TWI579554B - Preparation method of substrate for SERS detection of microfluidic channel type SDS, preparation method of substrate SERS detection substrate, preparation method of substrate for SERS detection, and detection method of organic pollutant - Google Patents
Preparation method of substrate for SERS detection of microfluidic channel type SDS, preparation method of substrate SERS detection substrate, preparation method of substrate for SERS detection, and detection method of organic pollutant Download PDFInfo
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本發明係關於一種微流體通道型SERS檢測用基材之製備方法、一種探針型SERS檢測用基材之製備方法、以及一種有機污染物之檢測方法;特別是關於一種微流體通道型或探針型表面增強拉曼散射(SERS)檢測用基材之製備方法,其包括:藉由光微影法,使用光罩,將包含至少一條以上的微通道圖案形成於玻璃基板上而製成微流道結構母模;將聚二甲基矽氧烷(Polydimethylsiloxane,PDMS)溶液塗布於前述之微流道結構母模,加熱固化並予以剝離而得到具有微流體通道之PDMS固化結構;將銀鏡反應試劑注入到PDMS固化結構之該微通道內,然後注入還原劑,以使產生銀鏡反應而還原形成銀奈米顆粒,並以去離子水沖洗而得到微流體通道型SERS檢測用基材。 The invention relates to a preparation method of a microfluidic channel type SERS detecting substrate, a preparation method of a probe type SERS detecting substrate, and a method for detecting organic pollutants; in particular, a microfluid channel type or probe A method for preparing a substrate for needle surface enhanced Raman scattering (SERS), comprising: forming a micro-channel pattern comprising at least one microchannel pattern on a glass substrate by a photolithography method using a photomask a flow channel structure master mold; a solution of polydimethylsiloxane (PDMS) is applied to the aforementioned microchannel structure master mold, heat curing and stripping to obtain a PDMS solidified structure having a microfluidic channel; The reagent is injected into the microchannel of the PDMS solidified structure, and then a reducing agent is injected to cause silver mirror reaction to be reduced to form silver nanoparticles, and rinsed with deionized water to obtain a microfluidic channel type SERS detecting substrate.
長期以來,國內外一直採用COD、BODs等有機污染綜合指標作為評價環境質量的參數,但是,它們僅僅反映某種籠統含量的數據,而無法提供有機污染物本身的性狀特征有效信息。 For a long time, comprehensive indicators of organic pollution such as COD and BOD have been used at home and abroad as parameters for evaluating environmental quality. However, they only reflect certain general content data, and cannot provide effective information on the characteristics of organic pollutants themselves.
例如,某些化合物如芳香族化合物的苯環上,存在大π鍵穩定結構的影響,難以氧化測定。而毒性的化合物能抑制微生物活動不能完全降解。即使TOC和TOD比較徹底地氧化有機物,對具體的有毒物質也不能進行定性、定量,尤其是三致性的有機物在環境中往往以低濃度水平出現,就足以對人類健康和生態環境產生極大的危害, 所以上述COD、BODs等常規分析方法已不能作為有機污染物的有效的控制方法。 For example, some compounds such as aromatic compounds have a large π bond-stabilized structure on the benzene ring, which is difficult to oxidize. Toxic compounds inhibit the microbial activity from being completely degraded. Even if TOC and TOD completely oxidize organic matter, the specific toxic substances cannot be qualitatively and quantitatively determined. Especially the triad organic substances often appear in low concentrations in the environment, which is enough for human health and ecological environment. harm, Therefore, the above conventional analytical methods such as COD and BODs cannot be used as an effective control method for organic pollutants.
近代氣相色譜(GC)、高效液相色譜(HPLC)、質譜(包括色質聯用)(NMR)等技術相繼應運而生,併在分析化學的前沿一環境監測領域中獲得了廣泛的應用,並取得了許多重要成果,為檢測環境中的有機污染物開闢了廣闊的前景。雖然歐美、中、日、蘇等國先後將色譜法(包括色質聯用)列為環境監測分析方法和標準分析方法,近數十年來被廣泛地應用在大氣、水質、土壤、生物、食品等環境監測上藉以分析痕量、複雜、多組分的有機污染物;然而色譜對於複雜體系的系統之分析方法上仍有相當大的限制。 Modern gas chromatography (GC), high performance liquid chromatography (HPLC), mass spectrometry (including color-mass spectrometry) (NMR) and other technologies have emerged, and have been widely used in the field of analytical chemistry, environmental monitoring. And has achieved many important results, opening up broad prospects for detecting organic pollutants in the environment. Although Europe, the United States, Japan, and the United States have successively classified chromatography (including color-mass spectrometry) as environmental monitoring analysis methods and standard analytical methods, they have been widely used in the atmosphere, water quality, soil, biology, and food in recent decades. Such environmental monitoring is used to analyze trace, complex, multi-component organic pollutants; however, chromatography still has considerable limitations on the analytical methods of complex systems.
例如,特別是存在於包括河川湖泊海洋等水體中之水與底泥、自來水、工業用水、家庭廢水、工業廢水、活性污泥、農田或溝渠中之底泥、或其他之水或污泥中之有機物,不僅種類繁多,同時濃度的變亦非常大,低的濃度可低至數千分之微ppm量級,因此分析鑑別益加困難。 For example, it is found in water and sediment, water, industrial water, domestic wastewater, industrial wastewater, activated sludge, sediment in farmland or ditch, or other water or sludge, especially in water bodies such as rivers, lakes and oceans. The organic matter is not only a wide variety, but also the concentration is very large, and the low concentration can be as low as several thousandths of micro ppm, so the analysis and identification are difficult.
尤其,以傳統檢測法分析鄰苯二甲酸酯類(PAs),例如,在檢測鄰苯二甲酸二甲酯(DMP)、鄰苯二甲酸二乙酯(DEP)、鄰苯二甲酸二丁酯(DBP)、鄰苯二甲酸丁基苯甲酯(BBP)、鄰苯二甲酸正二辛酯(DNOP)、鄰苯二甲酸二(2-乙基己基)酯(DEHP)時,需經由樣品前處理、萃取、濃縮、淨化和儀器分析等步驟,一般常需耗時約三天才可能知道結果。 In particular, the detection of phthalates (PAs) by conventional methods, for example, in the detection of dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), n-octyl phthalate (DNOP), di(2-ethylhexyl) phthalate (DEHP), before passing through the sample Processing, extraction, concentration, purification, and instrumental analysis often take about three days to know the results.
此外,在習用的檢測底泥中有機污染物之方法中,向來所使用的分析技術皆需要極其複雜處理程序,例如PBDEs、PAEs、PAHs、PCBs等有機污染物時,時常要經過長時間的萃取及繁複的淨化程序。例如,就以萃取方法而論,索氏萃取法需要花費12~24小時,微 波萃取法大約需要花費1小時;而超臨界流體萃取法也需要花費1小時;加速溶劑萃取法差不多需要花費0.5小時才能處理完成。不僅耗費時間、成本與人力,而且所得到分析數據在信賴度方面亦難令人滿意,的尚待更進一步的突破。 In addition, in the conventional method of detecting organic pollutants in sediments, the analytical techniques used in the past always require extremely complicated processing procedures, such as organic pollutants such as PBDEs, PAEs, PAHs, PCBs, etc., often undergo long-time extraction. And complicated purification procedures. For example, in terms of extraction method, Soxhlet extraction takes 12 to 24 hours, micro Wave extraction takes about 1 hour; supercritical fluid extraction also takes 1 hour; accelerated solvent extraction takes almost 0.5 hours to process. Not only is it time consuming, cost and manpower, but the analytical data obtained is also unsatisfactory in terms of reliability, and further breakthroughs are yet to be made.
又,例如,含溴原子的二苯醚化合物多溴二苯醚(polybrominated diphenyl ethers,PBDEs),在工業上常做為阻燃劑使用,高共有209種同源物。PBDEs脂溶性強、不易分解且會干擾生物體甲狀腺內分泌,因而對環境及人類的威脅日益升高。在PBDEs中對環境危害最大宗之污染物為使用量最大之十溴二苯醚(BDE-209),約佔總PBDEs之85%~95%。然而,由於目前檢測BDE-209處理程序,須經長時間萃取及繁複的淨化程序,耗費時間、成本與人力,因而需要研發出一種能夠快速且靈敏性高的偵測BDE-209之方法。 Further, for example, polybrominated diphenyl ethers (PBDEs) containing a bromine atom are often used as flame retardants in the industry, and have a total of 209 homologues. PBDEs are highly soluble, are not easily decomposed and interfere with the thyroid endocrine of the organism, and thus pose an increasing threat to the environment and humans. Among the PBDEs, the most harmful pollutants to the environment are the most used decabromodiphenyl ether (BDE-209), accounting for 85% to 95% of the total PBDEs. However, since the current detection of the BDE-209 processing procedure requires long time extraction and complicated purification procedures, which is time consuming, costly and labor intensive, it is necessary to develop a rapid and sensitive method for detecting BDE-209.
因而,期待開發出一種不需要複雜處理程序、能夠縮短前置作業時間、操作容易、節省成本、分析快迅、而且信賴度高之有效的有機污染物之檢測方法。 Therefore, it is expected to develop an effective method for detecting organic pollutants that does not require complicated processing procedures, can shorten the lead time, is easy to operate, saves cost, analyzes fast, and has high reliability.
有鑑於傳統的有機污染物檢測技術之上述問題點,本發明人等即潛心研究,應用表面增強拉曼散射(Surface Enhanced Raman Scattering,SERS)的特點,進而開發出能夠改善傳統檢測技術之問題點的新穎技術,因而完成本發明。 In view of the above problems of the conventional organic pollutant detection technology, the present inventors have devoted themselves to researching and applying the characteristics of Surface Enhanced Raman Scattering (SERS), thereby developing problems that can improve the conventional detection technology. The novel technology thus completes the present invention.
亦即,根據本發明之第一觀點可以提供一種微流體通道型SERS檢測用基材之製備方法,其為製備具有微流體通道的表面增強拉曼散射(SERS)晶片之方法,其包括:藉由光微影法,使用光罩,將包含至少一條以上的微通道圖案形成於玻璃基板上而製成微流道結構母模;將聚二甲基矽氧烷(Polydimethylsiloxane,PDMS)溶液塗布於 前述之微流道結構母模,加熱固化並予以剝離而得到具有微流體通道之PDMS固化結構;將銀鏡反應試劑注入到PDMS固化結構之該微通道內,然後注入還原劑,以使產生銀鏡反應而還原形成銀奈米顆粒,並以去離子水沖洗而得到微流體通道型SERS檢測用基材。 That is, according to the first aspect of the present invention, a method of preparing a substrate for a microfluidic channel type SERS detection, which is a method of preparing a surface enhanced Raman scattering (SERS) wafer having a microfluidic channel, includes: Forming a microchannel structure master mold by photolithography using a photomask to form at least one microchannel pattern on a glass substrate; applying a polydimethylsiloxane (PDMS) solution to the solution The aforementioned microchannel structure master mold is heat-cured and stripped to obtain a PDMS solidified structure having a microfluidic channel; a silver mirror reagent is injected into the microchannel of the PDMS solidified structure, and then a reducing agent is injected to generate a silver mirror reaction. The silver nanoparticles were reduced to form and washed with deionized water to obtain a microfluidic channel type SERS substrate.
其次,根據本發明之第二觀點可以提供一種探針型SERS檢測用基材之製備方法,其為製備具有針狀尖端的表面增強拉曼散射(SERS)晶片之方法,其包括:提供一不鏽鋼線以做為芯材,在該芯材之外層表面,藉由塗布聚二甲基矽氧烷(PDMS)溶液並予以加熱固化而形成探針型SERS檢測用PDMS基材;將所得到的該探針型SERS檢測用PDMS基材浸漬於銀鏡反應試劑中,接著注入甲醛使其表面發生均勻銀鏡反應而生成銀粒。 Secondly, according to a second aspect of the present invention, a method of preparing a probe type SERS detecting substrate, which is a method of preparing a surface enhanced Raman scattering (SERS) wafer having a needle tip, comprising: providing a stainless steel The wire is used as a core material, and a PDMS substrate for probe type SERS detection is formed by coating a polydimethylsiloxane (PDMS) solution on the surface of the outer layer of the core material; The probe type SERS detection PDMS substrate was immersed in a silver mirror reaction reagent, and then formaldehyde was injected to cause a uniform silver mirror reaction on the surface to generate silver particles.
再者,根據本發明之第三觀點可以提供一種探針型SERS檢測用基材之製備方法,其為製備具有針狀尖端的表面增強拉曼散射(SERS)晶片之方法,其包括:提供一不鏽鋼線以做為芯材,在該芯材之外層表面,藉由塗布聚甲基丙烯酸甲酯(PMMA)溶液並予以加熱固化而形成探針型SERS檢測用PMMA基材;將所得到的該探針型SERS檢測用PMMA基材浸漬於銀鏡反應試劑中,接著注入甲醛使其表面發生均勻銀鏡反應而生成銀粒。 Furthermore, according to a third aspect of the present invention, a method of preparing a probe type SERS detecting substrate can be provided, which is a method of preparing a surface enhanced Raman scattering (SERS) wafer having a needle tip, comprising: providing a a stainless steel wire is used as a core material, and a PMMA substrate for probe type SERS detection is formed by coating a polymethyl methacrylate (PMMA) solution on the surface of the outer layer of the core material; The probe type SERS detection was immersed in a silver mirror reaction reagent with a PMMA substrate, and then formaldehyde was injected to cause a uniform silver mirror reaction on the surface to generate silver particles.
又,根據本發明之第四觀點可以提供一種平面型SERS檢測用基材之製備方法,其為製備具有平板狀表面增強拉曼散射(SERS)晶片之方法,其包括:將經洗淨之矽晶基材浸漬於表面改質劑中,於一段時間後,以去離子水清洗潔淨而得到表面經修飾改質之矽晶基材;在該表面經修飾改質之矽晶基材、奈米金修飾劑置於離心機中,進行使用重力離心成膜,然後取出並自然乾燥而得到平面型SERS檢測用基材。 Further, according to a fourth aspect of the present invention, there can be provided a method for producing a substrate for planar SERS detection, which is a method for preparing a wafer having a flat surface enhanced Raman scattering (SERS), which comprises: washing the crucible The crystal substrate is immersed in the surface modifier, and after a period of time, it is cleaned with deionized water to obtain a surface-modified and modified crystal substrate; the surface modified and modified crystal substrate, nano The gold modifier was placed in a centrifuge, and the film was formed by gravity centrifugation, and then taken out and naturally dried to obtain a substrate for planar SERS detection.
另外,根據本發明之第五觀點可以提供一種平面型SERS檢測用基材之製備方法,其為製備具有平板狀表面增強拉曼散射(SERS)晶片之方法,其包括:將經洗淨之矽晶基材浸漬於表面改質劑中,於一段時間後,以去離子水清洗潔淨而得到表面經修飾改質之矽晶基材;在該表面經修飾改質之矽晶基材、奈米銀修飾劑置於離心機中,進行使用重力離心成膜,然後取出並自然乾燥而得到平面型SERS檢測用基材。 Further, according to a fifth aspect of the present invention, there can be provided a method for producing a substrate for planar SERS detection, which is a method for preparing a wafer having a flat surface enhanced Raman scattering (SERS), which comprises: washing the crucible The crystal substrate is immersed in the surface modifier, and after a period of time, it is cleaned with deionized water to obtain a surface-modified and modified crystal substrate; the surface modified and modified crystal substrate, nano The silver modifier was placed in a centrifuge, and formed into a film by gravity centrifugation, and then taken out and naturally dried to obtain a substrate for planar SERS detection.
此外,根據本發明之第五觀點可以提供一種有機污染物之檢測方法,其為可快速檢測出有機污染物之方法,其包括:將待檢測對象物中之有機污染物注入至由上述之製備方法所製得的微流體通道型SERS檢測用基材的微流體通道內;接著,對於上述之該微流體通道型SERS檢測用基材或探針型SERS檢測用基材,藉由三維奈米拉曼螢光顯微鏡系統進行偵測而得到表示拉曼訊號變化之拉曼光譜圖;經由該拉曼光譜圖比對分析並鑑別有機污染物之類別、濃度等。 Further, according to a fifth aspect of the present invention, a method for detecting an organic contaminant, which is a method for rapidly detecting an organic contaminant, comprising: injecting an organic contaminant in an object to be detected into the above-described preparation The microfluidic channel type SERS detecting substrate obtained by the method is in the microfluidic channel; and then, for the microfluidic channel type SERS detecting substrate or the probe type SERS detecting substrate, by the three-dimensional nanometer The Raman fluorescence microscope system performs detection to obtain a Raman spectrum indicating a change of the Raman signal; the Raman spectrum is compared and analyzed to identify the type and concentration of the organic pollutant.
此外,根據本發明之第六觀點可以提供一種有機污染物之檢測方法,其為可快速檢測出有機污染物之方法,其包括:將藉由如上述之製備方法所製得的探針型SERS檢測用基材或平面型SERS檢測用基材,置放於待檢測對象物中,以使有機污染物吸附於探針並放置預定的一段時間;接著,對於上述之該微流體通道型SERS檢測用基材或探針型SERS檢測用基材,藉由三維奈米拉曼螢光顯微鏡系統進行偵測而得到表示拉曼訊號變化之拉曼光譜圖;經由該拉曼光譜圖比對分析並鑑別有機污染物之類別、濃度等。 Further, according to a sixth aspect of the present invention, a method for detecting an organic contaminant, which is a method for rapidly detecting an organic contaminant, comprising: a probe type SERS prepared by the above-described preparation method, can be provided. a substrate for detection or a substrate for planar SERS detection, placed in an object to be detected, so that organic contaminants are adsorbed to the probe and left for a predetermined period of time; and then, for the microfluidic channel type SERS detection described above Using a substrate or probe type SERS detection substrate, a Raman spectrum representing a change in Raman signal is obtained by a three-dimensional nano-Raman fluoroscopy system; the Raman spectrum is analyzed and compared Identify the type and concentration of organic pollutants.
根據本發明之各種新穎觀點及研究成果,利用本發明所開發之微流體通道型、探針型表面增強拉曼散射(SERS)晶片、基樣, 藉由本發明之SERS拉曼光譜檢測有機污染檢測方法,不但能夠解決傳統氣相層析儀(GC)或高效能液態層析儀(HPLC)等之有機物檢測需要大量試樣、處理作業繁雜、分析時間長久、花費龐大人力及物力等之問題點,而且能夠分辨結構相似的有機物或菌株、以及同時偵測定性定量多種不同污染物。 According to various novel ideas and research results of the present invention, microfluidic channel type, probe type surface enhanced Raman scattering (SERS) wafers and substrates developed by the present invention are utilized. By detecting the organic contamination detection method by the SERS Raman spectroscopy of the present invention, it is possible to solve the problem that the organic gas detection such as the conventional gas chromatography (GC) or the high-performance liquid chromatography (HPLC) requires a large number of samples, and the processing operation is complicated and analyzed. It takes a long time, takes a lot of problems such as manpower and material resources, and can distinguish organic matter or strains with similar structure, and simultaneously detect qualitative and quantitative determination of many different pollutants.
更且,由於本發明之ERS拉曼光譜檢測有機污染檢測方法是一種檢測十分簡易、非常快速且分析精度、信賴度均高的方法,以及可以將本發明SERS檢測基材及有機污染檢測方法整併成為可攜式檢測系統或裝置,因而能夠廣泛應用於需要檢測有機污染之各種不同的科學用途與技術領域,藉以檢測物質與定量,包括在高分子聚合物、奈米材料、電化學、半導體、薄膜、礦物學、生物、醫學藥品、碳化物、線上過程監測、定量控制、刑事鑑定偵測:玻璃材料、氧化物、油漆和顏料、氫氧化物、高分子、硫化物、***物、碳酸鹽、纖維、硫酸鹽、化學殘留物、磷酸鹽、顆粒性包裹體、麻醉劑和可控制物質......等。 Moreover, since the ERS Raman spectroscopy detection organic contamination detecting method of the present invention is a method which is very simple, very rapid, and has high analytical precision and high reliability, and can be used for the SERS detecting substrate and the organic pollution detecting method of the present invention. And it becomes a portable detection system or device, so it can be widely used in various scientific applications and technical fields that need to detect organic pollution, thereby detecting substances and quantification, including in high molecular polymers, nano materials, electrochemistry, semiconductors. , film, mineralogy, biology, medical drugs, carbides, online process monitoring, quantitative control, criminal identification detection: glass materials, oxides, paints and pigments, hydroxides, polymers, sulfides, explosives, carbonic acid Salt, fiber, sulfate, chemical residues, phosphates, particulate inclusions, anesthetics and controllable substances...etc.
圖1為顯示本發明之製備例1的奈米金修飾型SERS檢測用Au-11-MUA基材之表面放大100,000倍的SEM照片。 Fig. 1 is a SEM photograph showing the surface magnified 100,000 times of the Au-11-MUA substrate for nano-modified SERS of Preparation Example 1 of the present invention.
圖2為顯示以Image J分析圖1所示之奈米金修飾型SERS檢測用Au-11-MUA基材所得到的銀粒徑分布圖。 Fig. 2 is a graph showing the particle size distribution of silver obtained by analyzing the Au-11-MUA substrate for nano-modified SERS shown in Fig. 1 by Image J.
圖3為顯示本發明之製備例2的奈米金修飾型SERS檢測用Au-6-MHA基材之表面放大100,000倍的SEM照片。 Fig. 3 is a SEM photograph showing a surface magnified 100,000 times of the surface of the Au-6-MHA substrate for nano-modified SERS of Preparation Example 2 of the present invention.
圖4為顯示以Image J分析圖3所示的奈米金修飾型SERS檢測用Au-6-MHA基材所得到的銀粒徑分布圖。 Fig. 4 is a graph showing the distribution of silver particle size obtained by analyzing the Au-6-MHA substrate for nano-modified SERS shown in Fig. 3 by Image J.
圖5為顯示本發明之製備例3的奈米金修飾型SERS檢測用Au-3-MPA基材之表面放大100,000倍的SEM照片。 Fig. 5 is a SEM photograph showing the surface magnified 100,000 times of the Au-3-MPA substrate for nano-gold-modified SERS detection of Preparation Example 3 of the present invention.
圖6為顯示以Image J分析圖5所示的奈米金修飾型SERS檢測用Au-MPA基材所得到的銀粒徑分布圖。 Fig. 6 is a graph showing the particle size distribution of silver obtained by analyzing the Au-MPA substrate for nano-modified SERS shown in Fig. 5 by Image J.
圖7為顯示本發明之製備例4的奈米銀修飾型SERS檢測用Ag-6-MHA基材之表面放大100,000倍的SEM照片。 Fig. 7 is a SEM photograph showing a surface magnification of 100,000 times of the surface of the Ag-6-MHA substrate for nano silver-modified SERS of Preparation Example 4 of the present invention.
圖8為顯示以Image J分析圖5所示的奈米銀修飾型SERS檢測用Ag-6-MHA基材所得到的銀粒徑分布圖。 Fig. 8 is a graph showing the particle size distribution of silver obtained by analyzing the nano silver-modified SERS for Ag-6-MHA substrate shown in Fig. 5 by Image J.
圖9為顯示實施例1所示以本發明之SERS檢測用基材偵測偵測鄰苯二甲酸酯類(PAs)所得到之拉曼光譜圖;其中(a)表示鄰苯二甲酸二丁酯(DBP)之拉曼光譜圖;(b)表示鄰苯二甲酸二乙酯(DEP)之拉曼光譜圖;(c)表示鄰苯二甲酸二甲酯(DMP)之拉曼光譜圖;(d)表示鄰苯二甲酸二正辛酯(DNOP)之拉曼光譜圖;(e)表示鄰苯二甲酸二(2-乙基己基)酯(DEHP)之拉曼光譜圖。 9 is a Raman spectrum diagram showing the detection of phthalate esters (PAs) by the substrate for SERS detection of the present invention shown in Example 1; wherein (a) represents dibutyl phthalate. Raman spectrum of ester (DBP); (b) shows the Raman spectrum of diethyl phthalate (DEP); (c) shows the Raman spectrum of dimethyl phthalate (DMP); (d) shows a Raman spectrum of di-n-octyl phthalate (DNOP); (e) shows a Raman spectrum of di(2-ethylhexyl) phthalate (DEHP).
圖10為顯示本發明之製備例4的奈米銀修飾微流體通道型SERS檢測用Ag基材之通道表面放大100,000倍的SEM照片。 Fig. 10 is a SEM photograph showing a 100,000-fold magnification of the channel surface of the Ag substrate for the nano silver-modified microfluidic channel type SERS detection of Preparation Example 4 of the present invention.
圖11為顯示本發明之製備例5的奈米銀修飾探針型SERS檢測用Ag基材之探針表面放大100,000倍的SEM照片。 Fig. 11 is a SEM photograph showing the surface of the probe of the Ag substrate for the silver-modified probe type SERS detection of the preparation example 5 of the present invention magnified 100,000 times.
圖12為顯示二仁溪底泥萃取液與空白萃取液樣品的表面增強拉曼光譜。 Figure 12 is a surface enhanced Raman spectrum showing a sample of Errenxi bottom mud extract and blank extract.
圖13為顯示二仁溪底泥樣品的表面增強拉曼光譜圖。 Figure 13 is a surface enhanced Raman spectrum showing a sample of Errenxi sediment.
圖14為顯示PDMS探針型SERS基材之十溴二苯醚之拉曼光譜圖。 Figure 14 is a Raman spectrum of decabromodiphenyl ether showing a PDMS probe type SERS substrate.
圖15為顯示PMMA探針型SERS基材之十溴二苯醚之拉曼光譜圖。 Figure 15 is a Raman spectrum diagram showing decabromodiphenyl ether of a PMMA probe type SERS substrate.
以下,列舉具體的實施例,一邊參照圖面一邊說明本發明的各種實施形態;但本發明並不受限於此等實施形態及實施例而已。對於熟知本項技術者而言,應理解到:只要其本身沒有脫離本發明之原理及精神,則該等實施形態及實施例分是可以修改或變更的,而且該等修改或變更皆落入本發明之適用範圍及其等效範圍之內,視同屬於本發明之範疇。 In the following, various embodiments of the present invention will be described with reference to the drawings, but the invention is not limited to the embodiments and examples. It will be understood by those skilled in the art that the embodiments and the embodiments may be modified or changed as long as they do not depart from the principles and spirit of the present invention, and such modifications or changes may fall into The scope of applicability of the invention and its equivalents are considered to be within the scope of the invention.
首先,說明表面增強拉曼散射(Surface Enhanced Raman Scattering,SERS)的特點。 First, the characteristics of Surface Enhanced Raman Scattering (SERS) are described.
拉曼光譜是一項重要的現代光譜技術,已經被廣泛地使用來探測分子結構及原子間交互作用力、及研究分子振動模式的技術,已成為研究分子結構和組態、確定晶體結構的對稱性、研究固體中的缺陷和雜質、環境污染物、生物分子和工業材料微觀結構的有力工具。 Raman spectroscopy is an important modern spectroscopy technique that has been widely used to detect molecular structures and interactions between atoms, and techniques for studying molecular vibration modes. It has become a study of molecular structure and configuration, and determination of crystal structure symmetry. A powerful tool for studying the defects and impurities in solids, environmental pollutants, biomolecules, and the microstructure of industrial materials.
Fleishmann等人[3]在研究電化學電池內銀電極上吸附的吡啶分子的拉曼光譜時,於1974年發現表面增強拉曼散射(Surface Enhanced Raman Scattering,SERS),他們發現其譜線強度有明顯增強,拉曼信號的增強可以達到104~106量級。雖然已知道在例如Ag、Au、Pt、Cu、Al和鹼金屬表面等能夠有效產生SERS,但並不是在任何表面都可以發生SERS的;在上述有限的幾種金屬表面以外,就算可發現SERS其強度也是非常微弱的,因而無法任意地應用於微量物質偵測,特別是有機污染物之檢測。 Fleishmann et al. [3] discovered Raman spectra of pyridine molecules adsorbed on silver electrodes in electrochemical cells. Surface Enhanced Raman Scattering (SERS) was discovered in 1974. increased significantly enhance the Raman signal can reach the order of 10 4 to 10 6. Although it is known that SERS can be efficiently produced on, for example, Ag, Au, Pt, Cu, Al, and alkali metal surfaces, SERS cannot occur on any surface; even on the limited metal surfaces mentioned above, even SERS can be found. Its intensity is also very weak, so it can not be arbitrarily applied to the detection of trace substances, especially the detection of organic pollutants.
為解決此等習用技術之問題點與困難點,本發明人等研究開發出能夠讓受測對象物中之化學物質良好地吸附或鍵結、且表面經修飾或特殊處理之微流體通道型SERS檢測用基材及探針型SERS檢測用基材;以及藉由本發明之SERS基材,利用拉曼散射光譜分析來檢測有機污染物之有效且快速的方法。 In order to solve the problems and difficulties of the conventional techniques, the present inventors have developed a microfluidic channel type SERS capable of adsorbing or bonding a chemical substance in a test object well and having a surface modified or specially treated. A substrate for detection and a substrate for probe type SERS detection; and an effective and rapid method for detecting organic contaminants by Raman scattering spectroscopy by the SERS substrate of the present invention.
例如,根據本發明之第一觀點可以提供一種微流體通道型SERS檢測用基材之製備方法,其為製備具有微流體通道的表面增強拉曼散射(SERS)晶片之方法,其包括:藉由光微影法,使用光罩,將包含至少一條以上的微通道圖案形成於玻璃基板上而製成微流道結構母模;將聚二甲基矽氧烷(Polydimethylsiloxane,PDMS)溶液塗布於前述之微流道結構母模,加熱固化並予以剝離而得到具有微流體通道之PDMS固化結構;將銀鏡反應試劑注入到PDMS固化結構之該微通道內,然後注入還原劑,以使產生銀鏡反應而還原形成銀奈米顆粒,並以去離子水沖洗而得到微流體通道型SERS檢測用基材。 For example, according to a first aspect of the present invention, a method of preparing a substrate for a microfluidic channel type SERS can be provided, which is a method of preparing a surface enhanced Raman scattering (SERS) wafer having a microfluidic channel, including: Photolithography method, using a photomask, forming at least one microchannel pattern on a glass substrate to form a microchannel structure master; applying a polydimethylsiloxane (PDMS) solution to the foregoing The microchannel structure master mold is heat-cured and stripped to obtain a PDMS cured structure having a microfluidic channel; a silver mirror reagent is injected into the microchannel of the PDMS solidified structure, and then a reducing agent is injected to generate a silver mirror reaction. The silver nanoparticles were reduced to form and washed with deionized water to obtain a microfluidic channel type SERS substrate.
其次,根據本發明之第二觀點可以提供一種探針型SERS檢測用基材之製備方法,其為製備具有針狀尖端的表面增強拉曼散射(SERS)晶片之方法,其包括:將探針型基材浸漬於銀銨溶液中,然後注入甲醛,以使產生銀鏡反應而還原形成銀奈米顆粒,並以去離子水沖洗而得到針型SERS檢測用基材。 Secondly, according to a second aspect of the present invention, a method for preparing a probe type SERS detecting substrate, which is a method of preparing a surface enhanced Raman scattering (SERS) wafer having a needle tip, comprising: a probe The substrate is immersed in a silver ammonium solution, and then injected with formaldehyde to cause a silver mirror reaction to be reduced to form silver nanoparticles, and rinsed with deionized water to obtain a needle-shaped SERS substrate.
根據本發明之一實施態樣,適用本發明之基板的種類並末特別限制;只要能夠在其表面或其內部形成流體通道的物質皆可以使用。舉例來說,例如,其可以是使用自聚二甲基矽氧烷(PDMS)、聚甲基丙烯酸甲酯(PMMA))構成群組中所選取之至少一種。 According to an embodiment of the present invention, the kind of the substrate to which the present invention is applied is particularly limited; as long as a substance capable of forming a fluid passage on the surface or inside thereof can be used. For example, it may be at least one selected from the group consisting of polydimethyl methoxy oxane (PDMS) and polymethyl methacrylate (PMMA).
又,根據上述本發明之第一觀點至第三觀點製備微流體通道型SERS檢測用基材、或探針型SERS檢測用基材之方法中,該聚二甲基矽氧烷(PDMS)溶液並未特別加以限制,可以是由末端含有乙烯基之PDMS單體、及含有Si-H的官能基之PDMS單體所構成。 Further, in the method for producing a microfluidic channel type SERS detecting substrate or a probe type SERS detecting substrate according to the first to third aspects of the present invention, the polydimethyloxane (PDMS) solution It is not particularly limited, and may be composed of a PDMS monomer having a vinyl group at the terminal and a PDMS monomer having a functional group containing Si-H.
此外,根據上述本發明之第一觀點至第三觀點製備微流體通道型SERS檢測用基材、或探針型SERS檢測用基材之方法中,該銀鏡反應試劑雖然是未特別加以限制,然而通常是銀銨溶液。 Further, in the method for producing a microfluidic channel type SERS detecting substrate or a probe type SERS detecting substrate according to the first to third aspects of the invention, the silver mirror reaction reagent is not particularly limited, however Usually a silver ammonium solution.
再者,根據上述本發明之第一觀點至第三觀點製備微流體通道型SERS檢測用基材、或探針型SERS檢測用基材之方法中,該銀銨溶液係由在硝酸銀溶液(AgNO3,2%)中加入數滴氫氧化鈉(NaOH,2.5M)溶液以使產生棕色的氧化銀,並且迅速於該溶液中滴加濃氨水,直至棕色沉澱恰好溶解為止而得到之銀銨溶液。 Further, in the method for producing a microfluidic channel type SERS detecting substrate or a probe type SERS detecting substrate according to the first to third aspects of the invention described above, the silver ammonium solution is obtained from a silver nitrate solution (AgNO) 3 , 2%), add a few drops of sodium hydroxide (NaOH, 2.5M) solution to produce brown silver oxide, and quickly add concentrated ammonia water to the solution until the brown precipitate just dissolves to obtain the silver ammonium solution. .
又,適用於本發明之一實施態樣的銀銨溶液之構成成分並末特別限制;舉例來說,例如,其可以是包括硝酸銀(AgNO3,2%)、氨水(NH4OH,2M)、氫氧化鈉(NaOH,2.5M)所構成,且該銀銨溶液之濃度為0.1重量%~5重量%。 Further, the constituent components of the silver ammonium solution applicable to one embodiment of the present invention are particularly limited; for example, it may include silver nitrate (AgNO 3 , 2%), ammonia water (NH 4 OH, 2 M), hydrogen. Sodium oxide (NaOH, 2.5 M) is used, and the concentration of the silver ammonium solution is 0.1% by weight to 5% by weight.
另外,根據上述本發明之第一觀點至第三觀點製備微流體通道型SERS檢測用基材、或探針型SERS檢測用基材之方法中,該還原劑並未特別加以限制,可以是自甲醛、乙醛、丙醛、其他醛類、甲酸、葡萄糖、葡萄糖酯、麥芽糖、果糖及其混合物構成群組中所選取之至少一種。然而,較佳者為甲醛、乙醛、丙醛、甲酸、葡萄糖、葡萄糖酯、麥芽糖、或果糖;更佳者為甲醛、乙醛、甲酸、葡萄糖、或果糖;特佳者為甲醛、乙醛、葡萄糖、或果糖;或者也可以是彼等之混合物。 Further, in the method of producing a microfluidic channel type SERS detecting substrate or a probe type SERS detecting substrate according to the first to third aspects of the present invention, the reducing agent is not particularly limited and may be Formaldehyde, acetaldehyde, propionaldehyde, other aldehydes, formic acid, glucose, glucose ester, maltose, fructose, and mixtures thereof constitute at least one selected from the group. However, preferred is formaldehyde, acetaldehyde, propionaldehyde, formic acid, glucose, glucose ester, maltose, or fructose; more preferably formaldehyde, acetaldehyde, formic acid, glucose, or fructose; particularly preferred is formaldehyde, acetaldehyde , glucose, or fructose; or it may be a mixture of them.
又,適用於本發明之一實施態樣的還原劑之配合比並末特別限制;舉例來說,例如,該甲醛之濃度可以是95容積%~99.99容積%。 Further, the mixing ratio of the reducing agent which is suitable for use in an embodiment of the present invention is particularly limited; for example, the concentration of the formaldehyde may be 95% by volume to 99.99% by volume.
再者,適用於本發明之一實施態樣的微流體通道之大小並末特別限制;舉例來說,該微流體通道之尺寸大小,例如,寬度可以是在0.01~0.5公分之範圍、深度可以是在0.01~0.5公分奈米之範圍。 Furthermore, the size of the microfluidic channel suitable for use in an embodiment of the present invention is particularly limited; for example, the size of the microfluidic channel, for example, the width may be in the range of 0.01 to 0.5 cm, and the depth may be It is in the range of 0.01~0.5 cm nanometer.
此外,根據本發明之第四觀點可以提供一種平面型SERS檢測用基材之製備方法,其為製備具有平板狀表面增強拉曼散射(SERS)晶片之方法,其包括:將經洗淨之矽晶基材浸漬於表面改質劑 中,於一段時間後,以去離子水清洗潔淨而得到表面經修飾改質之矽晶基材;在該表面經修飾改質之矽晶基材、奈米金修飾劑置於離心機中,進行使用重力離心成膜,然後取出並自然乾燥而得到平面型SERS檢測用基材。 Further, according to a fourth aspect of the present invention, a method of producing a substrate for planar SERS detection, which is a method for preparing a wafer having a flat surface-enhanced Raman scattering (SERS), comprising: a cleaned crucible Crystal substrate impregnated with surface modifier After a period of time, it is cleaned with deionized water to obtain a crystal-modified substrate whose surface has been modified and modified; the crystal substrate and the nano gold modifier which are modified and modified on the surface are placed in a centrifuge, The film was formed by gravity centrifugation, and then taken out and naturally dried to obtain a substrate for planar SERS detection.
又,適用於本發明的第四觀點之一實施態樣的表面改質劑之構成成分比並末特別限制;舉例來說,例如,該表面改質劑可以是由3-氨基丙基三乙氧基矽烷(APTMS)、乙醇所構成之溶液;該奈米金修飾劑為將由單寧酸、檸檬酸鈉、碳酸鉀溶解於去離子水形成之溶液A、四氯化金酸溶解於去離子水所形成之溶液B予以混合並充分攪拌而得到之奈米金膠體;接著以11-MUA(11-Mercaptoundecanoic acid)、6-MHA(6-mercaptohexanoic acid)、或3-MPA(3-Mercaptopropionic acid)修飾該所得到的奈米金膠體而成之奈米金修飾劑。 Further, the constituent ratio of the surface modifier which is applicable to one embodiment of the fourth aspect of the present invention is particularly limited; for example, the surface modifier may be 3-aminopropyltriethyl a solution of oxydecane (APTMS) and ethanol; the nanogold modifier is a solution A obtained by dissolving tannic acid, sodium citrate or potassium carbonate in deionized water, and tetrachloroauric acid is dissolved in deionized The solution B formed by water is mixed and thoroughly stirred to obtain a nano gold colloid; followed by 11-MUA (11-Mercaptooundecanoic acid), 6-MHA (6-mercaptohexanoic acid), or 3-MPA (3-Mercaptopropionic acid) a nano gold modifier prepared by modifying the obtained nano gold colloid.
此外,根據本發明之第五觀點可以提供一種平面型SERS檢測用基材之製備方法,其為製備具有平板狀表面增強拉曼散射(SERS)晶片之方法,其包括:將經洗淨之矽晶基材浸漬於表面改質劑中,於一段時間後,以去離子水清洗潔淨而得到表面經修飾改質之矽晶基材;在該表面經修飾改質之矽晶基材、奈米銀修飾劑置於離心機中,進行使用重力離心成膜,然後取出並自然乾燥而得到平面型SERS檢測用基材 Further, according to a fifth aspect of the present invention, a method of producing a substrate for planar SERS detection, which is a method for preparing a wafer having a flat surface enhanced Raman scattering (SERS), comprising: a cleaned crucible The crystal substrate is immersed in the surface modifier, and after a period of time, it is cleaned with deionized water to obtain a surface-modified and modified crystal substrate; the surface modified and modified crystal substrate, nano The silver modifier is placed in a centrifuge, and is formed into a film by gravity centrifugation, and then taken out and naturally dried to obtain a substrate for planar SERS detection.
又,適用於本發明的第五觀點之一實施態樣的表面改質劑之構成成分比並末特別限制;舉例來說,例如,該表面改質劑可以是由3-氨基丙基三乙氧基矽烷((3-Aminopropyl)trimethoxysilane,APTMS)、乙醇所構成之溶液;該奈米銀修飾劑為將予以檸檬酸鈉、去離子水、硝酸銀、硼氫化鈉混合並加熱充分攪拌而得到之奈米銀膠 體;接著,利用11-MUA、6-MHA、或3-MPA修飾該所得到的奈米銀膠體而成之奈米銀修飾劑。 Further, the constituent ratio of the surface modifier which is applicable to one embodiment of the fifth aspect of the present invention is particularly limited; for example, the surface modifier may be 3-aminopropyltriethyl a solution of (3-Aminopropyl) trimethoxysilane (APTMS) and ethanol; the nano silver modifier is obtained by mixing sodium citrate, deionized water, silver nitrate, sodium borohydride and heating and stirring well. Nano silver glue Next, the nano silver colloid obtained by modifying the obtained nano silver colloid is modified with 11-MUA, 6-MHA, or 3-MPA.
再者,根據本發明之第六觀點可以提供一種有機污染物之檢測方法,其為可快速檢測出有機污染物之方法,其包括:將待檢測對象物中之有機污染物注入至由如上述之製備方法所製得的微流體通道型SERS檢測用基材的微流體通道內;接著,對於上述之該微流體通道型SERS檢測用基材或探針型SERS檢測用基材,藉由三維奈米拉曼螢光顯微鏡系統進行偵測而得到表示拉曼訊號變化之拉曼光譜圖;經由該拉曼光譜圖比對分析並鑑別有機污染物之類別、濃度等。 Furthermore, according to a sixth aspect of the present invention, a method for detecting an organic contaminant can be provided, which is a method for rapidly detecting an organic contaminant, comprising: injecting an organic contaminant in an object to be inspected to be as described above The microfluidic channel type SERS detecting substrate obtained by the preparation method is in the microfluidic channel; and then, for the microfluidic channel type SERS detecting substrate or the probe type SERS detecting substrate, by three-dimensional The nano-Raman fluorescence microscope system detects the Raman spectrum representing the change of the Raman signal; the Raman spectrum is compared and analyzed to identify the type and concentration of the organic pollutant.
此外,根據本發明之第七觀點可以提供一種有機污染物之檢測方法,其為可快速檢測出有機污染物之方法,其包括:將藉由如上之製備方法所製得的探針型SERS檢測用基材或平面型SERS檢測用基材,置放於待檢測對象物中,以使有機污染物吸附於探針並放置預定的一段時間;接著,對於上述之該微流體通道型SERS檢測用基材或探針型SERS檢測用基材,藉由三維奈米拉曼螢光顯微鏡系統進行偵測而得到表示拉曼訊號變化之拉曼光譜圖;經由該拉曼光譜圖比對分析並鑑別有機污染物之類別、濃度等。 Further, according to a seventh aspect of the present invention, a method for detecting an organic contaminant, which is a method for rapidly detecting an organic contaminant, comprising: detecting a probe type SERS prepared by the above preparation method, can be provided. The substrate for substrate or planar SERS detection is placed in the object to be detected, so that the organic pollutants are adsorbed to the probe and left for a predetermined period of time; and then, for the above-mentioned microfluidic channel type SERS detection Substrate or probe type SERS detection substrate, which is detected by a three-dimensional nano-Raman fluorescence microscope system to obtain a Raman spectrum representing a change of Raman signal; the Raman spectrum is compared and analyzed by the Raman spectrum The type and concentration of organic pollutants.
根據本發明的一實施態樣,該電極板之電位差範圍並末特別限制;舉例來說,例如,較佳者可以是介於1.0至110伏特之範圍;更佳者可以是在10至110伏特之範圍;特佳者可以是在30至110伏特之範圍。 According to an embodiment of the present invention, the potential difference range of the electrode plate is particularly limited; for example, preferably, it may be in the range of 1.0 to 110 volts; more preferably, it may be in the range of 10 to 110 volts. The range; particularly good can be in the range of 30 to 110 volts.
根據本發明的一實施態樣之有機污染物之檢測方法,待檢測對象物並末特別限制;舉例來說,例如,包括河川湖泊海洋等水體中之水與底泥、自來水、工業用水、家庭廢水、工業廢水、活性污泥、農田或溝渠中之底泥、或其他之水或污泥等;或者是食物、飲料、化妝 品、保健用品、醫療器材、玩具、包裝材、兒童或老人護理品等皆能夠使用本發明來進行檢測。 According to an embodiment of the present invention, the method for detecting an organic pollutant is particularly limited in terms of the object to be detected; for example, water and sediment in a water body such as a river, a lake, and the like, tap water, industrial water, and a family. Waste water, industrial wastewater, activated sludge, sediment in farmland or ditch, or other water or sludge; or food, drink, makeup Products, health products, medical equipment, toys, packaging materials, children or elderly care products, etc. can be tested using the present invention.
根據本發明的一實施態樣之之檢測方法,檢測對象物之該有機污染物並末特別限制;舉例來說,例如,其可以是包括多溴二苯醚(polybrominated diphenylethers,PBDEs)、PAEs、多環芳香烴(polycyclic aromatic hydrocarbons,PAHs)、多氯聯苯(polychlorinated biphenyls,PCBs)、戴奧辛(dioxins)、氯化呋喃(chlorinated furans)、氯化苯類(chlorinated benzenes)、一氯苯、二氯苯、六氯苯、鄰苯二甲酸酯類(phthalates)、DMP、DEP、DBP、BBP、DNOP、DEHP、阿特靈(aldrin)、可氯丹(chlordane)、二氯二苯基三氯乙烷(4,4'-dichlorodiphenyltrichloroethane,DDT)及其衍生物、地特靈(dieldrin)、安特靈(endrin)、飛佈達(heptachlor)、毒殺芬(toxaphene)、安殺番(endosulfan)、甲萘威(carbaryl)、胸腺嘧啶(Thymine)、BDE-15、BDE-47、BDE-209、十溴二苯醚或其混合物等皆可以使用本發明來進行檢測。 According to an embodiment of the present invention, the detection of the organic contaminant of the object is particularly limited; for example, it may include polybrominated diphenyl ethers (PBDEs), PAEs, Polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), dioxins, chlorinated furans, chlorinated benzenes, monochlorobenzene, Chlorobenzene, hexachlorobenzene, phthalates, DMP, DEP, DBP, BBP, DNOP, DEHP, aldrin, chlordane, dichlorodiphenyl trichloride Ethyl (4,4'-dichlorodiphenyltrichloroethane, DDT) and its derivatives, dieldrin, endrin, heptachlor, toxaphene, endosulfan Carbaryl, thymine, BDE-15, BDE-47, BDE-209, decabromodiphenyl ether or mixtures thereof can be tested using the present invention.
以下,列舉實施例來進一步說明本發明,然而本發明當然是未限定於該等實施例而已。另外,在實施例中之特性的評價係以下述之方法來進行。 Hereinafter, the present invention will be further illustrated by the examples, but the present invention is of course not limited to the examples. Further, the evaluation of the characteristics in the examples was carried out in the following manner.
首先,製備由0.005g的單寧酸(美國Sigma-Aldrich公司製,試藥級99%)、0.4g的檸檬酸鈉(美國J.T.Backer公司製,試藥級99%)、0.0017g的碳酸鉀(台灣聯合化工,試藥級99%)溶解於去離子水 定量至200mL所形成的溶液A。接著,製備由0.10g的四氯化金酸(Hydrogen tetrachloro aurate(III)trihydrate,美國Alfa公司製,試藥級/99%)溶解於去離子水定量至200mL所形成的溶液B。將上述所製備的兩種溶液予以混合,於溫度為攝氏60度下持續烘烤4小時,使形成奈米金膠體。然後,將膠體放置冷卻至室溫,並保存於攝氏4度,防止團聚沉澱。然後,將上述溶液A與溶液B予以混合並以磁石充分攪拌而得到奈米金膠體溶液,粒徑約12nm。 First, 0.005 g of tannic acid (manufactured by Sigma-Aldrich, USA, 99% of the reagent grade), 0.4 g of sodium citrate (manufactured by JT Backer, Inc., 99% of the test grade), and 0.0017 g of potassium carbonate were prepared. (Taiwan United Chemical, 99% of the drug grade) dissolved in deionized water The solution A formed was quantified to 200 mL. Next, a solution B prepared by dissolving 0.10 g of Hydrogen tetrachloro aurate (III) trihydrate (manufactured by Alfa Co., Ltd., reagent grade/99%) in deionized water to 200 mL was prepared. The two solutions prepared above were mixed and baked at a temperature of 60 ° C for 4 hours to form a nano gold colloid. The gel was then placed to cool to room temperature and stored at 4 degrees Celsius to prevent agglomeration precipitation. Then, the above solution A and solution B were mixed and thoroughly stirred with a magnet to obtain a nano gold colloidal solution having a particle diameter of about 12 nm.
接著,將所製得的上述奈米金膠體溶液投入的11-Mercaptoundecanoic acid(11-MUA)(美國Sigma Aldrich公司製,試藥級99%)之修飾溶液中,使修飾溶液對奈米金膠體溶液的投入容積比成為10:1(V/V),並以去離子水進行調整pH=10.5,藉以對奈米金膠體進行表面修飾。 Next, the prepared nano gold colloid solution was put into a modification solution of 11-Mercaptoundecanoic acid (11-MUA) (manufactured by Sigma Aldrich Co., Ltd., 99%), and the modified solution was applied to the nano gold colloid. The ratio of the input volume of the solution was 10:1 (V/V), and the pH was adjusted to 10.5 with deionized water to thereby surface-modify the nano-gold colloid.
將矽晶片(台灣,虹惠光電製)予以清洗潔淨後,置入由雙氧水30%與硫酸以比例3:7(w/w)混合而成之HS溶液中進行表面初步改質處理,使成為Si-OH矽醇基的親水性經提高之表面;接著,用去離子水潤洗基板表面,再置入3-氨基丙基三乙氧基矽烷(APTMS)/乙醇溶液中,於浸泡8-12小時後,以去離子水超音波清洗而得到表面經修飾改質之矽晶基材。 After cleaning and cleaning the ruthenium wafer (made by Taiwan, Honghui Optoelectronics Co., Ltd.), the surface of the HS solution prepared by mixing 30% of hydrogen peroxide with sulfuric acid at a ratio of 3:7 (w/w) is used for preliminary surface modification. The hydrophilic surface of the Si-OH sterol group is improved; then, the surface of the substrate is rinsed with deionized water, and then placed in 3-aminopropyltriethoxydecane (APTMS)/ethanol solution for soaking in 8- After 12 hours, the surface was modified and modified to obtain a crystal substrate having a modified surface.
接著,將上述表面經修飾改質之矽晶基材、奈米金膠體及已修飾的溶液置入離心機中,使用重力離心643.2×g(Rcf)進行離心成膜。於成膜後,取出基材、自然乾燥並以氮***吹乾而得到奈米金修飾型SERS檢測用Au-11-MUA基材。 Next, the above-mentioned surface modified crystal substrate, nano gold colloid, and modified solution were placed in a centrifuge, and centrifuged to form a film by gravity centrifugation 643.2 × g (Rcf). After the film formation, the substrate was taken out, naturally dried, and blown dry with a nitrogen gas gun to obtain a Au-11-MUA substrate for nano-modified SERS.
經由以掃描式電子顯微鏡SEM放大100,000倍觀察,而得到圖1之放大100,000倍SEM照片。圖1為顯示本發明之製備例3的奈 米金修飾型SERS檢測用Au-MUA基材之表面放大100,000倍的SEM圖。如圖1之Au-11-MUA的SEM圖所示,可確認在奈米金修飾型SERS檢測用Au-11-MUA基材的表面上,具有銀顆粒均勻分布且均一堆疊在Au-11-MUA基材表面上,而兩個銀粒子間之間隙約為8-10nm左右。 The magnified 100,000-fold SEM photograph of Fig. 1 was obtained by magnifying 100,000 times with a scanning electron microscope SEM. 1 is a view showing the preparation of Example 3 of the present invention. The surface of the Au-MUA substrate for the Mikin-modified SERS detection was magnified 100,000 times the SEM image. As shown in the SEM image of Au-11-MUA in Fig. 1, it was confirmed that silver nanoparticles were uniformly distributed on the surface of the Au-11-MUA substrate for nano-modified SERS detection and uniformly stacked on Au-11- On the surface of the MUA substrate, the gap between the two silver particles is about 8-10 nm.
另外,經由Image J影像分析結果,如圖2所示。由圖2之以Image J分析所得到的銀粒徑分布圖,可確認奈米銀顆粒之粒徑平均為34.5nm。 In addition, the results of image analysis by Image J are shown in FIG. 2. From the silver particle size distribution obtained by Image J analysis in Fig. 2, it was confirmed that the average particle diameter of the nano silver particles was 34.5 nm.
然後,使用中興大學奈米科技中心之三維奈米拉曼螢光顯微鏡系統(雷射波長為632.8nm及488nm,最大功率為3.6mW),以羅丹明(Rhodamine B,R6G)為指標分子,在奈米金修飾平面型SERS檢測用Au-11-MUA基材上存在有R6G的情況、及R6G與T型二聚體銀的情況下,分別量測入射光633nm波長下之入射偵測訊號,利用式1計算出SERS增強效果,結果確認利用奈米金修飾平面型SERS檢測用Au-11-MUA基材可量測到T型銀二聚體(T-shape dimer)的偵測極限濃度最低為10-8M。 Then, using the three-dimensional nano-Raman fluorescence microscope system (the laser wavelength is 632.8nm and 488nm, the maximum power is 3.6mW) of the Nanotechnology Center of ZTE University, and Rhodamine B (R6G) is used as the indicator molecule. When the R6G is present on the Au-11-MUA substrate for the nano-modified SERS, and the R6G and T-dimer silver are used, the incident detection signals at the incident wavelength of 633 nm are respectively measured. The SERS enhancement effect was calculated using Equation 1. As a result, it was confirmed that the detection limit concentration of the T-shape dimer can be measured by using the Au-11-MUA substrate for the planar SERS detection using nanogold. 10 -8 M.
首先,以和製備例1同樣地操作所得到之奈米金膠體。接著,將所製得的奈米金膠體投入10:1(V/V,膠體:修飾溶液)的6-mercaptohexanoic acid(6-MHA)(美國Sigma Aldrich公司製,試藥級99%)之修飾溶液中,再以去離子水進行調整pH=10.5,藉以對奈米金膠體進行表面修飾,而得到Au-6-MHA修飾奈米金膠體。 First, the obtained nano gold colloid was operated in the same manner as in Production Example 1. Next, the prepared nano gold colloid was put into a modification of 6-mercaptohexanoic acid (6-MHA) (manufactured by Sigma Aldrich Co., Ltd., 99%) of 10:1 (V/V, colloid: modified solution). In the solution, the pH was adjusted to 10.5 with deionized water to thereby surface-modify the nano-gold colloid to obtain an Au-6-MHA-modified nano gold colloid.
接著,將製備例1中所得到的表面經修飾改質之矽晶基材、上述之Au-6-MHA修飾奈米金膠體、將已修飾的溶液置入離心機中,與製備例同地樣使用重力離心643.2×g(Rcf)進行離心成膜。於成膜後,取出基材、自然乾燥並以氮***吹乾而得到奈米金修飾型SERS檢測用Au-6-MHA基材。 Next, the surface-modified modified crystal substrate obtained in Preparation Example 1, the above-mentioned Au-6-MHA modified nano gold colloid, and the modified solution were placed in a centrifuge, in the same manner as the preparation example. The film was formed by centrifugation using gravity centrifugation 643.2 × g (Rcf). After the film formation, the substrate was taken out, naturally dried, and blown dry with a nitrogen gas gun to obtain a Au-6-MHA substrate for nano-modified SERS.
繼續,與製備例1同樣地經由以掃描式電子顯微鏡SEM放大100,000倍觀察,而得到圖3之放大100,000倍SEM照片。圖5為顯示本發明之製備例2的奈米金修飾型SERS檢測用Au-6-MHA基材之表面放大100,000倍的SEM圖。如圖3之Au-6-MHA的SEM圖所示,在該奈米金修飾型SERS檢測用Au-6-MHA基材的表面上,具有均勻分布且均一堆疊銀顆粒。 Further, in the same manner as in Preparation Example 1, the magnified 100,000-fold SEM photograph of FIG. 3 was observed by magnifying 100,000 times with a scanning electron microscope SEM. Fig. 5 is a SEM image showing the surface magnified 100,000 times of the Au-6-MHA substrate for nano-modified SERS of Preparation Example 2 of the present invention. As shown in the SEM image of Au-6-MHA in Fig. 3, on the surface of the Au-6-MHA substrate for the nanogold-modified SERS detection, there were uniformly distributed and uniformly stacked silver particles.
另外,經由Image J影像分析結果,如圖4所示。由圖4之以Image J分析所得到的銀粒徑分布圖,確認奈米銀顆粒之粒徑約為32.0nm。 In addition, the results of image analysis by Image J are shown in FIG. From the silver particle size distribution obtained by Image J analysis in Fig. 4, it was confirmed that the nano silver particles had a particle diameter of about 32.0 nm.
首先,以和製備例1同樣地操作所得到之奈米金膠體溶液。接著,將所製得的奈米金膠體投入10:1(V/V,膠體:修飾溶液)3-Mercaptopropionic acid(3-MPA)(美國Sigma Aldrich公司製,試藥級99%)之修飾溶液中,再以以去離子水進行調整pH=11.5,藉以對奈米金膠體進行表面修飾,而得到Au-3-MPA修飾奈米金膠體。 First, the obtained nano gold colloidal solution was operated in the same manner as in Production Example 1. Next, the prepared nano gold colloid was put into a modification solution of 10:1 (V/V, colloid: modified solution) 3-Mercaptopropionic acid (3-MPA) (manufactured by Sigma Aldrich Co., Ltd., test grade 99%). In the middle, the pH was adjusted to 11.5 with deionized water to surface-modify the nano-gold colloid to obtain a Au-3-MPA-modified nano gold colloid.
接著,將製備例1中所得到的表面經修飾改質之矽晶基材、上述之奈米Au-3-MPA膠體溶液及矽晶片置入離心機中,與製備例1同地樣使用重力離心643.2×g(Rcf)進行離心成膜。於成膜後,取 出基材、自然乾燥並以氮***吹乾而得到奈米金修飾型SERS檢測用Au-3-MPA基材。 Next, the surface-modified twin crystal substrate obtained in Preparation Example 1, the above-mentioned nano Au-3-MPA colloidal solution, and the ruthenium wafer were placed in a centrifuge, and gravity was used in the same manner as in Preparation Example 1. The film was formed by centrifugation at 643.2 × g (Rcf). After film formation, take The substrate was dried, naturally dried, and blown dry with a nitrogen gas gun to obtain a Au-3-MPA substrate for nano-modified SERS.
繼續,與製備例1同樣地經由以掃描式電子顯微鏡SEM放大100,000倍觀察,而得到圖5之放大100,000倍SEM照片。圖5為顯示本發明之製備例3的奈米金修飾型SERS檢測用Au-3-MPA基材之表面放大100,000倍的SEM圖。如圖5之Au-3-MPA的SEM圖所示,在該奈米金修飾型SERS檢測用Au-3-MPA基材的表面上,具有均勻分布且均一堆疊的銀顆粒。 Further, in the same manner as in Preparation Example 1, the SEM photograph was magnified 100,000 times by SEM magnification by a scanning electron microscope. Fig. 5 is a SEM image showing the surface magnification of the Au-3-MPA substrate for the nanogold-modified SERS detection of Preparation Example 3 of the present invention at 100,000 times. As shown in the SEM image of Au-3-MPA of Fig. 5, on the surface of the Au-3-MPA substrate for the nanogold-modified SERS detection, there were uniformly distributed and uniformly stacked silver particles.
另外,經由Image J影像分析結果,如圖6所示。由圖6之以Image J分析所得到的銀粒徑分布圖,確認奈米銀顆粒之粒徑約為29.1nm。 In addition, the results of image analysis by Image J are shown in FIG. 6. From the silver particle size distribution obtained by Image J analysis in Fig. 6, it was confirmed that the particle diameter of the nano silver particles was about 29.1 nm.
首先,將0.2g的檸檬酸鈉(美國J.T.Backer公司製,試藥級99%)溶解於去離子水,一邊加熱(溫度為50℃)一邊加入0.017g的硝酸銀(美國Sigma Aldrich公司製,試藥級99%)、0.2g的硼氫化鈉(美國Panreac公司製,試藥級99%),持續加熱約1小時以去除過剩的硼氫化鈉,以使形成含有預定粒徑之銀粒子的奈米銀膠體溶液(成長兩次粒徑30nm)。 First, 0.2 g of sodium citrate (manufactured by JT Backer Co., Ltd., a pharmaceutical grade of 99%) was dissolved in deionized water, and 0.017 g of silver nitrate (manufactured by Sigma Aldrich Co., Ltd.) was added while heating (temperature: 50 ° C). Pharmaceutical grade 99%), 0.2 g of sodium borohydride (manufactured by Panreac Co., USA, 99% of the test grade), and heating was continued for about 1 hour to remove excess sodium borohydride to form a nanoparticle containing silver particles of a predetermined particle diameter. Rice silver colloidal solution (grown twice with a particle size of 30 nm).
接著,將上述所製得的奈米銀膠體溶液投入6-mercaptohexanoic acid(6-MHA)之修飾溶液中,使修飾溶液對奈米銀膠體的投入容積比成為10:1(V/V,膠體:修飾),並以去離子水進行調整pH10.5,藉以對奈米銀膠體進行表面修飾。 Next, the above-mentioned nano silver colloid solution is put into a modification solution of 6-mercaptohexanoic acid (6-MHA), so that the input volume ratio of the modification solution to the nano silver colloid becomes 10:1 (V/V, colloid : Modified), and adjusted to pH 10.5 with deionized water to surface-modify the nano-silver colloid.
將矽晶片(台灣,虹惠光電製)予以清洗潔淨後,先以氧氣電漿機改質,通入氧氣間歇性改質10秒,停滯2分鐘重複3次後,再置入由雙氧水30%與硫酸以比例3:7(w/w)混合而成之HS溶液中進行表面初步改質處理,使成為Si-OH矽醇基的親水性經提高之表面;接著,用去離子水潤洗基板表面,再置入3-氨基丙基三乙氧基矽烷(APTMS)/乙醇溶液中10%(APTMS:Et 10:90 v/v),於浸泡12小時後,以去離子水超音波清洗而得到表面經修飾改質之矽晶基材。 After cleaning the enamel wafer (Taiwan, Honghui Optoelectronics), first upgrade it with an oxygen plasma machine, intermittently upgrade the oxygen for 10 seconds, stagnate for 2 minutes, repeat 3 times, and then place 30% of hydrogen peroxide. Surface preliminary modification with HS in a ratio of 3:7 (w/w) in a ratio of 3:7 (w/w) to make a hydrophilically enhanced surface of Si-OH sterol group; then, rinse with deionized water The surface of the substrate was placed in a 3-aminopropyltriethoxydecane (APTMS)/ethanol solution at 10% (APTMS:Et 10:90 v/v). After soaking for 12 hours, it was ultrasonically cleaned with deionized water. A crystal substrate having a surface modified and modified is obtained.
接著,將上述表面經修飾改質之矽晶基材、奈米銀膠體、聚二甲基矽氧烷(PDMS)溶液置入離心機中,使用重力離心643.2×g(Rcf)進行離心成膜。於成膜後,取出基材、自然乾燥並以氮***吹乾而得到奈米銀修飾平面型SERS檢測用Ag基材。經由以掃描式電子顯微鏡SEM觀察,而得到圖7之放大100,000倍SEM照片。 Next, the above-mentioned surface modified crystal substrate, nano silver colloid, and polydimethyloxane (PDMS) solution were placed in a centrifuge, and centrifuged to form a film by gravity centrifugation 643.2×g (Rcf). . After the film formation, the substrate was taken out, naturally dried, and blown dry with a nitrogen gas gun to obtain a nano substrate for nano silver-modified planar SERS detection. The magnified 100,000-fold SEM photograph of Fig. 7 was obtained by SEM observation with a scanning electron microscope.
圖7為顯示本發明之製備例4的奈米銀修飾型SERS檢測用Ag-6-MHA基材之表面放大100,000倍的SEM圖。如圖7之Ag-11-MUA的SEM圖所示,確認在該奈米銀修飾平面型SERS檢測用Ag基材的表面上,具有銀顆粒均勻分布且均一堆疊在Ag基材,而兩個銀粒子間之間隙約為1nm左右。 Fig. 7 is a SEM image showing the surface magnified 100,000 times of the Ag-6-MHA substrate for nano silver-modified SERS detection of Preparation Example 4 of the present invention. As shown in the SEM image of Ag-11-MUA in Fig. 7, it was confirmed that the surface of the Ag substrate for the nano silver-modified planar SERS was uniformly distributed and uniformly stacked on the Ag substrate, and two The gap between the silver particles is about 1 nm.
另外,經由Image J影像分析結果,如圖8所示。由圖8之以Image J分析所得到的銀粒徑分布圖,可確認奈米銀顆粒之粒徑約為34.5nm。 In addition, the results of analysis by Image J image are shown in FIG. From the silver particle size distribution obtained by Image J analysis in Fig. 8, it was confirmed that the nano silver particles had a particle diameter of about 34.5 nm.
首先,於無塵室(國立中興大學孟堯生物晶片中心Class 100)中,將清洗潔淨之玻璃基板放置於光阻塗布機(SCD-6000,詠欣 儀器,台灣)上塗布負光阻(日本JSR株式会社製,JSR THB 121N)光阻劑後,將玻璃基板置於紅外線雙面對準曝光機(OAI-500),利用光學投影以光微影蝕刻法,藉由光罩使微流體通道圖案形成於玻璃基板上。完成UV曝光後,將玻璃基板與光阻浸泡於顯影液(AD-TMAH 0.5)去除未曝光之光阻劑,然後烘烤退火去灰去除殘餘之溶劑,而得到微流道結構母模。 First, in the clean room (National Chung Hsing University Mengzi Biochip Center Class 100), the cleaned glass substrate is placed on the photoresist coater (SCD-6000, Yan Xin Instrument, Taiwan) After coating a photoresist with a negative photoresist (JSR THB 121N, manufactured by JSR Corporation, Japan), the glass substrate was placed in an infrared double-sided alignment exposure machine (OAI-500), and optical projection was performed by optical projection. In the etching method, the microfluidic channel pattern is formed on the glass substrate by the photomask. After the UV exposure is completed, the glass substrate and the photoresist are immersed in a developing solution (AD-TMAH 0.5) to remove the unexposed photoresist, and then baked and annealed to remove the residual solvent to obtain a microchannel structure master.
其次,將末端含有乙烯基之A劑(主劑,長的PDMS單體)及含有Si-H的官能基之B劑(硬化劑,短的PDMS單體),以重量比A:B=10:1(w/w)予以混合並充分攪拌至充滿氣泡後,將其放入真空乾燥鍋內以真空幫浦(GVD050A,台灣乾曜科技公司製)抽真空,以除去稠狀PDMS在混合攪拌時所產生之氣泡,而得到PDMS溶液。 Next, the A agent (main agent, long PDMS monomer) containing a vinyl group and the B agent (hardener, short PDMS monomer) containing a functional group of Si-H at a weight ratio of A: B = 10 :1 (w/w) was mixed and thoroughly stirred until it was filled with air bubbles, and then placed in a vacuum drying pot to vacuum the vacuum pump (GVD050A, manufactured by Taiwan Cognac Technology Co., Ltd.) to remove the thick PDMS in the mixing and stirring. The bubbles generated at the time gave a PDMS solution.
接著,將所得到PDMS溶液澆注於上述之微流道結構母模後,放置於85℃之加熱板(CO-PC600D,美國Corning公司製)上加熱固化3小時。待PDMS固化完成後,放置於室溫下充分冷卻,然後由該母模取下那已完全固化冷卻的PDMS,即可得到具有微流道結構之PDMS。 Next, the obtained PDMS solution was poured into the above-described microchannel structure master, and placed on a hot plate (CO-PC600D, manufactured by Corning, USA) at 85 ° C for 3 hours. After the PDMS curing is completed, it is sufficiently cooled at room temperature, and then the PDMS which has been completely solidified and cooled is removed from the master mold to obtain PDMS having a microchannel structure.
銀鏡反應試劑亦稱為多倫試劑、吐倫試劑、或土倫試劑(Tollens' reagent),指含有二氨合銀(I)離子([Ag(NH3)2]+)的水溶液,一般由硝酸銀或其他銀化合物與氨水反應而製得。理論上除所有含有醛基的物質以外,甲酸、葡萄糖、葡萄糖酯、麥芽糖、果糖亦皆可做為還原劑,使得銀鏡反應試劑發生銀鏡反應。一般比較常用的還原劑,例如,甲醛、乙醛和葡萄糖等。 Silver mirror reagents, also known as Duolun reagents, toluene reagents, or Tollens' reagents, refer to aqueous solutions containing silver (I) ions ([Ag(NH 3 ) 2 ]+), generally It is prepared by reacting silver nitrate or other silver compounds with ammonia water. Theoretically, in addition to all substances containing an aldehyde group, formic acid, glucose, glucose ester, maltose, and fructose can also be used as a reducing agent, so that the silver mirror reaction reagent undergoes a silver mirror reaction. Generally used reducing agents such as formaldehyde, acetaldehyde and glucose.
銀鏡反應試劑之製備方法,首先可於硝酸銀溶液中加入數滴氫氧化鈉溶液,如以下化學式所示,起初產生非常不穩定之氫氧化銀白色沉澱,旋即產生棕色的氧化銀沉澱。接著,於該溶液中滴加濃氨水,直至棕色沉澱剛好溶解,而得到含Ag(NH3)2NO3(aq)之銀銨溶液,此即為一種銀鏡反應試劑。 The silver mirror reaction reagent is prepared by first adding a few drops of sodium hydroxide solution to the silver nitrate solution. As shown in the following chemical formula, a very unstable silver hydroxide white precipitate is initially produced, which immediately produces a brown silver oxide precipitate. Next, concentrated ammonia water was added dropwise to the solution until the brown precipitate just dissolved, thereby obtaining a silver ammonium solution containing Ag(NH 3 ) 2 NO 3 (aq), which is a silver mirror reaction reagent.
AgNO3+NaOH→AgOH↓+NaNO3 AgNO 3 +NaOH→AgOH↓+NaNO 3
AgOH+2 NH3→[Ag(NH3)2]OH(aq) AgOH+2 NH 3 →[Ag(NH 3 ) 2 ]OH (aq)
繼續,將所得到的銀銨溶液(銀鏡反應試劑),以針筒注入上述所製備的PDMS微流道結構之流道入口端,藉由幫浦於另一端進行抽氣,使得氨溶液均勻分布於微流道內。然後,將甲醛注入於該流道內,使得整個微流道均勻發生銀鏡反應,藉以生成用來當做SERS訊號增強所需基材的銀粒,進而得到微流體通道型SERS檢測用基材。 Continuing, the obtained silver ammonium solution (silver mirror reaction reagent) is injected into the inlet end of the flow path of the PDMS micro-flow channel structure prepared by the syringe, and the pump is pumped at the other end to uniformly distribute the ammonia solution. In the micro flow channel. Then, formaldehyde is injected into the flow path, so that the silver mirror reaction occurs uniformly throughout the micro flow path, thereby generating silver particles for use as a substrate for SERS signal enhancement, thereby obtaining a microfluidic channel type SERS detection substrate.
經由以掃描式電子顯微鏡SEM觀察,而得到圖10之放大100,000倍SEM照片。圖10為顯示本發明之製備例5的奈米銀修飾微流體通道型SERS檢測用Ag基材之通道表面放大100,000倍的SEM圖。另外,經由Image J影像分析結果,可確認奈米銀顆粒之平均粒徑約為71.0±38.2nm。 The magnified 100,000-fold SEM photograph of Fig. 10 was obtained by SEM observation with a scanning electron microscope. Fig. 10 is a SEM image showing a 100,000-fold magnification of the channel surface of the Ag substrate for the nano silver-modified microfluidic channel type SERS detection of Preparation Example 5 of the present invention. Further, it was confirmed by Image J image analysis that the average particle diameter of the nano silver particles was about 71.0 ± 38.2 nm.
首先,以不鏽鋼為芯材,外層包覆單層的聚二甲基矽氧烷(PDMS)而製作成探針型SERS檢測用基材。外層包覆材料並未特別地限制,只要是能夠吸附污染物即可;舉例來說,例如,其可以是具 有疏水性的高分子材料。形狀亦未特別地限制;舉例來說,例如,其可以是細長狀。 First, a stainless steel was used as a core material, and a single layer of polydimethyl siloxane (PDMS) was coated on the outer layer to prepare a probe-type SERS substrate. The outer covering material is not particularly limited as long as it is capable of adsorbing contaminants; for example, it may be Hydrophobic polymer material. The shape is also not particularly limited; for example, it may be elongated.
其次,在包覆PDMS之後,繼續將它浸漬於由製備例5所得到的銀銨溶液(銀鏡反應試劑)中,接著注入甲醛使其表面發生均勻銀鏡反應,藉以生成用來當做SERS訊號增強所需基材的銀粒,進而得到探針型SERS檢測用PDMS基材。 Next, after coating the PDMS, it was further immersed in the silver ammonium solution (silver mirror reaction reagent) obtained in Preparation Example 5, followed by injecting formaldehyde to cause a uniform silver mirror reaction on the surface, thereby generating the SERS signal enhancement. The silver particles of the substrate are required to obtain a PDMS substrate for probe type SERS detection.
經由以掃描式電子顯微鏡SEM觀察,而得到放大50,000倍SEM照片。另外,經由Image J影像分析結果,可確認奈米銀顆粒之平均粒徑約為71.0±38.2nm。 A 50,000-fold SEM photograph was obtained by observation with a scanning electron microscope SEM. Further, it was confirmed by Image J image analysis that the average particle diameter of the nano silver particles was about 71.0 ± 38.2 nm.
首先,與製備例6同樣地以不鏽鋼為芯材,外層包覆單層的聚聚甲基丙烯酸甲酯(PMMA)而製作成探針型SERS檢測用PMMA基材。 First, in the same manner as in Preparation Example 6, stainless steel was used as a core material, and a single layer of polymethyl methacrylate (PMMA) was coated on the outer layer to prepare a probe-type SEMA substrate for SERS detection.
其次,在包覆PMMA之後,繼續將它浸漬於由製備例5所得到的銀銨溶液(銀鏡反應試劑)中,接著注入甲醛使其表面發生均勻銀鏡反應,藉以生成用來當做SERS訊號增強所需基材的銀粒,進而得到探針型SERS檢測用PMMA基材。 Next, after coating the PMMA, it was further immersed in the silver ammonium solution (silver mirror reaction reagent) obtained in Preparation Example 5, followed by injecting formaldehyde to cause a uniform silver mirror reaction on the surface, thereby generating a SERS signal enhancement station. The silver particles of the substrate are required to obtain a PMMA substrate for probe type SERS detection.
經由以掃描式電子顯微鏡SEM觀察,而得到圖11之放大50,000倍SEM照片。圖11為顯示本發明之製備例5的奈米銀修飾微流體通道型SERS檢測用Ag基材之通道表面放大100,000倍的SEM圖。另外,經由Image J影像分析結果,可確認奈米銀顆粒之平均粒徑約為71.0±38.2nm。 The magnified 50,000-fold SEM photograph of Fig. 11 was obtained by SEM observation with a scanning electron microscope. Fig. 11 is a SEM image showing a 100,000-fold magnification of the channel surface of the Ag substrate for the nano silver-modified microfluidic channel type SERS detection of Preparation Example 5 of the present invention. Further, it was confirmed by Image J image analysis that the average particle diameter of the nano silver particles was about 71.0 ± 38.2 nm.
使用在製備例1、製備例2、製備例3及製備例4分別所製得的銀修飾平面型SERS檢測用Ag-11-MUA基材、Ag-6-MHA基材、Ag-3-MPA基材、Ag基材,以約1cm2可偵測約10mL體積的對象物的比率裁切出適當大小,來偵測以甲醇為溶劑之三種不同濃度100mg/L、10mg/L、1mg/L的PAs(DMP、DEHP、DNOP、DBP及DEP)以建立鄰苯二甲酸酯類(PAs)有機污染物標準品拉曼光譜圖,結果如圖9所示。 Ag-11-MUA substrate, Ag-6-MHA substrate, Ag-3-MPA for silver-modified planar SERS detection prepared in Preparation Example 1, Preparation Example 2, Preparation Example 3, and Preparation Example 4, respectively. The substrate and the Ag substrate are cut to an appropriate size at a ratio of about 1 cm 2 to detect an object of about 10 mL in volume to detect three different concentrations of 100 mg/L, 10 mg/L, and 1 mg/L in methanol. The PAs (DMP, DEHP, DNOP, DBP, and DEP) were used to establish Raman spectra of phthalate ester (PAs) organic pollutant standards. The results are shown in Figure 9.
圖9為顯示實施例1所示以本發明之SERS檢測用基材偵測鄰苯二甲酸酯類(PAs)所得到之拉曼光譜圖;其中(a)表示鄰苯二甲酸二丁酯(DBP)之拉曼光譜圖;(b)表示鄰苯二甲酸二乙酯(DEP)之拉曼光譜圖;(c)表示鄰苯二甲酸二甲酯(DMP)之拉曼光譜圖;(d)表示鄰苯二甲酸二正辛酯(DNOP)之拉曼光譜圖;(e)表示鄰苯二甲酸二(2-乙基己基)酯(DEHP)之拉曼光譜圖。 Fig. 9 is a Raman spectrum diagram showing the detection of phthalic acid esters (PAs) by the substrate for SERS detection of the present invention shown in Example 1; wherein (a) represents dibutyl phthalate ( Raman spectrum of DBP); (b) shows the Raman spectrum of diethyl phthalate (DEP); (c) shows the Raman spectrum of dimethyl phthalate (DMP); ) represents a Raman spectrum of di-n-octyl phthalate (DNOP); (e) represents a Raman spectrum of di(2-ethylhexyl) phthalate (DEHP).
如圖9所示,可得知彼等PAs於403cm-1、654cm-1、1041cm-1、1126cm-1、1166cm-1、1581cm-1、1602cm-1和1727cm-1具有共同特徵峰,可做為判定偵測對象物中有無存在PAs(鄰苯二甲酸酯類)的依據。 As shown in FIG. 9, it can be seen that the PAs have a common characteristic peak at 403 cm -1 , 654 cm -1 , 1041 cm -1 , 1126 cm -1 , 1166 cm -1 , 1581 cm -1 , 1602 cm -1 and 1727 cm -1 . As a basis for determining the presence or absence of PAs (phthalates) in the object to be detected.
然後,將上述製備例1、製備例2、製備例3及製備例4的奈米金或奈米銀修飾平面型SERS檢測用基材所裁切之SERS基材,浸泡於採自環境樣品中的鄰苯二甲酸酯萃取液、取自二仁溪之底泥樣品(用以確認是否存在鄰苯二甲酸酯的有機污染)、萃取液空白樣品(以正己烷與丙酮為溶劑)中歷12小時後,量測SERS拉曼頻譜,每一個樣品至少量測500點並於不同區域各量測三次,讀取特徵峰635 cm-1、703cm-1、1013cm-1、1277cm-1、1607cm-1,結果如圖12、圖13所示。 Then, the SERS substrate cut by the substrate for the nano- or nano-silver-modified planar SERS detection of the above Preparation Example 1, Preparation Example 2, Preparation Example 3, and Preparation Example 4 was immersed in an environmental sample. a phthalate extract, a sediment sample from Errenxi (to confirm the presence or absence of organic contamination of phthalate), and a blank sample of the extract (with n-hexane and acetone as solvent) calendar after 12 hours, measured Raman SERS spectrum of each sample and the measurement of at least 500 points in each of the three measuring regions in different reading characteristic peak 635 cm -1, 703cm -1, 1013cm -1, 1277cm -1, 1607 cm -1 , the results are shown in Fig. 12 and Fig. 13.
圖12為顯示二仁溪底泥萃取液與空白萃取液樣品的表面增強拉曼光譜。圖13為顯示二仁溪底泥樣品的表面增強拉曼光譜圖。由該等圖12、圖13所示,可以確認銀修飾平面型SERS檢測用Ag-11-MUA基材、Ag-6-MHA基材、Ag-3-MPA基材及Ag基材均可用來快速檢測底泥等對象物鄰苯二甲酸酯類(PAs)之濃度。 Figure 12 is a surface enhanced Raman spectrum showing a sample of Errenxi bottom mud extract and blank extract. Figure 13 is a surface enhanced Raman spectrum showing a sample of Errenxi sediment. As shown in FIG. 12 and FIG. 13, it can be confirmed that the Ag-11-MUA substrate, the Ag-6-MHA substrate, the Ag-3-MPA substrate, and the Ag substrate for the silver-modified planar SERS detection can be used. Quickly measure the concentration of phthalates (PAs) in objects such as sediment.
使用在製備例5所製得的微流體通道型SERS檢測用基材,以約1cm2可偵測約10mL體積的對象物的比率裁切出適當大小,來偵測以乙酸乙酯為溶劑配製十溴二苯醚濃度各為0.09ppm、0.9ppm、9.0ppm、90ppm之樣品以建立十溴二苯醚(BDE-209)有機污染物標準品拉曼光譜圖,結果得到之拉曼光譜圖如圖14所示。 Using the microfluidic channel type SERS detection substrate prepared in Preparation Example 5, an appropriate size was cut at a ratio of about 1 cm 2 to detect an object of about 10 mL in volume to detect preparation with ethyl acetate as a solvent. A sample of 0.09 ppm, 0.9 ppm, 9.0 ppm, and 90 ppm of decabromodiphenyl ether was used to establish a Raman spectrum of the decabromodiphenyl ether (BDE-209) organic pollutant standard, and the resulting Raman spectrum was obtained. Figure 14 shows.
圖14為顯示PDMS探針型SERS基材之十溴二苯醚之拉曼光譜圖。由圖14所,可以確認約在1192cm-1及1227cm-1的波數位置具有醚鍵的波峰,且在波數約為1512cm-1的波數位置具有苯環結構的波峰。因此,根據本發明就十溴二苯醚檢測實施例可以確認微流體通道型SERS檢測用基材可用來快速檢測底泥等對象物中是否已有受到十溴二苯醚(BDE-209)之污染。 Figure 14 is a Raman spectrum of decabromodiphenyl ether showing a PDMS probe type SERS substrate. From Fig. 14, it was confirmed that a peak having an ether bond at a wavenumber position of about 1192 cm -1 and 1227 cm -1 has a peak of a benzene ring structure at a wave number position of a wave number of about 1512 cm -1 . Therefore, according to the present invention, the decabromodiphenyl ether detection example can confirm that the microfluidic channel type SERS detection substrate can be used for rapid detection of whether or not decabromodiphenyl ether (BDE-209) has been contained in objects such as sediment. Pollution.
另外,將各濃度之拉曼光譜圖於波數1620cm-1之訊號強度與濃度比較,可得一回歸方程式:y=14.452 x+664.732 R2=0.992(式中,y表示拉曼訊號強度;x表示濃度(ppm))且其R2大於0.99。 In addition, the Raman spectrum of each concentration is compared with the signal intensity of the wave number of 1620 cm -1 to obtain a regression equation: y=14.452 x+664.732 R 2 =0.992 (where y represents the Raman signal intensity; x represents the concentration (ppm) and its R 2 is greater than 0.99.
使用在製備例6所製得的探針型SERS檢測用PDMS基材、PMMA基材裁切出適當大小,於實驗室以乙醇為溶劑,配製十溴二苯醚濃度各為0.1ppm、0.2ppm、0.5ppm、1.0ppm、2.0ppm、4.0ppm之樣品,並於「三維奈米拉曼螢光顯微鏡系統」進行拉曼訊號偵測,藉以建立十溴二苯醚(BDE-209)有機污染物標準品拉曼光譜圖,結果得到如圖15所示之拉曼光譜圖。 The PDMS substrate and the PMMA substrate prepared by the probe type SERS prepared in Preparation Example 6 were cut to an appropriate size, and the concentration of decabromodiphenyl ether was 0.1 ppm and 0.2 ppm in the laboratory using ethanol as a solvent. , 0.5ppm, 1.0ppm, 2.0ppm, 4.0ppm samples, and Raman signal detection in the "3D Nano-Raman Fluorescence Microscope System" to establish decabromodiphenyl ether (BDE-209) organic pollutants The standard Raman spectrum shows the Raman spectrum as shown in FIG.
圖15為顯示PMMA探針型SERS基材之十溴二苯醚之拉曼光譜圖。由圖15所示,可以確認約在1808cm-1及1974cm-1為相對較明顯的波峰,並同時存在於各濃度的光譜圖上,且在約1750cm-1至2080cm-1的波數位置上具有苯環結構之倍頻(overtone)訊號。因此,就十溴二苯醚檢測實施例可以確認本發明之探針型SERS檢測用基材可用來快速檢測底泥等對象物中是否已受到十溴二苯醚(BDE-209)之污染。 Figure 15 is a Raman spectrum diagram showing decabromodiphenyl ether of a PMMA probe type SERS substrate. As shown in FIG. 15, it was confirmed 1808cm -1 to 1974cm -1 and a relatively distinct peaks, while the spectrum is present in each concentration, and at about 1750cm -1 to 2080cm -1 in wavenumber positions about An overtone signal having a benzene ring structure. Therefore, it can be confirmed that the probe type SERS detecting substrate of the present invention can be used for rapid detection of whether or not an object such as a sediment has been contaminated with decabromodiphenyl ether (BDE-209) in terms of the decabromodiphenyl ether detecting example.
根據本發明,分析大量之樣品,細針型SERS基材的製備時間加上進行拉曼訊號偵測的時間比傳統方法所需時間,可縮短90%以上的時間。此研究證實SERS可用於快速檢測底泥中多溴二苯醚之可能性,基於本研究成果,SERS應該可以應用於環境介質中微量疏水性污染物之快速檢測。 According to the present invention, a large number of samples can be analyzed, and the preparation time of the fine needle type SERS substrate plus the time required for Raman signal detection can be shortened by more than 90% over the time required for the conventional method. This study confirms the possibility that SERS can be used to rapidly detect PBDEs in sediments. Based on the results of this study, SERS should be applied to the rapid detection of trace hydrophobic contaminants in environmental media.
以上,雖然為了說明之目的而揭露本發明之較佳實施例,然而熟習本技術領域之具有通常知識者,將可以理解到:在不脫離附加於本發明的申請專利範圍所揭露之精神及範疇內是能夠有各種修改、 添加及替代方案的。此外,這些修改、添加及替代方案應該被視為落入本發明之範圍內。 The above description of the preferred embodiments of the present invention will be understood by those of ordinary skill in the art, There are various modifications within the Add and replace the program. In addition, such modifications, additions and substitutions should be considered as falling within the scope of the invention.
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