TWI499554B - Materials and method for sensing biomolecules with high selectivity - Google Patents
Materials and method for sensing biomolecules with high selectivity Download PDFInfo
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本發明係有關一種感測元件，特別是指一種具高度選擇性的生物感測材料及方法。The present invention relates to a sensing element, and more particularly to a highly selective biosensing material and method.
傳統的感測技術必須利用佈值特定的分子化合物在奈米金屬粒子表面，透過此一特定分子化合物與待測物間的選擇結合，而不與其他物質結合，藉此，可以達到對待測物的專一選擇性，避免產生感測干擾。然而，一般此一特定分子化合物與奈米金屬粒子的接合並不容易，其接合率可能不容易控制，一方面由於製程複雜可能造成感測元件成本的提高，另方面也因接合率的控制不易，可能產生感測元件訊號的不穩定。因此，如何找出一種有別於傳統技術並具有感測選擇性的感測方法，將是相當重要的課題。The traditional sensing technology must use the specific molecular compound of the cloth value on the surface of the nano metal particle, through the selection of the specific molecular compound and the object to be tested, and not combined with other substances, thereby achieving the object to be tested. Specific selectivity to avoid sensing interference. However, in general, the bonding of the specific molecular compound to the nano metal particles is not easy, and the bonding rate may not be easily controlled. On the one hand, the cost of the sensing element may be increased due to the complicated process, and the bonding rate is not easily controlled. , may cause instability of the sensing component signal. Therefore, how to find a sensing method that is different from traditional technology and has sensing selectivity will be a very important topic.
另外，許多奈米金屬粒子具有優異的表面電漿共振(Surface plasmon resonance；SPR)特性，且具有高表面積，因此被廣泛應用在生物感測領域作為感測材料。但奈米金屬粒子本質容易團聚，將造成可能喪失奈米粒子的獨特特性，因此，通常必須在奈米金屬粒子表面被覆界面活性劑(surfactants)，藉以提高奈米金屬粒子的分散性，來避免團聚及沉降的現象發生，但是，界面活性劑可能造成奈米金屬粒子原本具有的優異感測特 性的不利影響。In addition, many nano metal particles have excellent surface plasmon resonance (SPR) characteristics and high surface area, and thus are widely used as sensing materials in the field of biosensing. However, the nature of nano metal particles is easily agglomerated, which may result in the loss of the unique characteristics of the nanoparticles. Therefore, it is usually necessary to coat the surface of the nano metal particles with surfactants to improve the dispersibility of the nanoparticles. Agglomeration and sedimentation occur, but surfactants may cause excellent sensitivity to nano metal particles. Sexual adverse effects.
感測元件的訊號強度與靈敏度係和待測物及感測材料的接觸息息相關，一般而言，訊號的強度與待測物接觸感測材料的數量成正比，與二者接觸距離成反比，故在設計上須極大化感測材料的表面積及避免在感測材料上被覆其他物質。由此可見，待測物與奈米金屬粒子(例如奈米金)的間距將會對表面電漿共振訊號造成很大的影響，可能會降低感測元件的可偵測下限(low detection limit)，降低感測靈敏度；另一方面，奈米金屬粒子的聚集，使得奈米金屬粒子的表面積減少，也會造成感測靈敏度的下降。The signal intensity of the sensing component is closely related to the sensitivity of the sensing object and the sensing material. Generally, the intensity of the signal is proportional to the amount of the sensing material in contact with the object to be tested, and is inversely proportional to the contact distance between the two. It is designed to maximize the surface area of the sensing material and to avoid coating other materials on the sensing material. It can be seen that the distance between the analyte and the nano metal particles (such as nano gold) will have a great influence on the surface plasma resonance signal, which may reduce the low detection limit of the sensing element. To reduce the sensitivity of sensing; on the other hand, the aggregation of nano metal particles causes the surface area of the nano metal particles to decrease, which also causes a decrease in sensitivity.
鑑於以上的問題，本發明的主要目的在於提供一種具高度選擇性的生物感測材料及方法，利用操控測試環境pH值稍微小於待測生物分子的等電位點，則生物分子將帶有微弱正電荷，將可被吸附在帶負電荷的奈米金屬粒子表面，此一利用特別的靜電吸附特性，可以形成與待測生物分子有最佳的靜電吸附效果，從而達到具有專一選擇性的能力。In view of the above problems, the main object of the present invention is to provide a highly selective biosensing material and method. When the pH of the control test environment is slightly smaller than the equipotential point of the biomolecule to be tested, the biomolecule will have a weak positive The charge will be adsorbed on the surface of the negatively charged nano metal particles. This special electrostatic adsorption property can form an optimal electrostatic adsorption effect with the biomolecule to be tested, thereby achieving the ability to have specific selectivity.
本發明的另一目的在於提供一種具高度選擇性的生物感測材料及方法，是以奈米金屬粒子作為感測訊號的介質，並以幾丁聚醣高分子(carboxymethyl hexanoyl chitosan；CHC)自組裝形成的核殼結構作為載體，藉以取代在奈米金屬粒子上的界面活性劑，而能維持奈米金屬粒子的分散性，並增加奈米金屬粒子的感測靈敏度。Another object of the present invention is to provide a highly selective biosensing material and method, which is a medium for sensing signals using nano metal particles and is a carboxymethyl hexanoyl chitosan (CHC). The core-shell structure formed by assembly serves as a carrier to replace the surfactant on the nano metal particles, thereby maintaining the dispersibility of the nano metal particles and increasing the sensing sensitivity of the nano metal particles.
為達上述之目的，本發明提供一種具高度選擇性的生物感測材料，是由載體溶液和複數金屬奈米粒子混合形成為一幾丁聚醣-奈米金屬 粒子核殼結構複合溶液，其中載體溶液是由幾丁聚醣高分子自組裝形成核殼結構於其中之水溶液，金屬奈米粒子則混合於載體溶液中，並藉由靜電作用力吸附及分散於核殼結構的表面，由於幾丁聚醣高分子的水溶性良好，能避免奈米金屬粒子的沉澱與團聚，提高其感測靈敏度。並且，此幾丁聚醣-奈米金屬粒子核殼結構複合溶液之pH值設定為接近且小於特定生物分子的等電位點，將使受偵測之生物分子帶有微弱正電荷，而可與帶負電荷的奈米金屬粒子有最佳的靜電吸附效果，因而奈米金屬粒子對於此生物分子將具有專一選擇性吸附的能力。In order to achieve the above object, the present invention provides a highly selective biosensing material which is formed by mixing a carrier solution and a plurality of metal nanoparticles to form a chitosan-nano metal. A composite solution of a particle core-shell structure, wherein the carrier solution is an aqueous solution in which a chitosan polymer self-assembles to form a core-shell structure, and the metal nanoparticle is mixed in a carrier solution and adsorbed and dispersed by electrostatic force The surface of the core-shell structure, due to the good water solubility of the chitosan polymer, can prevent the precipitation and agglomeration of the nano metal particles and improve the sensing sensitivity. Moreover, the pH of the chitosan-nano metal particle core-shell composite solution is set to be close to and less than the equipotential point of the specific biomolecule, which will cause the detected biomolecule to have a weak positive charge, and The negatively charged nano metal particles have the best electrostatic adsorption effect, so the nano metal particles will have a specific selective adsorption ability for the biomolecule.
同時，本發明也提供一種具高度選擇性的生物感測方法，其步驟是使用生物感測材料來進行感測，此生物感測元件材料為一幾丁聚醣-奈米金屬粒子核殼結構複合溶液，並由載體溶液與複數奈米金屬粒子予以混合而成，載體溶液為幾丁聚醣高分子自組裝形成核殼結構於其中之水溶液，且奈米金屬粒子是藉由靜電作用力吸附並分散於核殼結構表面，然後，藉由調整幾丁聚醣-奈米金屬粒子核殼結構複合溶液之pH值至接近並小於特定生物分子的等電位點，而讓奈米金屬粒子對於此生物分子具有專一選擇性吸附能力，之後，則將一個或多個待測物加入幾丁聚醣-奈米金屬粒子核殼結構複合溶液中，若待測物恰可吸附於奈米金屬粒子表面，則判定此待測物即為該特定生物分子，反之，則判定待測物並非該特定生物分子。Meanwhile, the present invention also provides a highly selective biosensing method, the step of which is to perform sensing using a biosensing material, which is a chitosan-nano metal particle core-shell structure. The composite solution is prepared by mixing a carrier solution and a plurality of nano metal particles, and the carrier solution is a self-assembled chitosan polymer to form an aqueous solution in which a core-shell structure is formed, and the nano metal particles are adsorbed by electrostatic force. And dispersed on the surface of the core-shell structure, and then, by adjusting the pH of the chitosan-nano metal particle core-shell composite solution to be close to and less than the equipotential point of the specific biomolecule, the nano metal particles are The biomolecule has a specific selective adsorption capacity, and then one or more analytes are added to the chitosan-nano metal particle core-shell composite solution, if the analyte is adsorbed on the surface of the nanoparticle Then, it is determined that the object to be tested is the specific biomolecule, and conversely, it is determined that the object to be tested is not the specific biomolecule.
底下藉由具體實施例詳加說明，當更容易瞭解本發明之目的、技術內容、特點及其所達成之功效。The purpose, technical content, features and effects achieved by the present invention will be more readily understood by the detailed description of the embodiments.
10‧‧‧幾丁聚醣核殼結構10‧‧‧ chitosan core-shell structure
20‧‧‧奈米金屬粒子20‧‧‧Nano metal particles
第1圖，為本發明所提供之具高度選擇性的生物感測材料及方法之流程圖。1 is a flow chart of a highly selective biosensing material and method provided by the present invention.
第2圖，為本發明所提供之具高度選擇性的生物感測材料之結構示意圖。Fig. 2 is a schematic view showing the structure of a highly selective biosensing material provided by the present invention.
第3A~3D圖，分別為本發明之實施例將AuNR-CHC複合溶液調整為不同pH值(pH4、pH5、pH6、pH9)的情況下，以人血清白蛋白(Human serum albumin；HSA)和從雞蛋清提取的溶菌酶(Lysozyme)為待測物來進行感測之示意圖。3A-3D, respectively, in the case where the AuNR-CHC complex solution is adjusted to different pH values (pH 4, pH 5, pH 6, pH 9) according to an embodiment of the present invention, human serum albumin (HSA) and A schematic diagram of sensing by using lysozyme (Lysozyme) extracted from egg white as a test object.
請參照第1圖，顯示本發明具高度選擇性的生物感測方法的流程圖。以下針對本發明之各個步驟進行詳細描述：首先，如步驟S100，提供一生物感測材料，此生物感測材料為一幾丁聚醣-奈米金屬粒子核殼結構複合溶液，由幾丁聚醣高分子自組裝(self assembly)形成核殼(core-shell)結構10於水溶液所形成的載體溶液中，將奈米金屬粒子20予以混合，使奈米金屬粒子20得以藉由靜電作用力吸附在幾丁聚醣核殼結構10表面，藉此，可避免奈米金屬粒子20在沒有界面活性劑條件下的沉澱與團聚，又因為幾丁聚醣高分子具有很好的水溶性，能輕易均勻溶解分散於水中，將有助於奈米金屬粒子20得以在幾丁聚醣核殼結構10表面保持長時間的分散性，其結構如第2圖所示。Referring to Figure 1, a flow chart of a highly selective biosensing method of the present invention is shown. The following is a detailed description of each step of the present invention. First, in step S100, a biosensing material is provided, which is a chitosan-nano metal particle core-shell composite solution, which is composed of chitin The sugar polymer self-assembly forms a core-shell structure 10 in a carrier solution formed by an aqueous solution, and the nano metal particles 20 are mixed to allow the nano metal particles 20 to be adsorbed by electrostatic force. On the surface of the chitosan core-shell structure 10, thereby preventing precipitation and agglomeration of the nano metal particles 20 in the absence of a surfactant, and because the chitosan polymer has good water solubility, it can be easily Dispersion and uniform dispersion in water will help the nano metal particles 20 to maintain long-term dispersibility on the surface of the chitosan core-shell structure 10, and the structure is as shown in Fig. 2.
然後，如步驟S200，將幾丁聚醣-奈米金屬粒子核殼結構複合溶液之pH值調整為接近但小於特定生物分子的等電位點(Isoelectric point；IEP)，而讓奈米金屬粒子對於此生物分子具有專一選擇性吸附能力。Then, in step S200, the pH of the chitosan-nano metal particle core-shell composite solution is adjusted to be close to but less than the isoelectric point (IEP) of the specific biomolecule, and the nano metal particles are allowed to This biomolecule has a specific selective adsorption capacity.
最後，如步驟S300，將一個或多個待測物加入幾丁聚醣-奈米金屬粒子核殼結構複合溶液中，若待測物呈現帶有微弱正電荷，將恰可吸附於奈米金屬粒子表面，則可判定此待測物即為該特定生物分子，反之，無法予以吸附的待測物則可判定為並非該特定生物分子。Finally, in step S300, one or more analytes are added to the chitosan-nano metal particle core-shell composite solution, and if the analyte exhibits a weak positive charge, it will adsorb to the nano metal. On the surface of the particle, it can be determined that the analyte is the specific biomolecule, and conversely, the analyte that cannot be adsorbed can be determined not to be the specific biomolecule.
因此，本發明乃特別適合應用於同時存在多種感測物之感測，藉由簡單的調整幾丁聚醣-奈米金屬粒子核殼結構複合溶液之pH值，來製得對於某特定生物分子具有專一選擇性吸附能力之生物感測材料，並可藉由待測物是否可吸附於生物感測材料之奈米屬粒子表面，來判定及選擇生物分子。Therefore, the present invention is particularly suitable for the sensing of simultaneous presence of a plurality of sensing materials, by simply adjusting the pH of the chitosan-nano metal particle core-shell composite solution to produce a specific biomolecule. The biosensing material has a specific selective adsorption capability, and the biomolecule can be determined and selected by whether the analyte can be adsorbed on the surface of the nanoparticle of the biosensing material.
本發明生物感測材料的組成中，幾丁聚醣高分子可為經雙性改質之幾丁聚醣高分子，奈米金屬粒子之材質可為金、銀或鉑，且奈米金屬粒子可為球體、棒狀、或其他形狀。而生物感測材料可感測的生物分子包含人血清白蛋白(human serum albumin,HSA)或溶菌酶(Lysozyme)等蛋白質，亦可為三聚氰胺或咖啡因等其他物質。In the composition of the biosensing material of the present invention, the chitosan polymer may be a chitosan polymer modified by amphoteric modification, and the material of the nano metal particles may be gold, silver or platinum, and the nano metal particles. It can be a sphere, a rod, or other shape. The biomolecules that the biosensing material can sense include proteins such as human serum albumin (HSA) or lysozyme (Lysozyme), and may also be other substances such as melamine or caffeine.
在此，以雙性幾丁聚醣與金奈米粒子的組成為實施例，簡單說明本發明如何製作幾丁聚醣-奈米金屬粒子核殼結構(CHC-AuNR)複合溶液之生物感測材料，其製作流程包含下列步驟：Here, taking the composition of the amphoteric chitosan and the gold nanoparticles as an example, how to make the biosensing of the chitosan-nano metal particle core-shell structure (CHC-AuNR) complex solution will be briefly explained. The material, its production process consists of the following steps:
(1)金奈米粒子(AuNRs)的合成：本實施例是利用種晶促進長成法(seed-mediated growth)來製備金奈米粒子。首先，利用硼氫化鈉(NaBH4 )(0.6mL,0.1M)為還原劑，在三甲基溴化銨(cetyl trimethylammonium bromide,CTAB)(5mL,0.2M)的存在與攪拌條件下，將四氯金酸(HAuCl4 )(5mL,510-4 M)還原。經過1.5小時後，當溶液 的顏色逐漸變成棕黃色後，即反應完成而製得金種晶溶液。此棕黃色溶液將用於合成金奈米粒子。金奈米粒子合成過程，是先取2.5mL,0.01M的四氯金酸、47.5mL,0.1M的CTAB和0.6mL,0.01M的硝酸銀水溶液混合。緩慢混合溶液，並逐漸加入0.275mL,0.1M的L-抗壞血酸(L-ascorbic acid,LAA)，在35℃的溫度下在不斷攪拌，然後將0.06mL的金種晶溶液加到該混合物中，當溶液顏色改變時，即形成金奈米粒子。這些金奈米粒子需在攪拌條件下老化24小時，再通過離心分離方式，除去過量的CTAB。(1) Synthesis of Gold Nanoparticles (AuNRs): In this example, gold nanoparticles were prepared by seed-mediated growth. First, using sodium borohydride (NaBH 4 ) (0.6 mL, 0.1 M) as a reducing agent, in the presence and stirring conditions of cetyl trimethylammonium bromide (CTAB) (5 mL, 0.2 M) Reduction of chloroauric acid (HAuCl 4 ) (5 mL, 510 -4 M). After 1.5 hours, when the color of the solution gradually turned brownish yellow, the reaction was completed to prepare a gold seed crystal solution. This brownish yellow solution will be used to synthesize the gold nanoparticles. The gold nanoparticle particle synthesis process is first mixed with 2.5 mL, 0.01 M tetrachloroauric acid, 47.5 mL, 0.1 M CTAB and 0.6 mL, 0.01 M silver nitrate aqueous solution. The solution was slowly mixed, and 0.275 mL of 0.1 M L-ascorbic acid (LAA) was gradually added thereto, stirring was continued at a temperature of 35 ° C, and then 0.06 mL of a gold seed crystal solution was added to the mixture. When the color of the solution changes, the gold nanoparticles are formed. These gold nanoparticles were aged under agitation for 24 hours and then centrifuged to remove excess CTAB.
(2)幾丁聚醣-奈米金屬粒子核殼結構(CHC-AuNR)複合溶液的合成：首先，將0.8mL去離子水加入0.2mL已製備好的金奈米粒子溶液中，並在7000rpm的轉速下離心15分鐘。之後，加入0.1mL雙性幾丁聚醣(CHC)溶液，並使用0.9mL乙醇，除去金奈米粒子表面的CTAB。然後，在12000rpm的轉速下離心分離20分鐘，除去溶液中的乙醇上清液。於是，形成凝膠狀的幾丁聚醣-奈米金屬粒子核殼結構奈米粒子，然後加入1mL去離子水(DI water)，使凝膠狀的幾丁聚醣-奈米金屬粒子核殼結構奈米粒子再次分散(re-disperse)，以形成分散良好的懸浮溶液，即為本發明作為生物感測材料之CHC-AuNR奈米核殼結構複合溶液。最後，將得到的CHC-AuNR奈米核殼結構複合溶液一次取0.5mL，再用0.5ml的各種緩衝溶液予以混合，來獲得不同pH值之CHC-AuNR奈米核殼結構複合溶液，並利用紫外光-可見光(UV-Vis)光譜儀監測之。(2) Synthesis of chitosan-nano metal particle core-shell structure (CHC-AuNR) composite solution: First, 0.8 mL of deionized water was added to 0.2 mL of prepared gold nanoparticle solution, and at 7000 rpm Centrifuge at the speed for 15 minutes. Thereafter, 0.1 mL of amphoteric chitosan (CHC) solution was added, and 0.9 mL of ethanol was used to remove CTAB on the surface of the gold nanoparticles. Then, the mixture was centrifuged at 12,000 rpm for 20 minutes to remove the ethanol supernatant from the solution. Thus, a gelatinous chitosan-nano metal particle core-shell nanoparticle is formed, and then 1 mL of deionized water (DI water) is added to make a gelatinous chitosan-nano metal particle core shell. The structural nanoparticles are re-dispersed to form a well-dispersed suspension solution, which is a CHC-AuNR nano-core-shell composite solution of the present invention as a biosensing material. Finally, the obtained CHC-AuNR nano-core-shell composite solution was taken once in 0.5 mL, and then mixed with 0.5 ml of various buffer solutions to obtain CHC-AuNR nano-core-shell composite solution with different pH values, and utilized. It is monitored by an ultraviolet-visible (UV-Vis) spectrometer.
其中，緩衝溶液係可選用檸檬酸與檸檬酸鈉混合溶液、乙酸與乙酸鈉混合溶液、醋酸與醋酸鈉混合溶液、或檸檬酸、磷酸氫鉀、硼酸與二乙基巴比妥酸混合溶液；使用緩衝溶液可用以減緩CHC-AuNR奈米核 殼結構複合溶液pH值的劇烈變化。Wherein, the buffer solution may be a mixed solution of citric acid and sodium citrate, a mixed solution of acetic acid and sodium acetate, a mixed solution of acetic acid and sodium acetate, or a mixed solution of citric acid, potassium hydrogen phosphate, boric acid and diethyl barbituric acid; Use buffer solution to slow down CHC-AuNR nanonuclei The pH of the composite solution of the shell structure changes drastically.
接著，再針對本發明具高度選擇性的生物感測材料及方法的使用原理詳加說明。請參照第3A~3D圖，分別顯示本發明之實施例將CHC-AuNR奈米核殼結構複合溶液調整為不同pH值(pH4、pH5、pH6、pH9)的情況下，以人血清白蛋白(HSA)和從雞蛋清提取的溶菌酶(Lysozyme)為待測物來進行感測。Next, the principle of use of the highly selective biosensing materials and methods of the present invention will be further described. Referring to FIGS. 3A-3D, respectively, the embodiment of the present invention adjusts the CHC-AuNR nano core-shell composite solution to different pH values (pH 4, pH 5, pH 6, pH 9), and human serum albumin ( HSA) and lysozyme (Lysozyme) extracted from egg white were sensed for the analyte.
如第3A圖所示，AuNR-CHC複合溶液之pH值為4時，雙性幾丁聚醣-金奈米粒子核殼結構(CHC-AuNR)奈米粒子表面帶正電荷，待測物HSA的表面帶正電荷，待測物Lysozyme的表面也帶正電荷，故，基於庫侖定律之同性電荷相斥的原理，待測物HSA和Lysozyme皆無法吸附於AuNR-CHC奈米粒子。As shown in Fig. 3A, when the pH value of the AuNR-CHC composite solution is 4, the surface of the amphoteric chitosan-gold nanoparticle core-shell structure (CHC-AuNR) nanoparticles is positively charged, and the analyte HSA is measured. The surface of the sample is positively charged, and the surface of the analyte Lysozyme is also positively charged. Therefore, based on the principle of the same charge repulsive by Coulomb's law, the analytes HSA and Lysozyme cannot adsorb to the AuNR-CHC nanoparticle.
如第3B圖所示，CHC-AuNR奈米核殼結構複合溶液之pH值為5時，此pH值接近但小於待測物HSA的等電位點(5.3)，CHC-AuNR奈米粒子表面接近電中性，其中CHC表面帶正電荷，AuNR表面帶負電荷，待測物HSA的表面帶微弱正電荷，待測物Lysozyme的表面帶正電荷，故，基於庫侖定律之同性電荷相斥及異性電荷相吸的原理，CHC會和待測物Lysozyme相斥，AuNR無法吸附待測物Lysozyme，而是容易吸引待測物HSA吸附於AuNR表面。As shown in Fig. 3B, when the pH value of the CHC-AuNR nano core-shell composite solution is 5, the pH is close to but less than the isoelectric point of the HSA of the analyte (5.3), and the surface of the CHC-AuNR nanoparticle is close. Electroneutral, in which the surface of CHC is positively charged, the surface of AuNR is negatively charged, the surface of HSA of test object has a weak positive charge, and the surface of the analyte Lysozyme is positively charged. Therefore, the same charge repulsive and heterosexual based on Coulomb's law The principle of charge attraction, CHC will be repelled by the analyte Lysozyme, AuNR can not adsorb the analyte Lysozyme, but it is easy to attract the analyte HSA to the AuNR surface.
如第3C圖所示，CHC-AuNR奈米核殼結構複合溶液之pH值為6時，CHC-AuNR奈米粒子表面轉為帶負電荷，待測物HSA的表面帶負電荷，待測物Lysozyme的表面帶正電荷，故，基於庫侖定律之同性電荷相斥的原理，待測物HSA無法吸附於AuNR表面，而且，CHC也會和待測物 Lysozyme相斥，AuNR無法吸附待測物Lysozyme。As shown in Fig. 3C, when the pH value of the CHC-AuNR nano core-shell composite solution is 6, the surface of the CHC-AuNR nanoparticle is negatively charged, and the surface of the analyte HSA is negatively charged. The surface of Lysozyme is positively charged. Therefore, based on the principle of the same charge repulsive by Coulomb's law, the HSA of the analyte cannot be adsorbed on the AuNR surface, and the CHC and the analyte are also detected. Lysozyme repelled that AuNR could not adsorb the analyte Lysozyme.
如第3D圖所示，CHC-AuNR奈米核殼結構複合溶液之pH值為9時，此pH值接近但小於待測物Lysozyme的等電位點(10.1)，CHC-AuNR奈米粒子表面帶負電荷，待測物HSA的表面帶負電荷，待測物Lysozyme的表面帶微弱正電荷，故，基於庫侖定律之同性電荷相斥及異性電荷相吸的原理，待測物HSA無法附著於AuNR表面，但AuNR可以很輕易地吸引待測物Lysozyme而附著於表面上。As shown in Fig. 3D, when the pH value of the CHC-AuNR nano core-shell composite solution is 9, the pH is close to but less than the isoelectric point of the analyte Lysozyme (10.1), and the surface band of the CHC-AuNR nanoparticle is as shown in Fig. 3D. Negative charge, the surface of the analyte HSA is negatively charged, and the surface of the analyte Lysozyme has a weak positive charge. Therefore, based on the principle of Coulomb's law of homologous charge repulsion and heterogeneous charge attraction, the HSA of the analyte cannot be attached to AuNR. Surface, but AuNR can easily attract the analyte Lysozyme to attach to the surface.
由上述說明可以理解，當CHC-AuNR奈米核殼結構複合溶液pH值為比待測物的等電位點稍微小的pH值時，對於待測物將呈現最佳的感測靈敏度。因此，本發明生物感測材料利用對於載體溶液pH值的操控，的確可使奈米金屬粒子對於特定生物分子具有專一選擇性吸附能力，而不需如傳統方法利用接合分子化合物的方式方能達到感測選擇性的目的。It can be understood from the above description that when the pH value of the CHC-AuNR nano core-shell structure composite solution is slightly lower than the isoelectric point of the analyte, the sensing sensitivity will be optimal for the analyte. Therefore, the biosensing material of the present invention utilizes the manipulation of the pH of the carrier solution to ensure that the nano metal particles have a specific selective adsorption capacity for a specific biomolecule, without the need to use a molecular compound as in the conventional method. The purpose of sensing selectivity.
底下更利用數個實施例的實驗結果，來輔助說明本發明可達到對於特定生物分子的感測具有高選擇性及高靈敏性。The experimental results of several examples are further utilized to assist in illustrating that the present invention achieves high selectivity and sensitivity to sensing of specific biomolecules.
(一)以HSA感測為例，在不同pH值的CHC-AuNR奈米核殼結構複合溶液下測試其紫外光-可見光(UV-Vis)特性，如表一所示，可以很清楚的觀察到在HSA的等電位點(等電位點為5.3)附近的pH值(pH 5)呈現較靈敏的峰值位移(peak shift)。因此，可以利用pH值的操控來達到感測靈敏度，並藉由各種生物分子可能具有的等電位點特性來達到選擇性的效果。(1) Taking HSA sensing as an example, the UV-Vis characteristics of the CHC-AuNR nano-core-shell composite solution at different pH values were tested, as shown in Table 1, which can be clearly observed. The pH value (pH 5) near the isoelectric point of HSA (equal potential point is 5.3) exhibits a more sensitive peak shift. Therefore, pH sensing can be utilized to achieve sensing sensitivity, and selective effects can be achieved by the isopotential characteristics that various biomolecules may have.
(二)以Lysozyme感測為例，在不同pH值的CHC-AuNR奈米核殼結構複合溶液下測試其紫外光-可見光(UV-Vis)特性，如表二所示，可以很清楚的觀察到在Lysozyme的等電位點(等電位點為10.1)附近的pH值(pH 9)呈現較靈敏的峰值位移(peak shift)。因此，可以利用pH值的操控來達到感測靈敏度，並藉由各種生物分子可能具有的等電位點特性來達到選擇性的效果。(2) Taking the sensitivity of Lysozyme as an example, the UV-Vis characteristics of the CHC-AuNR nano-core-shell composite solution at different pH values were tested, as shown in Table 2, which can be clearly observed. The pH peak (pH 9) near the isopotential point of Lysozyme (the isoelectric point is 10.1) exhibits a more sensitive peak shift. Therefore, pH sensing can be utilized to achieve sensing sensitivity, and selective effects can be achieved by the isopotential characteristics that various biomolecules may have.
(三)另方面而言，一般的金奈米粒子，為了維持其分散性， 一般都需被覆一層界面活性劑，但表面被覆將影響金奈米粒子的表面電漿共振(SPR)，因此其靈敏度將會受到影響。本發明實施例所使用的CHC-AuNR奈米核殼結構複合溶液，乃將金奈米粒子的表面被覆以酒精清洗後，以CHC水溶液為載體溶液，提供金奈米粒子予以吸附並避免金奈米粒子的聚集，在HSA感測的特性上，利用傳統的UV-Vis光譜儀測試，其可偵測下限(low detection limit)上有很大的提升，如表三所示。相較被覆CTAB的金奈米粒子，即使在HSA濃度達100μg/c.c.，UV-Vis光譜的峰值位移仍然無規律性，但在CHC-AuNR奈米核殼結構複合溶液中，HSA濃度在大於10ng/c.c.峰值位移即呈現穩定增加的特性，即其可偵測下限有顯著降低，顯示其感測靈敏度(sensitivity)大幅被提升。(3) On the other hand, in order to maintain the dispersibility of the general gold nanoparticles, Generally, a layer of surfactant is required, but the surface coating will affect the surface plasma resonance (SPR) of the gold nanoparticles, so the sensitivity will be affected. The CHC-AuNR nano-core shell structure composite solution used in the embodiment of the present invention is characterized in that the surface of the gold nanoparticle is coated with alcohol and then the CHC aqueous solution is used as a carrier solution to provide gold nanoparticles for adsorption and avoiding Chennai. The aggregation of the rice particles, in the characteristics of the HSA sensing, is improved by the conventional UV-Vis spectrometer, and the low detection limit is greatly improved, as shown in Table 3. Compared with the coated gold nanoparticles of CTAB, even if the HSA concentration reaches 100μg/cc, the peak displacement of UV-Vis spectrum is still irregular, but in the CHC-AuNR nano-core-shell composite solution, the HSA concentration is greater than 10ng. The /cc peak displacement exhibits a steadily increasing characteristic, that is, its detectable lower limit is significantly reduced, indicating that its sensing sensitivity is greatly improved.
綜上所述，根據本發明所提供之具高度選擇性的生物感測材料及方法，以幾丁聚醣高分子水溶液為載體溶液，取代在奈米金屬粒子上的界面活性劑，藉以維持奈米金屬粒子的分散性，並可以增進奈米金屬粒子原有的感測特性。同時，為了增強生物感測材料之靈敏度與選擇性，本發明利用操控環境pH值來控制待測物與感測元件的表面電位(zeta potential)。將pH值調整到接近但小於某特定生物分子之等電位點，使特定生物分子恰巧可突破幾丁聚醣核殼結構所產生的電子屏蔽，並因電荷與奈 米金屬粒子表面電荷相異而易吸附於奈米金屬粒子表面上，而非幾丁聚醣核殼結構表面。相對於其他待測物質在此特定的pH值下，則無法穿透屏蔽或是吸附到奈米金屬粒子表面上，因此不會造成奈米金屬粒子的紫外光-可見光(UV-vis)吸收訊號造成偏移。本發明可穩定奈米金屬粒子的分散性及提升奈米金屬粒子表面對於待測物之靈敏度，且同時賦予生物感測元件對於待測物的良好選擇性，而增強生物感測元件之感測能力。In summary, according to the highly selective biosensing material and method provided by the present invention, an aqueous solution of chitosan polymer is used as a carrier solution to replace the surfactant on the nano metal particles, thereby maintaining the naphthalene The dispersibility of the metal particles can enhance the original sensing characteristics of the nano metal particles. At the same time, in order to enhance the sensitivity and selectivity of the biosensing material, the present invention utilizes the operating environment pH to control the zeta potential of the analyte and the sensing element. Adjusting the pH to be close to but less than the isopotential point of a particular biomolecule, so that a specific biomolecule can happen to break through the electron shield generated by the chitosan core-shell structure, and The surface charge of the rice metal particles is different and easily adsorbed on the surface of the nano metal particles, not the surface of the chitosan core-shell structure. Compared with other substances to be tested at this specific pH value, it cannot penetrate the shield or adsorb on the surface of the nano metal particles, so it does not cause ultraviolet-visible (UV-vis) absorption signals of the nano metal particles. Causes an offset. The invention can stabilize the dispersibility of the nano metal particles and enhance the sensitivity of the surface of the nano metal particles to the object to be tested, and at the same time impart good selectivity to the biological sensing element for the object to be tested, and enhance the sensing of the biological sensing element. ability.
唯以上所述者，僅為本發明之較佳實施例而已，並非用來限定本發明實施之範圍。故即凡依本發明申請範圍所述之特徵及精神所為之均等變化或修飾，均應包括於本發明之申請專利範圍內。The above is only the preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Therefore, any changes or modifications of the features and spirits of the present invention should be included in the scope of the present invention.
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