張濾器吸除液滴。使該柵接觸DI水滴 之表面歷經數秒鐘，以在空氣中乾燥之前移除鹽類。TEΜ 觀察係使用具有 UHR極之 JEM 2010或201 OF ( Japan Electron Optics laboratories)進行。SEM 分析：掃描式電 -45- 200535246 (42) 子顯微鏡（SEM )分析係於掃描式電子顯微鏡（ Hitachi)下檢測COIN粒子。試樣製備方法如下·· 晶圓基材（1 X 1厘米2 )以一滴（2 0微升）聚-L -離 0 . 1 % )潤濕；5分鐘後，基材以去離子水（DI水） 於氮流下乾燥；2 0微升膠體試樣沈積於塗覆有聚 酸之基材上。該基材最後以DI水淋洗，並在SEM 前於空氣中乾燥。拉曼光譜分析：在溶液中之所窄 及COIN檢測皆使用裝置有514奈米氬離子雷射（ )之拉曼顯微鏡（Renishaw, UK )。一般，將一滴 2 0 〇微升）試樣放置於鋁表面上。雷射光束聚焦於 面之頂表面上，收集光子歷經1 0至2 0秒。拉曼系 在10秒鐘收集時間內於1 040厘米-1自甲醇生成約 數。固定於表面上之分析物之拉曼光譜偵測中，使 於屋內之拉曼顯微鏡記錄拉曼光譜。此拉曼顯微鏡 連續波模式操作之水冷式氬離子雷射、二色型反射 息凹口濾波器、Czerny-Turner光譜儀及液態氮 C CD (電荷耦合裝置）照相機構成。光譜組件係連 鏡，使得顯微鏡物鏡將雷射光束聚焦於試樣上，並 向散射之拉曼發射。試樣上之雷射功率〜60毫瓦。 曼光譜皆於5 1 4奈米激發波長收集。 吸收光譜分析：拉曼標記及膠體懸浮液之激發 以 UV-Vis 光譜儀（Model 8453，Agilent Technolog 錄。 COIN粒子與抗體之軛合：5 00微升在1 mM檸 S-4500, 小片矽 丨胺酸（ 淋洗並 -L -離胺 觀察之 r SERS 25毫瓦 (50至 試樣液 統一般 60 0計 用內建 係由在 器、全 冷卻型 接顯微 收集反 所有拉 光譜係 i e s )記 檬酸鈉 -46- 200535246 (43) (pH 9 )中含2毫微克生物素化抗人類IL-2或IL-8抗體 (抗- IL-2或抗- IL-8 )之溶液與5 00微升COIN溶液（使 用8-氮雜-腺嘌呤或N-苄醯基-腺嘌呤製得）混合；形成之 溶液於室溫下培育1小時，之後添加100微升PEG-400 ( 聚乙二醇-400 )。溶液於室溫下培育另外30分鐘，之後 將 200微升1% Tween-20㊣添加於該溶液中。該溶液於 2 000 X g下離心10分鐘。移除上淸液之後，片粒再懸浮 於1毫升含有0.5% BSA、0.1 % Tween-20及1 mM檸檬酸 鈉（BS AT)之溶液中。該溶液隨後於1 000 X g下離心10 分鐘。重複該B SAT洗滌程序總共3次。最終片粒再懸浮 於 700 微升稀釋溶液（0.5% BSA, IX PBS, 0.05% Tween-20® )中。使用拉曼顯微鏡（其於10秒鐘收集時間內於 1 040厘米°下自甲醇產生約600計數）測量COIN之拉曼 活性，並將其調整爲每微升每1 〇秒約500個光子計數之 比活性。 抗體-COIN軛合之確認：爲得到標準曲線，根據製造 商指示（BD BioSciences)使用固定之捕集抗體、固定分 析物濃度（5毫微克/毫升IL-2蛋白質）及連續稀釋之偵 測抗體（〇, 〇.〇1，〇·1, 1及10微克/毫升）進行ELISA (酶 鍵合免疫吸收檢測）實驗。偵測抗體鍵結之後，鏈親和 素-HRP (辣根過氧酶）隨之與生物素化偵測抗體反應，施 加TMB (四甲基聯苯胺）受質，之以進行UV吸光度測量 。藉著相對於抗體濃度繪出吸光度値而產生標準曲線。爲 評估可連接於COIN粒子上之抗體分子的量，隨之使用軛 -47- 200535246 (44) 合有偵測抗體之COIN進行相同ELISA實驗。收集ELISA 數據，COIN-抗體軛合物之鍵結活性與標準曲線比較，以 估計抗體於COIN-抗體軛合物中之當量。假設在鍵結於固 定分析物之COIN粒子上僅軛合一個抗體分子，則所有 COIN粒子所具之生物素部分皆藉鏈親和素-HRP鍵結。最 後，藉將COIN-抗體中之抗體當量除以估計之COIN粒子 數目，則估計出每個COIN之抗體分子數目。吾人估計 COIN粒子上可有多達50個抗體分子。Sciences' via VWR) to thicken particles in the range of 50 to loo nanometers. The COIN particles can be coated with a protective agent (eg, BSA, antibodies) before being enriched. Some of the prepared batches of COIN (not further processed in the synthesis) remain stable for more than 3 months at room temperature without significant physical and chemical changes. Particle size measurement: The size of silver and gold seed grains and COIN are determined using photon correlation spectroscopy (PCS, Zetasizer3 3 000 HS or Nano-ZS, Malvern). All measurements were performed at 25 ° C using He-Ne laser at 63 3 nm. The samples were diluted with DI water as needed. TEM analysis: Transmission electron microscope (TEM) analysis uses carbon-coated copper grids to prepare samples. The sample suspension was sprayed onto the grid using a full glass sprayer (Ted Pella). Or sample suspension droplets (20 μl) are deposited on the grid. After five minutes, the droplets were aspirated through a filter. The grid was exposed to DI water droplets for several seconds to remove salts before drying in air. TEM observations were performed using JEM 2010 or 201 OF (Japan Electron Optics laboratories) with UHR poles. SEM analysis: Scanning Electron-45- 200535246 (42) A sub-microscope (SEM) analysis is performed using a scanning electron microscope (Hitachi) to detect COIN particles. The sample preparation method is as follows: · The wafer substrate (1 X 1 cm 2) is wetted with one drop (20 microliters) of poly-L-0.1%); after 5 minutes, the substrate is deionized water ( DI water) dried under a stream of nitrogen; 20 microliters of colloidal samples were deposited on a substrate coated with polyacid. The substrate was finally rinsed with DI water and dried in air before SEM. Raman spectroscopy: Raman microscopes (Renishaw, UK) with a 514 nm argon ion laser () are used to measure the narrowness and COIN in the solution. Generally, a drop of 200 microliters) of the sample is placed on the aluminum surface. The laser beam is focused on the top surface of the surface, and photons are collected over 10 to 20 seconds. The Raman system produced approximately a fraction from methanol at 1,040 cm-1 in a 10-second collection time. In the detection of the Raman spectrum of the analyte fixed on the surface, a Raman microscope in the room is used to record the Raman spectrum. This Raman microscope consists of a water-cooled argon ion laser operating in continuous-wave mode, a dichroic reflectance notch filter, a Czerny-Turner spectrometer, and a liquid nitrogen C CD (charge coupled device) camera. The spectral components are connected to a mirror so that the microscope objective focuses the laser beam on the specimen and emits it to the scattered Raman. Laser power on the sample is ~ 60 mW. The Mann spectra were collected at an excitation wavelength of 5 1 4 nm. Absorption spectrum analysis: Raman labeling and colloidal suspension excitation using a UV-Vis spectrometer (Model 8453, recorded by Agilent Technolog. COIN particle and antibody conjugation: 5 00 μl in 1 mM lemon S-4500, small piece of silicon amine) Acid (eluate and -L-ionamine for observation SERS 25 milliwatts (50 to 60% of the sample system is generally used in the built-in system by the microscope, full cooling type to collect all anti-pull spectrum ies) Remember sodium citrate-46-200535246 (43) (pH 9) containing 2 nanograms of biotinylated anti-human IL-2 or IL-8 antibody (anti-IL-2 or anti-IL-8) solution with 5 00 μl of COIN solution (made using 8-aza-adenine or N-benzyl-adenine) was mixed; the resulting solution was incubated at room temperature for 1 hour, and then 100 μl of PEG-400 (polyethylene Glycol-400). The solution was incubated at room temperature for another 30 minutes, after which 200 microliters of 1% Tween-20㊣ was added to the solution. The solution was centrifuged at 2 000 X g for 10 minutes. The supernatant was removed The tablets were then resuspended in 1 ml of a solution containing 0.5% BSA, 0.1% Tween-20 and 1 mM sodium citrate (BS AT). The solution was then centrifuged at 1,000 X g for 10 minutes Bell. Repeat this B SAT washing procedure a total of 3 times. The final pellets were resuspended in 700 μl of the diluted solution (0.5% BSA, IX PBS, 0.05% Tween-20®). Using a Raman microscope (which was under 10 seconds Approximately 600 counts were generated from methanol at 1 040 cm at collection time) Raman activity of COIN was measured and adjusted to a specific activity of approximately 500 photon counts per microliter per 10 seconds. Antibody-COIN Conjugate Confirmation: To obtain the standard curve, use a fixed capture antibody, a fixed analyte concentration (5 ng / ml IL-2 protein), and a serially diluted detection antibody (〇, 〇.〇.) According to the manufacturer's instructions (BD BioSciences). 1.0, 1.1 and 10 μg / ml) for ELISA (enzyme-linked immunosorbent detection) experiments. After detecting antibody binding, streptavidin-HRP (horseradish peroxidase) is then biotinylated To detect the antibody response, a TMB (tetramethylbenzidine) substrate was applied for UV absorbance measurement. A standard curve was generated by plotting the absorbance 値 relative to the antibody concentration. To evaluate antibody molecules that can be attached to COIN particles Amount, followed by using yoke -47- 200535246 (44) The same ELISA experiment was performed on the COIN with the detection antibody. The ELISA data was collected, and the binding activity of the COIN-antibody conjugate was compared with the standard curve to estimate the equivalent of the antibody in the COIN-antibody conjugate. Assuming that only one antibody molecule is conjugated to the COIN particle bound to the fixed analyte, the biotin portion of all COIN particles is bound by streptavidin-HRP. Finally, the number of antibody molecules per COIN can be estimated by dividing the antibody equivalent in the COIN-antibody by the estimated number of COIN particles. We estimate that there can be up to 50 antibody molecules on COIN particles.

免疫夾層檢測：（1 )檢測擔體製備：Xen〇bindTM醛 載片（Poly sciences，Inc·，PA，USA )作爲供免疫檢測使用 之基材；使用之後，位於載片上之井洞係藉著覆上一片1 毫米厚之固化 PDMS ( D· Duffy， J. McDonald, 0. S chuel 1 er, and G. Whitesides, Rapid Prototyping of Microfluidic Systems in Poly (dime thy l sil ox an e) o Anal. Chem·，1 998， 70(23): p. 4974-4984 )而製備。該 PDMS 具有直徑5毫米之孔洞。（2 )捕集抗體鍵結：抗-人類 IL-2抗體（9微克/毫升）係於0.33XPBS中製備。將一份 5〇微升抗體添加於位在載片上之井洞中，該載片於37t 潮濕槽中培育2小時。（3 )表面封阻：移除抗體溶液之 後，將50微升在1〇 mM甘胺酸溶液中之1% BSA添加於 各井洞中，以驟冷該醛基。載片於3 7 °C培育另1小時，之 後井洞洗滌4次，每次皆使用50微升PBST洗滌溶液（ 1XPBS，補充以 〇·〇5°/。Tween-20®) 。 （4)蛋白質鍵結： 於稀釋緩衝劑（1 X P B S，0.5 % B S A，0 . 〇 5 % T w e e η - 2 0 )中 200535246 (45) 製備各種濃度（自〇至5 0微克/毫升，視實驗而定）之 IL-2及IL-8蛋白質溶液。將含有40微升抗體溶液之試樣 添加於井洞中；鍵結係於3 7 °C下進行數小時（進行隔夜以 確定完成鍵結）。含試樣之井洞係以5 0微升P B S T溶液洗 滌總共4次。（5 )偵測抗體鍵結：結合等量之個別軛合 有抗-IL-2偵測抗體及抗-IL-8偵測抗體之COIN試樣，之 後添加於各PDMS井洞；溶液隨後於37t下培育1小時。 移除軛合溶液後，井洞洗滌四次，各使用5 0微升稀釋緩 衝溶液，之後以5 0微升DI水洗滌一次。最後，於偵測拉 曼信號之前將3 0微升DI水添加於各井洞。 實施例2 COIN合成及分析：平均粒徑12奈米之銀膠體溶液（ 50毫升）係自2 mM AgN03，0.3 mM NaBH4製得且補充有 4 mM檸檬酸Na3。該溶液於添加8-氮雜-腺嘌呤（AA)達 最終2 // Μ之前加熱至沸騰。沸騰5分鐘後，添加附加之 0.5 mM AgN03。隨後降低溫度並保持於95 + l°C。於指定 時間間隔聚出部分（各1毫升）溶液，以在使用1 mM檸 檬酸鈉以1 : 3 0稀釋後測定光譜。如圖2 A所示，所取出之 試樣份的吸收光譜顯示在較大波長（ >45 0奈米）有波峰 位移及增高之吸光度。在取出試樣份（各5 0微升，置入 位於白光箱上之陪氏培養皿中）之時間間隔（小箭號表示 進一步分析吸光度變化之位置）拍照，顯示隨著反應加熱 時間之時間相依性顏色變化。以反應（加熱）時間函數表 -49 - 200535246 (46) 示之吸光度及拉曼活性係出示於圖2 B。在6 5分鐘後之 7 0 0奈米吸光度降低係因形成在溶液中迅速沉降之大型聚 集體。 實施例3 有機化合物誘發之金屬粒子聚集··使用在1 mM檸檬 酸Na3中之如本發明所述般製備之金屬粒子（金15奈米 ，Abs52〇nm = 〇.37;銀 60 奈米，Abs42()nm = 0.3) •，各有機化 合物（參照表1中之縮寫關鍵字）於光譜測定之前與所示 濃度之金屬膠體溶液試樣混合1 0分鐘。就各試樣而言， 主峰之吸光度係使用波峰1値，而較高波長（600奈米至 7 00奈米）之較高吸光度係作爲波峰2値；波峰2/波峰1 之比例係相對於有機化合物濃度繪圖；高値比例表示高度 之金屬粒子聚集。圖6A出示由有機化合物誘發之金粒子 聚集。相對低濃度之有機化合物即足以使銀粒子聚集。如 圖6B所示，需要相對高濃度之有機化合物來誘發銀粒子 聚集。 實施例4 以8-氮雜-腺嘌呤濃度之函數表示的銀粒子ζ電位··銀 粒子係藉著於95°C至l〇(TC以檸檬酸鈉還原硝酸銀而製備 。該粒子以 PCS ( Zetasizer Nan o-ZS，Malvern)測量之 ζ· 平均尺寸係爲47奈米。銀總濃度係藉著用以測定ζ電位之 1.00 Mm檸檬酸鈉懸浮介質而固定於〇·1〇 mM。使用相同 -50- 200535246 (47) 銀濃度及懸浮介質’測量於2 0 // Μ 8 -氮雜-腺曝玲存在下 之聚集體尺寸（ζ -平均値）發展。圖7 Α及Β個別顯示絕 對ζ電位及聚集動力學。預測在使用遠較爲高之銀濃度（1 至4.5 mM )及較小粒子（小於20奈米）之COIN合成條 件下，會有較高之絕對ζ電位及較慢之聚集動力學。 實施例5 銀粒子之ΤΕΜ分析係於四種製備條件下進行：銀膠 體係藉本發明所述方法合成。1 ·試樣保持於室溫歷經1 週，之以藉透射式電子顯微鏡（ΤΕΜ )分析，顯示大部分 粒子皆小於1 〇奈米。2 .來自相同來源之銀試樣係沸騰40 分鐘，之後冷卻至室溫，之後進行ΤΕΜ分析，顯示粒徑 無明顯變化。3 ·來自相同來源之銀試樣係於室溫下使用 8-氮雜-腺嘌呤（最終濃度20 /z Μ )培育兩週，之後進行 ΤΕΜ分析，顯示部分粒子開始聚集並熔合。4 ·在20 μ Μ 8-氮雜-腺嘌呤存在下沸騰19分鐘後以ΤΕΜ分析銀粒子顯 示出現小型粒子（小於1 0奈米）及大型粒子（大於1 〇奈 米）。此等結果（亦參照圖2 )導致長時間沸騰導致團簇 形成之結論。 實施例6 塗覆有BSA之COIN的合成Immune sandwich detection: (1) Preparation of detection support: Xen〇bindTM aldehyde slide (Poly sciences, Inc., PA, USA) as a substrate for immunoassay; after use, the wells located on the slide are by Cover with a 1 mm thick cured PDMS (D. Duffy, J. McDonald, 0. Schuel 1 er, and G. Whitesides, Rapid Prototyping of Microfluidic Systems in Poly (dime thy l sil ox an e) o Anal. Chem ·, 1 998, 70 (23): p. 4974-4984). The PDMS has a 5 mm diameter hole. (2) Capture antibody binding: anti-human IL-2 antibody (9 μg / ml) was prepared in 0.33 × PBS. A 50 microliter portion of the antibody was added to a well located on a slide, which was incubated for 2 hours in a 37t humidified bath. (3) Surface blocking: After removing the antibody solution, 50 microliters of 1% BSA in a 10 mM glycine solution was added to each well to quench the aldehyde group. The slides were incubated at 37 ° C for another 1 hour, after which the wells were washed 4 times, each time using 50 microliters of PBST washing solution (1XPBS, supplemented with 0.05 ° /. Tween-20®). (4) Protein binding: 200535246 in dilution buffer (1 XPBS, 0.5% BSA, 0.05% T wee η-20) (45) Prepare various concentrations (from 0 to 50 μg / ml, depending on Experiment dependent) IL-2 and IL-8 protein solutions. A sample containing 40 microliters of the antibody solution was added to the well; bonding was performed at 37 ° C for several hours (overnight to confirm completion of bonding). The wells containing the samples were washed 4 times with 50 μl of P B S T solution. (5) Detection antibody binding: Combine equal amounts of individual COIN samples conjugated with anti-IL-2 detection antibody and anti-IL-8 detection antibody, and then add them to each PDMS well; the solution is then Incubate at 37t for 1 hour. After the conjugate solution was removed, the wells were washed four times, each using 50 microliters of the diluted buffer solution, and then once with 50 microliters of DI water. Finally, 30 microliters of DI water was added to each well before Raman signals were detected. Example 2 COIN synthesis and analysis: A silver colloidal solution (50 ml) with an average particle size of 12 nm was prepared from 2 mM AgN03, 0.3 mM NaBH4 and supplemented with 4 mM citrate Na3. This solution was heated to boiling before 8-aza-adenine (AA) was added to a final 2 // M. After boiling for 5 minutes, additional 0.5 mM AgN03 was added. The temperature was then lowered and maintained at 95 + 1 ° C. A portion (1 ml each) of the solution was collected at specified time intervals to measure the spectrum after 1: 30 dilution with 1 mM sodium citrate. As shown in Fig. 2A, the absorption spectrum of the removed sample portion showed a peak shift at a larger wavelength (> 450 nm) and an increased absorbance. Take photos at the time interval (50 microliters each, placed in a petri dish on a white light box) (small arrow indicates the position for further analysis of absorbance changes), showing the time with the reaction heating time Dependency color changes. As a function of reaction (heating) time, the absorbance and Raman activity shown in Table -49-200535246 (46) are shown in Figure 2B. The decrease in absorbance at 700 nm after 65 minutes was due to the formation of large aggregates that settled rapidly in the solution. Example 3 Aggregation of Metal Particles Induced by Organic Compounds ·· Metal particles prepared as described in the present invention in 1 mM citric acid Na3 (15nm gold, Abs52nm = 0.37; silver 60nm, Abs42 () nm = 0.3) • Each organic compound (refer to the abbreviated keywords in Table 1) is mixed with a sample of the metal colloid solution of the indicated concentration for 10 minutes before the spectral measurement. For each sample, the absorbance of the main peak uses peak 1 値, and the higher absorbance at higher wavelengths (600 nm to 700 nm) is used as peak 2 値; the ratio of peak 2 / peak 1 is relative to Mapping of organic compound concentrations; high radon ratios indicate high levels of metal particle aggregation. Fig. 6A shows aggregation of gold particles induced by an organic compound. Relatively low concentrations of organic compounds are sufficient to aggregate silver particles. As shown in Fig. 6B, a relatively high concentration of an organic compound is required to induce aggregation of silver particles. Example 4 The zeta potential of silver particles as a function of 8-aza-adenine concentration. The silver particles were prepared by reducing silver nitrate with sodium citrate at 95 ° C to 10 ° C. The particles were prepared with PCS ( Zetasizer Nan o-ZS, Malvern) measured the average zeta size of 47 nm. The total silver concentration was fixed at 0.10 mM by a 1.00 Mm sodium citrate suspension medium used to determine the zeta potential. The same was used -50- 200535246 (47) Ag concentration and suspension medium 'measured at the size of aggregates (ζ-average 値) developed in the presence of 20 // M 8 -aza-gland exposed. Figure 7 A and B individually show absolute Zeta potential and aggregation kinetics. It is predicted that under the conditions of COIN synthesis using much higher silver concentration (1 to 4.5 mM) and smaller particles (less than 20 nm), there will be higher absolute zeta potential and slower Aggregation kinetics. Example 5 The TEM analysis of silver particles was performed under four preparation conditions: the silver colloid system was synthesized by the method described in the present invention. 1 · The sample was kept at room temperature for 1 week, and the transmission method was adopted. Electron microscope (TEM) analysis showed that most of the particles were smaller than 10 nm. 2. The silver sample from the same source was boiled for 40 minutes, then cooled to room temperature, and then the TEM analysis showed no significant change in particle size. 3 · Silver samples from the same source were used at room temperature using 8-aza- Adenine (final concentration 20 / z Μ) was incubated for two weeks, and then TEM analysis was performed, showing that some particles began to aggregate and fuse. 4 · After boiling for 19 minutes in the presence of 20 μM 8-aza-adenine, silver was analyzed by TEM The particles show the appearance of small particles (less than 10 nanometers) and large particles (more than 10 nanometers). These results (see also Figure 2) lead to the conclusion that long-term boiling leads to the formation of clusters. Example 6 Coating with BSA Synthesis of COIN

粒子塗覆BSA: COIN粒子藉著在達到所需之COIN 尺寸時將0.2% BSA添加於COIN合成溶液而塗覆以BSA -51 - 200535246 (48) 之吸附層。B S A之添加抑制進一步聚集。 交聯該BSA塗層：該BSA吸附層與戊二醛交聯，之 後以NaBH4還原。交聯係藉著將12毫升塗覆有BSA之 COIN (銀總濃度約1 .5 mM )移入1 5毫升離心管中並添加 0.36克7 0%戊二醛及213微升1 mM檸檬酸鈉而完成。將 溶液充分混合並於室溫下放置約1 〇分鐘，之後放置於4°C 冷藏庫中。溶液保持4°C歷經至少4小時，之後添加275 微升之新製備NaBH4 ( 1M)。將溶液混合並於室溫下保持 · 30分鐘。溶液隨後於5 000 rpm下離心60分鐘。使用吸量 管移除上淸液，在離心管中留下約1 .2毫升液體及片粒。 COIN藉著添加0.8毫升1 mM檸檬酸鈉再懸浮，以產生最 終體積2.0毫升。 封包之COIN的FPLC純化：經塗覆之COIN於交聯 瓊脂糖尺寸排斥型管柱上藉FPLC (快速蛋白質液體層析 )純化。濃縮之COIN反應混合物懸浮液（2.0毫升）以 AKTA純化器上之Superose 6 FPLC管柱純化。COIN混合 ^ 物以〇. 5毫升批量注射，將1毫升/分鐘之1 mM檸檬酸鈉 等濃度流施加於管柱。偵測215奈米、2 8 0奈米及5 00奈 米處之吸光度，以收集波峰。封包之COIN於約7至9分 鐘溶離出來，而BSA/交聯BSA溶離份於約9至11分鐘溶 · 離出來。戊二醛、硼氫化鈉及拉曼標記於約20分鐘後溶 離出來。結合來自多個FPLC實驗之溶離份。 實施例6 -52- 200535246 (49) 電子顯微相片顯示團簇形成對於COIN之拉曼信號的影響 1 .作爲起始物質之銀種晶的透射式電子顯微鏡（ TEM)分析顯示大部分粒子係<10奈米；未測得SERS效 應。 2.藉著於有機拉曼標記存在下加熱銀種晶粒子所形 成之放大銀粒子（在此特定試樣中，拉曼標記爲2.5 // Μ 8-氮雜-腺嘌呤，5.0//Μ亞甲基藍及2.5//Μ 9-胺基吖啶， 顯示大部分粒子> 1 〇奈米，團簇物極少；其他拉曼標記產 生類似結果）的ΤΕΜ ;拉曼信號弱。 3 ·在如同第2預之條件（5 · 0 // Μ 8 -氮雜-腺嘌呤， 5.0//Μ亞甲基藍及7.5//Μ 9-胺基吖啶））下製得之拉曼 活性團簇奈米粒子的ΤΕΜ顯示形成大量團簇物，測得來 自此試樣之強拉曼信號，即使該試樣在形成團簇物之前產 生弱拉曼信號亦然。 4 ·於拉曼標記（例如1 0 // Μ腺嘌呤或2 0 μ Μ 8 -氮 雜-腺嘌呤）存在下製得具有類似尺寸及形態之金種晶粒 子。 5·具有金核心之銀粒子（自含有0.25 mM AuHC14及 1 ·25 mM AgN03之溶液製得）；金核心係於1 0 // Μ腺嘌|]令 存在下自金離子製得，僅在使用鹽（即，100 mM Li C1 ) 誘發聚集時產生可偵測之拉曼信號。 6 .掃描式電子顯微相片顯示在如同5之條件下使用5 // Μ N -苄醯基腺嘌呤製備拉曼活性銀團簇，不同處係不添 加附加AgN03 ( 0.75 mM )誘使團簇形成。 -53- 200535246 (50) 實施例7 SERS及COIN之拉曼信號的比較·就SERS試驗而言 ，1 〇 〇微升含有8 -氮雜-腺嘌呤（A A ’最終4 // Μ )之銀膠 體與1 〇 〇微升選自下列者之試驗試劑混合：水（對照組） ，Ν-苄醯基腺嘌呤（BA，1〇#Μ) 5 B S A ( 1 % ) J Tween- 2 0⑧（Twn，1% )，乙醇（eth，100% );形成之 200 微升 混合物隨之與100微升水（-Li)或100微升0.34 M Lici (+ L i )混合，之後藉拉曼顯微鏡測量拉曼散射信號。拉 曼信號係使用任意單位且經標準成個別最大値。使用相同 方法以測試COIN (使用20 μ Μ 8-氮雜-腺嘌呤製得），不 同處係不使用附加之8-氮雜-腺嘌呤。圖9Α出示以Ν-苄 醯基腺嘌呤（ΒΑ)爲試驗試劑之8-氮雜-腺嘌呤（ΑΑ)的 SERS光譜，顯示SERS信號需要鹽，而ΑΑ信號被ΒΑ信 號抑制；圖9B出示使用BA作爲試驗試劑之COIN拉曼光 譜，顯示產生COIN信號不需要鹽，而鹽減弱AA信號。 添加鹽時僅偵測到微弱之BA信號。圖9C出示使用牛血 淸蛋白（BSA )作爲試驗試劑時之8-氮雜-腺嘌呤（AA) 的SERS光譜，顯示SERS信號被BSA所抑制；圖9D出 示使用B S A爲試驗試劑時來自C Ο IN之拉曼光譜，顯示 BSA對於 COIN幾乎不具有負面影響，且實際上可使 COIN安定化。圖9E出示以Tween-20® ( Twn )爲試驗試 劑時之8-氮雜-腺嘌呤（AA )的SERS光譜，顯示在無鹽 時偵測到相對強之SERS信號；圖9F顯示使用Tween-20® -54- 200535246 (51) 爲試驗試劑時來自C 0 1N之拉曼光譜，顯示T w e e η - 2 0抑 制一部分C O IN信號，但另一方面可部分補償鹽之負面影 響；圖9G出示以乙醇（eth )爲試驗試劑時之8-氮雜-腺 嘌呤（AA )的SERS光譜，顯示SERS信號需要鹽，而乙 醇可增強3個波峰（以箭號表示）；圖9 η顯示使用乙醇 爲試驗試劑時來自COIN之拉曼光譜，顯示鹽對COIN信 號具有負面影響，且並無顯著增強之波峰。 實施例8 使用COIN作爲多重分析物偵測之標籤。使用圖5A 所示之偵測流程，其中省略在藉抗體軛合C Ο IN鍵結分析 物之後的放大反應步驟，使用.8-氮雜-腺嘌呤COIN作爲 標籤自IL-2之免疫夾層檢測收集一組50個光譜（圖5B 主峰位置爲1 3 40厘米」）。40微升1 pg/ml之IL-2添加 於塗覆有固觸之IL-2捕集抗體的5毫米井洞；藉著連續 地移除電動台架而自一試樣收集50個光譜；每個光譜各 表示在1 00毫秒週期所收集之資料。雷射光束尺寸直徑約 4微米。扣除背景信號·，偏移光譜之X及Y兩軸以顯示個 別光譜。圖5C係爲分析物信號之長條圖；使用含有1或 2個不同比例（5 : 0，4 : 1，1 : 1，1 : 4及0 : 5 )之分析物IL 2 及IL8 (兩者皆具有約20 kDa之分子量）的試樣進行實驗 ;於個別容器中試驗試樣，各試樣之結合分析物濃度係爲 5〇 pg/ml ; IL-2偵測抗體係軛合於使用8-氮雜-腺嘌呤（ AA)製備之C0IN，而IL-8偵測抗體係軛合於使用N-苄 -55- 200535246 (52) 醯基腺嘿吟（B A )製得之c ΟIN，比例爲1 : 1 ;自每個試 樣共400個數據點收集數據。在預測拉曼位移位置上顯示 正信號之光譜係計數爲所測量之信號點（圖5 C ;寬條） ’以兩分析物在對應之試樣中的整體正信號之百分比表示 。預測値（2標記共1 〇 〇 % )係以狹條表示（圖5 c )。 雖然參照前述實施例描述本發明，但應明瞭該等修飾 及變化係涵盡於本發明精神及範圍內。是故，本發明僅受 限於以下申請專利範圍。 · 【圖式簡單說明】 圖1說明可使用SERS作爲放大方法，以根據其拉曼 特徵偵測標的分子”A”及”B”之方法，此方法與含COIN之 分子”A”及”B”之使用對照，以偵測分子”c”及”D”。 圖2A及B係爲出示COIN (複合型有機-無機奈米團 簇）吸收光譜及拉曼活性之圖，該COIN係由使用8-氮雜 腺嘌呤（A A )合成平均粒徑1 2奈米之銀膠體（5 0毫升） 在以檸檬酸鈉1 : 3 0稀釋後製得。圖2 A出示在所示時間自 9 5 °C溶液取出之試樣份（1毫升）的吸收光譜，出示在較 高波長（大於45 0奈米）之波峰位移及增加之吸光度。小 Stf號表不進一步分析吸光度變化之位置。插圖表不試樣隨 著熱暴露時間而變暗。圖2B係爲出示以反應（加熱）時 間函數表示之吸光度及拉曼活性的圖。Y軸値係爲針對個 別最大値標準化之任意單位；使用420/3 95奈米之吸光度 比來偵測主要吸收峰之位移（3 9 5奈米—420奈米）。拉 •56- 200535246 (53) 曼散射信號係直接自相同稀釋試樣測量’而不使用鹽來誘 發膠體聚集。在65分鐘後於700奈米處之吸光度降低係 因形成迅速於溶液中沉降之大型聚集體所致。 圖3 A至D提供SERS及COIN之拉曼信號的對照。 就各個SERS試驗而言，100微升包含8-氮雜-腺嘌 呤（AA )之銀膠體與1 00微升選自下列者之試驗試劑混 合：水（對照組）、N-苄醯基腺嘌呤（B A，1 0 // Μ )； B S A ( 1 % ) ; Tween-2 03 ( Twn，1 % );乙醇（Et h，1 0 0 0/〇 )。形成之200微升混合物隨後在測量拉曼信號之前與 1〇〇 微升水（-Li)或 100 微升 0.34 M LiCl(+Li)混合。 拉曼信號強度係爲任意單位，且針對個別最大値標準化。 COIN (使用20 # M 8-氮雜-腺嘌呤製得）試驗使用相同方 法，不同處係不使用附加之8 -氮雜-腺嘌呤。圖3 A出示以 水作爲試驗試劑之8 -氮雜-腺嘌呤的拉曼光譜，顯示需要 鹽且測得多重主峰；箭號出示較COIN強之波峰。圖3B 出示使用水作爲試驗試劑之C Ο IN拉曼光譜，箭號出示較 SERS低之波峰。圖3C出示在不同試驗條件下於134〇厘 米_1之SERS信號強度長條圖。圖3D出示在不同試驗條 件下於1 340厘米-1之COIN信號強度長條圖。 圖4A及B出示多重偵測之COIN特徵。COIN係由濃 度爲2.5 // Μ至2 0 # Μ之個別拉曼標記或其混合物製得， 視所需特徵而定·· 8 -氮雜-腺嘌n令（A A ) 、9 -胺基D 丫 n 定（ AN )、亞甲基藍（MB )。代表性波峰係以箭號表示；波 峰強度値係針對個別最大値加以標準化；γ軸値係爲任意 -57- 200535246 (54) 單位；光譜係彼此偏移1單位。圖4 A出示以三個拉曼標 記製得之COIN的特徵，顯示每個標記產生獨特之特徵。 圖4B出示自產生所示特徵之濃度的3個拉曼標記之混合 物製得之COIN特徵：HLL之AA ( H)表示高波峰強度， AN ( L )與MB ( L )兩者爲低波峰強度；LHL之AA ( L ) 表示低波峰強度，AN ( Η )爲高波峰強度，而MB ( L )爲 低；LLH之AA ( L )及AN ( L )兩者表示低，而MB ( Η )表示高。應注意波峰高度可藉著改變標記濃度而調整， 但可能並非必然與所用之標記濃度成比例，因爲拉曼標記 於金屬表面上之吸附親和性不同。 圖5 A至C說明C Ο IN作爲多重分析物偵測之標籤的 應用。圖5 A係爲例示偵測流程圖，出示使用抗體-軛合 COIN之分析物結合。圖5B出示使用8-氮雜-腺嘌呤COIN 作爲標籤（主峰位置1 240厘米-1 )之il-2免疫夾層檢測 所收集之一組5 0個光譜。扣除背景信號；光譜在X及γ 軸皆偏移，以出示個別光譜。圖5 C係爲分析物信號之長 條圖；分析物係爲IL-2及IL-8 (兩者皆具有約20 KDa之 分子量）；實驗係使用含有不同比例（5:0，4: 1，1 : 1， 1 :4及0:5 )之1或2種分析物的試樣進行。IL-2偵測抗 體係軛合於使用8 -氮雜-腺嘌呤（A A )製備之C Ο IN，而 IL-8偵測抗體係軛合於使用N-苄醯基腺嘌呤（BA )製得 之COIN。其使用比例爲1··1 ;每個試樣自總共400個數據 點收集數據；出示位於預測拉曼位移位置之正信號的光譜 係以測量之信號點（寬條）來計數，且以對應試樣中兩分 -58- 200535246 (55) 析物之正信號總量的百分比來表示。細條表示預測値（2 標記總共100%)。 圖6A及B係爲出示金屬粒子（金爲15奈米，Abs520 奈米= 0.37 ;銀爲60奈米，Aba42〇奈米，在1 mM棒字冡 酸N a3中）之有機標記誘發聚集的圖。在測量光譜之前， 每種有機化合物（參照表1中之縮寫索引）與所示濃度之 金屬膠體溶液試樣混合1 0分鐘。每種試樣皆使用主峰之 吸光度作爲波峰1値，而較高波長（600奈米至奈米 )之較高吸光度係作爲波峰2値；波峰2/波峰1之比例係 相對於有機化合物濃度繪圖；該比例値高表示金屬粒子高 度聚集。 圖7A及B個別顯示原始i z-平均粒徑47奈米之銀粒 子（0.10 mM)的ζ電位測量値，懸浮介質爲1.00 mM檸檬 酸鈉，於1〇 // Μ 8-氮雜-腺嘌呤存在下發展聚集物尺寸（ Ζ-平均）。 圖8Α至D出示SERS光譜與COIN光譜之對照。所 示之有機化合物的實例係用於COIN合成；出示8個拉曼 標記之化學結構。COIN ( C )之拉曼光譜與SERS所得之 光譜重疊，顯示C0IN光譜在與個別SERS比較下可具有 不同主峰。在某些情況下，部分波峰係於COIN中增寬； 光譜針對個別最大値（任意單位）標準化以顯示相對波峰 強度；應注意光譜之主要特色並非分析物濃度相依性。 圖9A至Η出示SERS與COIN之拉曼信號對照。就 SERS試驗而言’含有8-氮雜-腺嘌呤之銀膠體與試驗試劑 -59- 200535246 (56) 混合’之後在測量拉曼散射信號之前與水（_ L i )或L i C 1 (+Li )混合。含8-氮雜-腺嘌呤之COIN使用相同方法。 BA = N-苄醯基腺嘌呤；BS A=牛血淸蛋白；Twn = Tween-20TM; eth=乙醇；COIN之拉曼光譜（C)與得自SERS之 光譜（S )重疊；顯示COIN光譜在與個別SERS比較下可 具有不同之主峰。 圖10出示拉曼標記之吸收光譜。15 μ Μ 8-氮雜-腺嘌 啥（ΑΑ )與5 // Μ Ν-苄醯基腺嘌呤（ΒΑ )個別用以製得 COIN ;在COIN合成後，該COIN溶液經300 kDa濾器（ Pall Life Sciences，VWR)單元藉離心（1 000 X g 歷經 5 分鐘）過濾，澄淸溶液用於測量吸光度；亦出示2 5 // Μ ΑΑ及5 // MBA及1 mM檸檬酸Na3之吸收光譜；數據 顯示溶液中之游離拉曼標記分子耗盡。 圖11A及B出示多重分析中所得之COIN特徵（續圖 7) 。COIN係藉前述爐式培育（oven incubation)法使用Particle coated BSA: COIN particles are coated with an adsorption layer of BSA -51-200535246 (48) by adding 0.2% BSA to the COIN synthesis solution when the desired COIN size is reached. The addition of B S A inhibited further aggregation. Cross-linking the BSA coating: The BSA adsorption layer is cross-linked with glutaraldehyde and then reduced with NaBH4. Cross-linking by moving 12 ml of BSA-coated COIN (total silver concentration of about 1.5 mM) into a 15 ml centrifuge tube and adding 0.36 g of 70% glutaraldehyde and 213 μl of 1 mM sodium citrate carry out. The solution was thoroughly mixed and left at room temperature for about 10 minutes before being placed in a refrigerator at 4 ° C. The solution was held at 4 ° C for at least 4 hours, after which 275 μl of freshly prepared NaBH4 (1M) was added. The solution was mixed and held at room temperature for 30 minutes. The solution was then centrifuged at 5 000 rpm for 60 minutes. Remove the supernatant with a pipette, leaving approximately 1.2 ml of liquid and pellets in the centrifuge tube. COIN was resuspended by adding 0.8 ml of 1 mM sodium citrate to produce a final volume of 2.0 ml. FPLC purification of encapsulated COIN: The coated COIN was purified by FPLC (Fast Protein Liquid Chromatography) on a cross-linked agarose size exclusion type column. The concentrated COIN reaction mixture suspension (2.0 ml) was purified on a Superose 6 FPLC column on an AKTA purifier. The COIN mixture was injected in batches of 0.5 ml, and an equal concentration of 1 mM sodium citrate at 1 ml / min was applied to the column. Detect absorbance at 215 nm, 280 nm, and 500 nm to collect peaks. The COIN of the packet dissolves in about 7 to 9 minutes, and the BSA / crosslinked BSA dissolves in about 9 to 11 minutes. The glutaraldehyde, sodium borohydride and Raman labels were dissolved after about 20 minutes. Combine dissociations from multiple FPLC experiments. Example 6 -52- 200535246 (49) Electron micrograph showing the effect of cluster formation on the Raman signal of COIN 1. Transmission electron microscopy (TEM) analysis of silver seed crystals as the starting material shows most of the particle systems < 10 nm; no SERS effect was measured. 2. Magnified silver particles formed by heating silver seed particles in the presence of an organic Raman label (in this particular sample, the Raman label is 2.5 // Μ 8-aza-adenine, 5.0 // Μ Methylene blue and 2.5 // M 9-aminoacridine showed most of the particles> 10 nm with very few clusters; other Raman labels produced similar results); TEM; weak Raman signal. 3. Raman active group prepared under the same conditions as in the second step (5 · 0 // M 8 -aza-adenine, 5.0 // M methylene blue and 7.5 // M 9-aminoacridine)) The TEM of the clustered nanoparticle showed the formation of a large number of clusters, and a strong Raman signal from this sample was measured, even if the sample produced a weak Raman signal before cluster formation. 4. In the presence of a Raman label (for example, 1 0 // Μ adenine or 20 μ Μ 8-aza-adenine), gold seed crystals with similar size and morphology are prepared. 5. · Silver particles with a gold core (made from a solution containing 0.25 mM AuHC14 and 1.25 mM AgN03); the gold core is made from gold ions in the presence of 1 0 // Μ adenine |] order, only The use of salt (ie, 100 mM Li C1) produces detectable Raman signals when aggregation is induced. 6. Scanning electron micrographs show that 5 // MN-benzyl adenine was used to prepare Raman-active silver clusters under the same conditions as in 5. No additional AgN03 (0.75 mM) was used to induce cluster formation under different conditions. . -53- 200535246 (50) Example 7 Comparison of Raman Signals of SERS and COIN · For the SERS test, 100 μl of silver containing 8-aza-adenine (AA 'final 4 // M) The colloid was mixed with 1000 microliters of a test reagent selected from the group consisting of water (control group), N-benzylidene adenine (BA, 10 # Μ) 5 BSA (1%) J Tween- 2 0⑧ (Twn , 1%), ethanol (eth, 100%); 200 microliters of the resulting mixture was then mixed with 100 microliters of water (-Li) or 100 microliters of 0.34 M Lici (+ L i), and then the Raman microscope was used to measure the pull Man scattered signals. Raman signals use arbitrary units and are standardized as individual maximum chirps. The same method was used to test COIN (made with 20 μM 8-aza-adenine), with no additional 8-aza-adenine being used. FIG. 9A shows the SERS spectrum of 8-aza-adenine (AA) using N-benzyl adenine (ΒAA) as a test reagent, showing that the SERS signal requires a salt, and the Α signal is suppressed by the ΒA signal; FIG. 9B shows the use of The COIN Raman spectrum of BA as a test reagent shows that no salt is needed to generate the COIN signal, and the salt weakens the AA signal. Only weak BA signals were detected when salt was added. FIG. 9C shows the SERS spectrum of 8-aza-adenine (AA) when using bovine blood prion protein (BSA) as a test reagent, which shows that the SERS signal is suppressed by BSA; FIG. 9D shows that when BSA is used as the test reagent, it is from C 〇 The Raman spectrum of IN shows that BSA has almost no negative effect on COIN and can actually stabilize COIN. Figure 9E shows the SERS spectrum of 8-aza-adenine (AA) with Tween-20® (Twn) as the test reagent, showing that a relatively strong SERS signal was detected in the absence of salt; Figure 9F shows the use of Tween- 20® -54- 200535246 (51) is a Raman spectrum from C 0 1N when the test reagent is used, showing that T wee η-2 0 suppresses a part of the CO IN signal, but on the other hand can partially compensate for the negative effects of the salt; shown in Figure 9G The SERS spectrum of 8-aza-adenine (AA) with ethanol (eth) as the test reagent shows that the SERS signal requires salt, while ethanol can enhance 3 peaks (indicated by arrows); Figure 9 η shows the use of ethanol The Raman spectrum from COIN when used as a test reagent shows that salt has a negative effect on the COIN signal and does not have significantly enhanced peaks. Example 8 Using COIN as a label for multiple analyte detection. The detection process shown in FIG. 5A is used, in which the amplification reaction step after conjugating the analyte by binding the antibody with C Ο IN is omitted, and .8-aza-adenine COIN is used as the tag for immunosuppression detection from IL-2 A set of 50 spectra was collected (the main peak position in Fig. 5B is 1 3 40 cm "). 40 microliters of 1 pg / ml IL-2 was added to a 5 mm well coated with solid-touch IL-2 capture antibody; 50 spectra were collected from a sample by continuously removing the motorized stand; Each spectrum represents data collected during a 100 millisecond period. The laser beam is about 4 microns in diameter. Subtract the background signal, and shift the X and Y axes of the spectrum to display individual spectra. Figure 5C is a bar graph of the analyte signal; using the analytes IL 2 and IL8 (two with 1 or 2 different ratios (5: 0, 4: 1,1: 1,1: 4, and 0: 5)) (Both of which have a molecular weight of about 20 kDa) are tested; the samples are tested in individual containers, and the concentration of the bound analyte in each sample is 50 pg / ml; the IL-2 detection antibody system is conjugated to use COIN prepared from 8-aza-adenine (AA), and the IL-8 detection system is conjugated to c ΟIN prepared using N-benzyl-55-200535246 (52) adenyl adenine (BA), The ratio is 1: 1; data are collected from a total of 400 data points per sample. A spectrum showing a positive signal at the predicted Raman shift position is counted as the measured signal point (Figure 5C; wide bar) ′ is expressed as a percentage of the overall positive signal of the two analytes in the corresponding sample. Prediction 値 (100% of 2 marks) is shown as a narrow bar (Figure 5c). Although the present invention is described with reference to the foregoing embodiments, it should be understood that such modifications and variations are intended to be within the spirit and scope of the present invention. Therefore, the present invention is limited only by the following patent applications. · [Brief description of the figure] Figure 1 illustrates the method that SERS can be used as an amplification method to detect the target molecules "A" and "B" based on its Raman characteristics. "" To detect molecules "c" and "D". Figures 2A and B are graphs showing the absorption spectrum and Raman activity of COIN (composite organic-inorganic nanoclusters). The COIN is synthesized by using 8-azaadenine (AA) with an average particle size of 12 nm. Silver colloid (50 ml) was prepared by diluting 1:30 with sodium citrate. Figure 2A shows the absorption spectrum of the sample portion (1 ml) taken from the 95 ° C solution at the indicated time, showing the peak shift and increased absorbance at higher wavelengths (greater than 450 nm). The small Stf table does not further analyze the position of the absorbance change. The inset indicates that the specimen darkens with the time of heat exposure. Fig. 2B is a graph showing absorbance and Raman activity as a function of reaction (heating) time. The Y-axis unit is an arbitrary unit standardized for the individual largest unit; the absorbance ratio of 420/3 95 nm is used to detect the shift of the main absorption peak (395 nm to 420 nm). • 56- 200535246 (53) The Mann scattering signal is measured directly from the same diluted sample ’without using salt to induce colloidal aggregation. The decrease in absorbance at 700 nm after 65 minutes was due to the formation of large aggregates that quickly settled in solution. Figures 3 to D provide a comparison of the Raman signals for SERS and COIN. For each SERS test, 100 microliters of silver colloid containing 8-aza-adenine (AA) was mixed with 100 microliters of test reagents selected from the group consisting of water (control group), N-benzylidene gland Purines (BA, 10 // M); BSA (1%); Tween-2 03 (Twn, 1%); Ethanol (Et h, 1000 / 〇). The 200 microliters of the resulting mixture was then mixed with 100 microliters of water (-Li) or 100 microliters of 0.34 M LiCl (+ Li) before measuring the Raman signal. Raman signal strength is in arbitrary units and is normalized for individual maximum chirps. The COIN (made with 20 # M 8-aza-adenine) test uses the same method, except that the additional 8-aza-adenine is not used. Figure 3A shows the Raman spectrum of 8-aza-adenine using water as the test reagent, showing that salt is needed and the main peak is much more measured; the arrow shows a peak stronger than COIN. FIG. 3B shows the CO IN Raman spectrum using water as a test reagent, and the arrow shows a lower peak than SERS. FIG. 3C shows a bar graph of SERS signal intensity at 134 cm-1 under different test conditions. Figure 3D shows a bar graph of COIN signal strength at 1 340 cm-1 under different test conditions. Figures 4A and B show the COIN characteristics of multiple detections. COIN is made from individual Raman labels or mixtures thereof at a concentration of 2.5 // Μ to 2 0 # Μ, depending on the required characteristics ... 8-Aza-Adenine (AA), 9-Amine D y n 定 (AN), methylene blue (MB). Representative peaks are indicated by arrows; peak intensities are normalized for individual maxima; the γ-axis is arbitrary -57- 200535246 (54) units; the spectrum is offset by 1 unit from each other. Figure 4A shows the characteristics of a COIN made with three Raman marks, showing that each mark produces a unique feature. FIG. 4B shows the COIN characteristics made from a mixture of 3 Raman-labeled mixtures that produce the concentrations shown, with HLA and AA (H) representing high peak intensities, and AN (L) and MB (L) both having low peak intensities. ; AA (L) of LHL indicates low peak intensity, AN (Η) is high peak intensity, and MB (L) is low; both AA (L) and AN (L) of LLH indicate low, and MB (Η) Means high. It should be noted that the peak height can be adjusted by changing the label concentration, but it may not necessarily be proportional to the label concentration used, because Raman labels have different adsorption affinities on the metal surface. Figures 5 to A illustrate the application of CO IN as a tag for multiple analyte detection. Figure 5 A is an exemplary detection flowchart showing the analyte binding using antibody-conjugated COIN. Fig. 5B shows il-2 immunosandwich detection using 8-aza-adenine COIN as a tag (main peak position 1 240 cm-1). One set of 50 spectra was collected. The background signal is subtracted; the spectra are shifted on the X and γ axes to show individual spectra. Figure 5 C is a bar graph of the analyte signal; the analytes are IL-2 and IL-8 (both of which have a molecular weight of about 20 KDa); the experimental system uses different ratios (5: 0, 4: 1 , 1: 1, 1: 4, and 0: 5). The IL-2 detection antibody was conjugated to C IO IN using 8-aza-adenine (AA), while the IL-8 detection antibody was conjugated to N-benzyl adenine (BA). Get the COIN. Its usage ratio is 1 ·· 1; each sample collects data from a total of 400 data points; the spectrum showing the positive signal at the predicted Raman shift position is counted by the measured signal point (wide bar), and the corresponding The two points in the sample are expressed as a percentage of the total positive signal of -58- 200535246 (55). Thin bars represent predictions (2 marks total 100%). Figures 6A and B show the aggregation induced by the organic labeling of metal particles (15nm gold, Abs520 nanometer = 0.37; silver 60nm, Aba42nm, in 1 mM clavulanic acid N a3). Illustration. Before measuring the spectrum, each organic compound (refer to the abbreviated index in Table 1) was mixed with a sample of the metal colloidal solution at the indicated concentration for 10 minutes. Each sample uses the absorbance of the main peak as peak 1 値, and the higher absorbance at higher wavelengths (600 nm to nanometers) is used as peak 2 値; the ratio of peak 2 / peak 1 is plotted against the concentration of organic compounds ; The high ratio indicates that the metal particles are highly aggregated. Figures 7A and B individually show the zeta potential measurement of the original i z-silver particles (0.10 mM) with an average particle size of 47 nm. The suspension medium is 1.00 mM sodium citrate at 10 // M 8-aza-gland. Aggregate size (Z-mean) developed in the presence of purines. 8A to 8D show the comparison between the SERS spectrum and the COIN spectrum. Examples of the organic compounds shown are for COIN synthesis; 8 Raman-labeled chemical structures are shown. The Raman spectrum of COIN (C) overlaps with the spectrum obtained by SERS, showing that the COIN spectrum can have different main peaks when compared with individual SERS. In some cases, some of the peaks are broadened in COIN; the spectrum is normalized to the individual maximum radon (arbitrary unit) to show the relative peak intensity; it should be noted that the main feature of the spectrum is not the analyte concentration dependency. Figures 9A to Η show the Raman signal comparison between SERS and COIN. For the SERS test, 'silver colloid containing 8-aza-adenine and test reagent -59- 200535246 (56)' are mixed with water (_L i) or L i C 1 ( + Li). The same method is used for COIN containing 8-aza-adenine. BA = N-benzylidene adenine; BS A = bovine hemoglobin; Twn = Tween-20TM; eth = ethanol; Raman spectrum (C) of COIN overlaps with spectrum (S) obtained from SERS; shows COIN spectrum It may have different main peaks when compared with individual SERS. Fig. 10 shows a Raman-labeled absorption spectrum. 15 μ Μ 8-aza-adenine (AA) and 5 // Μ Ν-Benzyl adenyl adenine (ΒΑ) were used to prepare COIN; after COIN synthesis, the COIN solution was passed through a 300 kDa filter (Pall The Life Sciences, VWR) unit was filtered by centrifugation (1 000 X g for 5 minutes), and the clear solution was used to measure absorbance; the absorption spectra of 2 5 // ΜΑΑ and 5 // MBA and 1 mM citrate Na3 were also shown; The data showed that the free Raman-labeled molecules in the solution were depleted. Figures 11A and B show the COIN characteristics obtained in the multiplex analysis (continued Figure 7). COIN is used by the aforementioned oven incubation method

2或3種濃度爲2·5至20 // Μ之拉曼標記混合物製得，視 所需之特徵而定。該3種拉曼標記爲8-氮雜-腺嘌呤（ΑΑ )、9 -胺基π丫 π定（A Ν )、亞甲基藍（Μ B )。主峰位置係 以箭號表示；波峰高度（以任意單位表示）針對於個別最 大値加以標準化；光譜彼此偏移1單位。圖1 1 Α出示使用 得到所示相對波峰高度之濃度的2種拉曼標記（AA及MB )製得之 COIN 的特徵：AA = MB(HH) ，AA>MA(HL) 且AA<MB(LH)。圖1 1B出示自產生所示特徵之濃度的 3種拉曼標記混合物製得之COIN的拉曼特徵：HHL之AA -60- 200535246 (57) (Η)及AN ( Η)表示高波峰強度，而Mb ( L)表示低波 峰強度；HLH之AA ( H)表示高波峰強度，an ( Μ表示 低波峰強度，且MB ( Η )表示高波峰強度。其他特色可由 電腦分析顯露。 圖1 2係爲例示含C Ο IN微球的示意圖。 圖13係爲說明製造含COIN微球之方法（包含法） 的流程圖。 圖14說明製造含COIN微球之備擇方法（浸入法） 〇 圖1 5說明製造含COIN微球之另一備擇方法（內建 法）。 圖1 6說明製造含C Ο IN微球之另一備擇方法（外加 法）。2 or 3 Raman labeled mixtures with a concentration of 2.5 to 20 // M, depending on the desired characteristics. The three kinds of Raman labels are 8-aza-adenine (AA), 9-amino π-yridine (A N), and methylene blue (MB). The main peak position is indicated by an arrow; the peak heights (in arbitrary units) are normalized for the individual maximum chirps; the spectra are shifted by 1 unit from each other. Figure 1 1 A shows the characteristics of a COIN made using two types of Raman markers (AA and MB) that give the concentrations shown in the relative peak heights: AA = MB (HH), AA > MA (HL), and AA < MB ( LH). Fig. 1B shows the Raman characteristics of the COIN prepared from the three types of Raman labelled mixtures producing the concentrations of the characteristics shown: HHL's AA -60- 200535246 (57) (Η) and AN (Η) represent high peak intensities, Mb (L) indicates low peak intensity; AA (H) of HLH indicates high peak intensity, an (M indicates low peak intensity, and MB (Η) indicates high peak intensity. Other characteristics can be revealed by computer analysis. Figure 1 2 Series An example is a schematic diagram of C Ο IN containing microspheres. Figure 13 is a flowchart illustrating a method (including method) for manufacturing COIN containing microspheres. Figure 14 illustrates an alternative method (immersion method) for manufacturing COIN containing microspheres. Figure 1 5 illustrates another alternative method of manufacturing microspheres containing COIN (built-in method). Figure 16 illustrates another alternative method of manufacturing microspheres containing CO IN (additive method).

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Claims (1)

200535246 (1) 十、申請專利範圍 1· 一種複合型有機-無機奈米團簇，其包含由多個金 屬粒子形成之聚集體，該金屬粒子聚集體內部吸附有多個 拉曼（Raman )活性有機化合物。 2. 如申請專利範圍第1項之奈米團簇，其中至少一 個拉曼活性有機化合物係位於由兩個或多個金屬粒子之鄰 近處所產生的接合點中。 3. 如申請專利範圍第1項之奈米團簇’其中該聚集 體係包含兩種不同之拉曼活性有機化合物。 4. 如申請專利範圍第1項之奈米團簇，其中該金屬 粒子係包含金、銀、鈾、銅或鋁。 5. 如申請專利範圍第1項之奈米團簇，其中該金屬 粒子係包含金或銀。 6 ·如申請專利範圍第1項之奈米團簇，其進一步包 含異於第一種金屬之第二種金屬，其中該第二種金屬係形 成覆蓋該奈米團簇之表層。 7 ·如申請專利範圍第6項之奈米團簇，其中該第一 及第二種金屬係選自金、銀、鉑、銅或鋁。 8 ·如申請專利範圍第1項之奈米團簇，其進一步包 含有機層。 9.如申請專利範圍第1項之奈米團簇，其中該奈米 團簇亦包含專一性地鍵結於已知分析物的探針。 10·如申請專利範圍第9項之奈米團簇，其中該探針 係選自抗體、抗原、聚核苷酸、寡聚核苷酸、受體、胜肽 -62- 200535246 (2) 、核酸、醣類及配位體。 1 1 ·如申請專利範圍第1項之奈米團簾’其中該拉受 活性有機化合物係選自腺嘌呤、心胺基-吼哗并（3,4-d ) 嘧啶、2-氟腺嘌呤、N6-苄醯基腺嘌11令、激動素（kinet111 )、二甲基-烯丙基-胺基-腺嘌呤、玉米素（Zeatin )、溴· 腺嘌呤、8 -氮雜-腺嘌呤、8 -氮雜鳥嘌呤、6 -氫硫基嘌呤、 4-胺基-6-氫硫基吡唑并（3,4-d )嘧啶、8·氫硫基腺嘌11令及 9-胺基-吖啶。 12. 如申請專利範圍第1項之奈米團簇’其中該拉曼 活性化合物係包含螢光標記。 13. 如申請專利範圍第1項之奈米團簇’其中該奈米 團簇係具有約50奈米至約200奈米之平均直徑。 14. 一種製造複合型有機-無機奈米團簇之方法，其 包含： 將包含拉曼活性有機化合物、金屬離子來源、還原劑 及金屬種晶粒子之液體組成物加熱足以生成增大之金屬粒 子的時間，使該拉曼活性有機化合物吸附於其表層，並於 該液體組成物中形成增大之粒子的奈米團簇。 1 5 ·如申請專利範圍第1 4項之方法，其中該方法係 進一步包含將形成之奈米團簇塗覆以有機層。 16.如申請專利範圍第14項之方法，其中該方法中 進一步包含將該奈米團簇塗覆以牛血淸蛋白。 1 7 ·如申請專利範圍第1 4項之方法，其中該加熱係 維持足以使液體組成物之主要吸收峰位移之時間。 -63- 200535246 (3) 1 8 ·如申請專利範圍第1 4項之方法，其中該形成之 奈米團簇係具有約50至約200奈米之平均直徑。 19.如申請專利範圍第1 4項之方法，其中該金屬係 選自金、銀、鉑、銅、鋁及其組合物。 20·如申請專利範圍第1 4項之方法，其中該金屬係 爲銀或金。 2 1 ·如申請專利範圍第1 4項之方法，其中該至少一 種拉曼活性有機化合物係爲螢光。 2 2 ·如申請專利範圍第1 4項之方法，其中該方法係 在每次重複時各使用不同之拉曼活性有機化合物的情況下 重複數次，以生成一組奈米團簇，而該組之每個成員各具 有獨特之拉曼特徵。 23 .如申請專利範圍第1 4項之方法，其中該液體組 成物係包含至少兩種不同之拉曼活性有機化合物。 24· —種拉曼活性金屬奈米團簇組，其具有約50奈 米至約200奈米之平均直徑，該組中每個成員各具有由摻 入金屬奈米團簇中之至少一種拉曼活性有機化合物所產生 的獨特拉曼特徵。 2 5 ·如申請專利範圍第2 4項之拉曼活性金屬奈米團 簇組’其中該組中至少一成員係具有由摻入該組奈米團簇 中之各個至少一成員中的不同拉曼活性有機化合物組合產 生之獨特拉曼特徵。 2 6 ·如申請專利範圍第2 4項之拉曼活性金屬奈米團 簇組，其中該不同組合係爲不同莫耳比之拉曼活性有機化 -64 - 200535246 (4) 合物。 2 7 ·如申請專利範圍第2 4項之拉曼活性金屬奈米團 簇組，其中該組之各成員係進一步包含專一性地鍵結於已 知生物分析物之探針。 2 8 · —種偵測試樣中之分析物的方法，其包含： 使含有分析物之試樣與多個金屬粒子之聚集體接觸， 該聚集體內吸附有多個拉曼活性有機化合物，且亦包含探 針，其中該探針係專一性地鍵結於該分析物；及 偵測由該奈米團簇所發射之SERS信號，其中該信號 係指示分析物之存在。 2 9·如申請專利範圍第2 8項之方法，其中該試樣係 爲氣體試樣，且接觸係包含使該氣體試樣與含有該奈米團 簇之溶液接觸。 3 0·如申請專利範圍第28項之方法，其中該試樣係 爲液體試樣。 3 1 ·如申請專利範圍第2 8項之方法，其中該試樣係 爲生物試樣。 3 2.如申請專利範圍第2 8項之方法，其中該奈米團 簇係埋置於聚合物珠粒內，且其中該珠粒係包含選自聚烯 烴、聚苯乙烯、聚丙烯酸酯及聚（甲基）丙烯酸酯之聚合 物。 3 3 · —種辨識試樣中之生物分析物的方法，該方法係 包含= 在適於使連接於該金屬奈米團簇組之探針專一性地鍵 -65- 200535246 (5) 結於存在於試樣中之分析物而形成複合物的條件下，使包 含多個生物分析物之試樣與一平均直徑約50奈米至約200 奈米之拉曼活性金屬奈米團簇組接觸，該組之每個成員各 具有由摻入其中之至少一種拉曼活性有機化合物所產生之 獨特拉曼特徵； 分離已鍵結之複合物； 以多重方式偵測由已鍵結複合物中之有機拉曼活性化 合物所發射之拉曼特徵，其中各個拉曼特徵係指示試樣中 已知之生物分析物的存在。 3 4·如申請專利範圍第33項之方法，其中該生物分 析物係爲蛋白質，而該組中之探針係爲抗體，其中各個抗 體係專一性地鍵結於不同之已知蛋白質。 3 5 ·如申請專利範圍第3 3項之方法，其中該檢測係 爲不使用信號放大之夾層免疫檢測。 36· —種微球，其包含聚合物珠粒及多個奈米團簇， 該奈米團簇包含多個金屬粒子之聚集體及至少一個拉曼活 性有機化合物，其中該拉曼活性有機化合物係吸附於金屬 粒子之聚集體內，其中該奈米團簇係埋置於該聚合物珠粒 內。 3 7.如申請專利範圍第3 6項之微球，其中該聚合物 珠粒係包含聚烯烴。 3 8.如申請專利範圍第3 6項之微球，其中該聚合物 珠粒係包含聚苯乙烯。 3 9·如申請專利範圍第3 6項之微球，其中該聚合物 -66- 200535246 (6) 珠粒係包含聚丙烯酸酯。 40. 如申請專利範圍第3 6項之微球，其中該聚合物 珠粒係包含聚（甲基）丙烯酸酯。 41. 一種製造含有埋置之奈米團簇的聚合物微球之方 法，其包含 a )藉著使用至少一種界面活性劑將水均質化而產生 微胞； b )將如申請專利範圍第1項或如申請專利範圍第1 3 項之奈米團簇與疏水劑一起導入該微胞內； c )添加抗-聚集安定化劑； d )導入一對極性及非極性有機單體；及 e )導入自由基起始劑以起始聚合反應，而產生內部 埋置有奈米團簇之聚合物微球。 42 · —種製造含有埋置之拉曼活性奈米團簇之微球的 方法，其包含： a )使一對微胞形成性之有機極性及非極性有機單體 於丙烯酸存在下在有機溶液中共聚，以經由乳化聚合形成 均勻尺寸之聚合物微球； b )使該微球與至少一種拉曼活性分子於液體非溶劑 中接觸，以將該分子導入該微球內； c )將金屬膠體懸浮液導至b )所得之混合物中，以形 成內部埋置有如申請專利範圍第1項或如申請專利範圍第 1 3項之奈米團簇的聚合物微球。 4 3 · —種製造含有埋置之奈米團簇的聚合物微球之方 -67- 200535246 (7) 法，其包含： a )使帶正電之聚合物粒子與帶負電之如申請專利範 圍第1項或如申請專利範圍第1 3項之奈米團簇接觸，以 形成聚合物-奈米團簇複合物； b )使該複合物與可交聯之聚合物接觸；及 c )使用鍵合劑分子使該可交聯之聚合物交聯，以形 成內部埋置有奈米團簇之不可溶聚合物微球。 44· 一種製造含有埋置之奈米團簇之聚合物微球的方 法，其包含： a )使一對微胞形成性之極性及非極性有機單體於丙 嫌酸存在下共聚，以經由乳化聚合形成均勻尺寸之微球； b )使在至少一種有機溶劑中之該微球與至少一種拉 曼活性分子接觸，以使該分子擴散至該微球內； c )添加金屬膠體於該有機溶劑，以形成內部封包有 如申請專利範圍第1項或如申請專利範圍第1 3項之奈米 團簇的微球。 45 · —種用以偵測生物分析物之組套，其包含： 多種位於固體擔體上之如申請專利範圍第1項或如申 請專利範圍第1 3項之奈米團簇，及 生物作用劑。 4 6.如申g靑專利範圍第4 5項之組套，其中該生物作 用劑係爲胜肽、聚胜肽、蛋白質、抗體或聚核苷酸。 4 7 *如申請專利範圍第4 5項之組套，其中該固體擔 體係爲粒子陣列。 -68-200535246 (1) 10. Scope of patent application 1. A composite organic-inorganic nano-cluster, which includes aggregates formed by a plurality of metal particles, and a plurality of Raman activities are adsorbed inside the metal particle aggregates Organic compounds. 2. For the nano-cluster of item 1 of the patent application scope, at least one Raman-active organic compound is located in a junction generated by the vicinity of two or more metal particles. 3. The nano-cluster of item 1 of the patent application scope, wherein the aggregation system comprises two different Raman-active organic compounds. 4. The nano-cluster of item 1 of the patent application scope, wherein the metal particles include gold, silver, uranium, copper or aluminum. 5. The nano-cluster of claim 1, wherein the metal particles include gold or silver. 6. The nano-cluster according to item 1 of the patent application scope, further comprising a second metal different from the first metal, wherein the second metal system forms a surface layer covering the nano-cluster. 7. The nano-cluster of item 6 of the patent application, wherein the first and second metals are selected from gold, silver, platinum, copper or aluminum. 8 · If the nano-cluster of item 1 of the patent application scope further includes an organic layer. 9. The nano-cluster of claim 1, wherein the nano-cluster also includes a probe that specifically binds to a known analyte. 10. The nanoclusters according to item 9 of the scope of the patent application, wherein the probe is selected from the group consisting of antibodies, antigens, polynucleotides, oligonucleotides, receptors, and peptides-62-200535246 (2), Nucleic acids, sugars and ligands. 1 1 · The nano-group curtain according to item 1 of the patent application, wherein the active organic compound is selected from the group consisting of adenine, aminamine-pyrido (3,4-d) pyrimidine, and 2-fluoroadenine , N6-benzyl adenine adenine 11 kinetin (kinet111), dimethyl-allyl-amino-adenine, Zeatin, bromine adenine, 8-aza-adenine, 8-Azaguanine, 6-Hydrothiopurine, 4-Amino-6-Hydrothiopyrazolo (3,4-d) pyrimidine, 8 · Hydrothioadenine, and 9-Amine -Acridine. 12. The nanocluster of item 1 of the patent application scope, wherein the Raman active compound comprises a fluorescent label. 13. The nano-cluster of item 1 of the patent application range, wherein the nano-cluster system has an average diameter of about 50 nm to about 200 nm. 14. A method of manufacturing a composite organic-inorganic nano-cluster, comprising: heating a liquid composition containing a Raman-active organic compound, a source of metal ions, a reducing agent, and metal seed particles to generate enlarged metal particles The Raman-active organic compound is adsorbed on its surface layer within a period of time, and nanoparticle clusters of enlarged particles are formed in the liquid composition. 15. The method according to item 14 of the scope of patent application, wherein the method further comprises coating the formed nanoclusters with an organic layer. 16. The method of claim 14 wherein the method further comprises coating the nanocluster with bovine blood prion protein. [17] The method according to item 14 of the scope of patent application, wherein the heating is maintained for a time sufficient to shift the main absorption peak of the liquid composition. -63- 200535246 (3) 1 8 · The method according to item 14 of the patent application range, wherein the formed nanoclusters have an average diameter of about 50 to about 200 nanometers. 19. The method of claim 14 in which the metal is selected from the group consisting of gold, silver, platinum, copper, aluminum, and combinations thereof. 20. The method of claim 14 in the scope of patent application, wherein the metal is silver or gold. 2 1 · The method according to item 14 of the scope of patent application, wherein the at least one Raman-active organic compound is fluorescent. 2 2 · The method according to item 14 of the scope of patent application, wherein the method is repeated several times in the case of using different Raman-active organic compounds each time to generate a group of nanoclusters, and the Each member of the group has unique Raman characteristics. 23. A method according to item 14 of the patent application, wherein the liquid composition comprises at least two different Raman-active organic compounds. 24 · A Raman-active metal nano-cluster group having an average diameter of about 50 nm to about 200 nm, each member of the group has a pull by at least one of the metal nano-cluster incorporated Unique Raman characteristics produced by MAN active organic compounds. 25. The Raman-active metal nano-cluster group according to item 24 of the patent application, wherein at least one member of the group has a different pull from each at least one member incorporated in the nano-cluster of the group. Unique Raman characteristics resulting from the combination of MAN active organic compounds. 2 6. The Raman-active metal nano-cluster group according to item 24 of the patent application range, wherein the different combination is a Raman-active organicated -64-200535246 (4) compound with different mol ratios. 27. The Raman-active metal nano-cluster group according to item 24 of the patent application, wherein each member of the group further comprises a probe specifically bonded to a known biological analyte. 28. A method for detecting an analyte in a sample, comprising: contacting the sample containing the analyte with an aggregate of a plurality of metal particles, the aggregate having a plurality of Raman-active organic compounds adsorbed therein, and It also includes a probe, wherein the probe is specifically bonded to the analyte; and detecting a SERS signal emitted by the nano-cluster, wherein the signal is indicative of the presence of the analyte. 29. The method of claim 28, wherein the sample is a gas sample, and the contact system comprises contacting the gas sample with a solution containing the nano-cluster. 30. The method of claim 28, wherein the sample is a liquid sample. 3 1 · The method according to item 28 of the scope of patent application, wherein the sample is a biological sample. 3 2. The method according to item 28 of the scope of patent application, wherein the nano-cluster system is embedded in a polymer bead, and wherein the bead system comprises a material selected from the group consisting of polyolefin, polystyrene, polyacrylate and Polymer of poly (meth) acrylate. 3 3 · —A method for identifying a biological analyte in a sample, the method comprising: -65- 200535246 (5) binding to a probe specifically adapted to attach to a metal nanocluster Under the condition that the analyte present in the sample forms a complex, a sample containing a plurality of biological analytes is brought into contact with a Raman-active metal nanocluster group having an average diameter of about 50 nm to about 200 nm. Each member of the group has unique Raman characteristics resulting from at least one Raman-active organic compound incorporated therein; separation of bonded complexes; detection of multiple compounds from bonded complexes in multiple ways Raman signatures emitted by organic Raman active compounds, where each Raman signature indicates the presence of a known biological analyte in the sample. 34. The method according to item 33 of the patent application, wherein the bioanalyte is a protein, and the probes in the group are antibodies, in which each anti-system is specifically bonded to a different known protein. 3 5 · The method according to item 33 of the scope of patent application, wherein the detection is a sandwich immunoassay that does not use signal amplification. 36 · A microsphere comprising polymer beads and a plurality of nano-clusters, the nano-clusters comprising an aggregate of a plurality of metal particles and at least one Raman-active organic compound, wherein the Raman-active organic compound The nano-cluster system is adsorbed in the aggregate of metal particles, and the nano-cluster system is embedded in the polymer beads. 37. The microspheres according to item 36 of the application, wherein the polymer beads comprise a polyolefin. 38. The microspheres according to claim 36, wherein the polymer beads comprise polystyrene. 39. The microspheres according to item 36 of the patent application range, wherein the polymer -66- 200535246 (6) the bead system comprises polyacrylate. 40. The microspheres according to claim 36, wherein the polymer beads comprise poly (meth) acrylate. 41. A method of manufacturing polymer microspheres containing embedded nanoclusters, comprising a) generating cells by homogenizing water with at least one surfactant; b) as described in the first patent application Nano clusters of item 13 or item 13 of the patent application are introduced into the cell together with the hydrophobic agent; c) adding an anti-aggregation stabilizer; d) introducing a pair of polar and non-polar organic monomers; and e ) A free radical initiator is introduced to initiate the polymerization reaction to produce polymer microspheres with nano-cluster embedded therein. 42. A method of manufacturing microspheres containing embedded Raman-active nanoclusters, comprising: a) making a pair of microcell-forming organic polar and non-polar organic monomers in an organic solution in the presence of acrylic acid Copolymerization in China to form polymer microspheres of uniform size through emulsion polymerization; b) contacting the microspheres with at least one Raman active molecule in a liquid non-solvent to introduce the molecules into the microspheres; c) metal The colloidal suspension is led to the mixture obtained in b) to form polymer microspheres with nanoclusters embedded therein such as in the scope of claim 1 or claim 13 in the scope of patent application. 4 3 · —A method for manufacturing polymer microspheres containing embedded nano-cluster-67- 200535246 (7) method, which includes: a) Applying a patent for polymer particles with positive charge and negative charges Contact nano-cluster of scope item 1 or patent application scope item 13 to form a polymer-nano-cluster complex; b) contact the composite with a crosslinkable polymer; and c) The crosslinkable polymer is crosslinked using a bonder molecule to form insoluble polymer microspheres with nanoclusters embedded therein. 44. A method for manufacturing polymer microspheres containing embedded nanoclusters, comprising: a) copolymerizing a pair of microcell-forming polar and non-polar organic monomers in the presence of propionic acid to pass Emulsion polymerization to form microspheres of uniform size; b) contacting the microspheres in at least one organic solvent with at least one Raman-active molecule to diffuse the molecules into the microspheres; c) adding a metal colloid to the organic Solvent to form microspheres with nano-cluster inside encapsulated as in item 1 of the scope of patent application or as item 13 in the scope of patent application. 45 · —A kit for detecting biological analytes, comprising: a plurality of nanoclusters on a solid support, such as those in the scope of patent application No. 1 or those in the scope of patent application No. 13, and biological effects Agent. 46. The kit according to item 45 of the patent scope of claim 6, wherein the biological agent is a peptide, a peptide, a protein, an antibody or a polynucleotide. 4 7 * As set of item 45 in the scope of patent application, wherein the solid support system is a particle array. -68-