CN103361398A - Method for detecting thrombin activity and screening thrombin inhibitor in plasma by using polypeptide microarray chip - Google Patents
Method for detecting thrombin activity and screening thrombin inhibitor in plasma by using polypeptide microarray chip Download PDFInfo
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Abstract
本发明公开了一种多肽微阵列芯片在血浆中检测凝血酶活性及筛选凝血酶抑制剂的方法，解决了现有分析化学中凝血酶的检测方法不适用于血液凝结环境中凝血酶的检测及凝血酶抑制剂的筛选的技术问题。本发明将多肽微阵列芯片与激活的血浆或者与含有凝血酶抑制剂的激活的血浆在36-38°C反应40min以上，然后与亲和素反应1h以上，再与多肽修饰的金纳米粒子反应1h以上，检测多肽微阵列芯片上金纳米粒子的共振光散射信号，实现凝血酶活性的检测及凝血酶抑制剂的筛选。本发明可直接在血浆中检测凝血酶活性，初步区分普通血浆、高凝血浆和低凝血浆；并且在血液凝结环境下，筛选凝血酶抑制剂，为凝血酶抑制剂类药物的开发和应用提供基础。
The invention discloses a method for detecting thrombin activity and screening thrombin inhibitors in blood plasma by a polypeptide microarray chip, which solves the problem that the existing thrombin detection method in analytical chemistry is not suitable for the detection of thrombin in the blood coagulation environment. Technical issues in the screening of thrombin inhibitors. In the present invention, the polypeptide microarray chip is reacted with activated plasma or activated plasma containing a thrombin inhibitor at 36-38°C for more than 40 minutes, then reacted with avidin for more than 1 hour, and then reacted with polypeptide-modified gold nanoparticles For more than 1 hour, the resonance light scattering signal of gold nanoparticles on the polypeptide microarray chip is detected to realize the detection of thrombin activity and the screening of thrombin inhibitors. The present invention can directly detect the thrombin activity in the blood plasma, preliminarily distinguish normal plasma, hypercoagulable plasma and hypocoagulable plasma; and screen thrombin inhibitors under the blood coagulation environment, which provides a basis for the development and application of thrombin inhibitor drugs. Base.
Description
技术领域technical field
本发明涉及一种多肽微阵列芯片在血浆中检测凝血酶活性及筛选凝血酶抑制剂的方法，属于多肽微阵列芯片技术领域。The invention relates to a polypeptide microarray chip for detecting thrombin activity in blood plasma and a method for screening thrombin inhibitors, belonging to the technical field of polypeptide microarray chips.
背景技术Background technique
凝血酶是一种丝氨酸蛋白酶，在凝血途径中它一方面将纤维蛋白原转化为纤维蛋白，促进血小板聚集，加速血液凝固，另一方面又激活抗凝机制，防止血栓形成。凝血酶的形成是整个凝血途径中各因子相互作用的结果，低水平的凝血酶活性与出血有关，高水平的凝血酶活性与血栓前状态相关(ThrombinFunctions During Tissue Factor-Induced Blood CoaguLation,Blood,2002,100,148-152；ABriefHistoricalReviewoftheWaterfall/CascadeofBloodCoaguLation,J.Biol.Chem.,2003,278,50819-50832)。Thrombin is a serine protease. In the blood coagulation pathway, on the one hand, it converts fibrinogen into fibrin, promotes platelet aggregation, and accelerates blood coagulation. On the other hand, it activates the anticoagulant mechanism to prevent thrombus formation. The formation of thrombin is the result of the interaction of various factors in the whole coagulation pathway. Low-level thrombin activity is related to bleeding, and high-level thrombin activity is related to the prothrombotic state (ThrombinFunctions During Tissue Factor-Induced Blood CoaguLation, Blood, 2002 , 100, 148-152; A Brief Historical Review of the Waterfall/Cascade of Blood CoaguLation, J. Biol. Chem., 2003, 278, 50819-50832).
血液凝结是一个受多种因子调控的复杂网络过程，某一因子浓度的改变都会改变体系中其他因子的浓度。凝血酶活性检测能更准确地反映凝血途径中各因子的组合效应(In Vivo Imaging of Thrombin Activity in Experimental Thrombiwith Thrombin-Sensitive Near-Infrared MolecuLar Probe,Arterioscler.Thromb.Vasc.Biol.,2002,22,1929-1935)。现有分析化学技术中检测血浆中的凝血酶多采用加标实验，引入的凝血酶会改变体系中其他因子的浓度，从而导致凝血酶本身的浓度也发生改变，所以分析化学技术中凝血酶的检测不适用于血液凝结环境。Blood coagulation is a complex network process regulated by multiple factors, and changes in the concentration of a certain factor will change the concentration of other factors in the system. Thrombin activity detection can more accurately reflect the combined effect of each factor in the coagulation pathway (In Vivo Imaging of Thrombin Activity in Experimental Thrombiwith Thrombin-Sensitive Near-Infrared MolecuLar Probe, Arterioscler.Thromb.Vasc.Biol.,2002,22,1929 -1935). In existing analytical chemistry techniques, the detection of thrombin in plasma mostly uses standard addition experiments. The introduced thrombin will change the concentration of other factors in the system, resulting in changes in the concentration of thrombin itself. Therefore, the thrombin in analytical chemistry techniques The test is not suitable for blood clotting environment.
多肽微阵列芯片技术是一种广泛用于研究抗原表位分析，蛋白与底物的相互作用，细胞内含物与多肽的相互作用，以及小分子物质与蛋白的相互作用的高通量分析方法(Microarray-Based Detection of Protein Binding and Functionalityby Gold Nanoparticle Probes，Anal.Chem.,2005,77,5770-5774)。但现有技术中，还没有利用多肽微阵列芯片在血浆中检测凝血酶活性和筛选凝血酶抑制剂的方法。Peptide microarray chip technology is a high-throughput analysis method widely used in the study of antigen epitope analysis, protein-substrate interaction, cell content and polypeptide interaction, and small molecule substance-protein interaction (Microarray-Based Detection of Protein Binding and Functionality by Gold Nanoparticle Probes, Anal. Chem., 2005, 77, 5770-5774). However, in the prior art, there is no method for detecting thrombin activity and screening thrombin inhibitors in plasma by using a polypeptide microarray chip.
发明内容Contents of the invention
为解决现有分析化学中检测凝血酶的方法不适用于血液凝结环境中凝血酶的检测及凝血酶抑制剂的筛选的技术问题，本发明提供一种多肽微阵列芯片在血浆中检测凝血酶活性及筛选凝血酶抑制剂的方法。In order to solve the technical problem that the existing method for detecting thrombin in analytical chemistry is not suitable for the detection of thrombin in the blood coagulation environment and the screening of thrombin inhibitors, the present invention provides a polypeptide microarray chip for detecting thrombin activity in plasma And a method for screening thrombin inhibitors.
本发明的多肽微阵列芯片在血浆中检测凝血酶活性的方法，包括以下步骤：The polypeptide microarray chip of the present invention detects the method for thrombin activity in blood plasma, comprises the following steps:
(1)将多肽微阵列芯片与激活的血浆在36-38°C反应40min以上，得到反应后的多肽微阵列芯片；(1) reacting the polypeptide microarray chip with the activated plasma at 36-38° C. for more than 40 minutes to obtain the reacted polypeptide microarray chip;
所述多肽微阵列芯片以生物素修饰的多肽为底物，所述多肽的氨基酸序列为Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys，Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys，Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys，Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys，Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys，Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys，Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys或者Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys，其中，生物素均修饰在末端赖氨酸上，D-Phe为D型苯丙氨酸，其它氨基酸均为L型氨基酸；The polypeptide microarray chip uses a biotin-modified polypeptide as a substrate, and the amino acid sequence of the polypeptide is Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys , Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg -Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg -Ser-Phe-Lys-Val-Val-Lys, Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys, Cys-Ala-Glu-Gly-Gly -D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys or Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala -Leu-Phe-Asp-Lys, wherein biotin is modified on the terminal lysine, D-Phe is D-type phenylalanine, and other amino acids are L-type amino acids;
(2)将反应后的多肽微阵列芯片与亲和素(avidin)反应1h以上，再与多肽修饰的金纳米粒子反应1h以上，得到金纳米粒子标记的多肽微阵列芯片；(2) reacting the reacted polypeptide microarray chip with avidin (avidin) for more than 1 hour, and then reacting with polypeptide-modified gold nanoparticles for more than 1 hour to obtain a gold nanoparticle-labeled polypeptide microarray chip;
所述多肽修饰的金纳米粒子为Cys-Ala-Leu-Asn-Asn与Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly修饰的金纳米粒子，其中，Lys上修饰有生物素；The polypeptide-modified gold nanoparticles are Cys-Ala-Leu-Asn-Asn and Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly modified gold nanoparticles, wherein Lys is modified with biotin;
(3)检测金纳米粒子标记的多肽微阵列芯片上金纳米粒子的共振光散射信号，实现对凝血酶活性的检测。(3) detecting the resonant light scattering signal of the gold nanoparticle on the peptide microarray chip labeled with the gold nanoparticle, so as to realize the detection of thrombin activity.
优选的是，所述多肽微阵列芯片的多肽点阵浓度为0.1-2mg/mL。Preferably, the peptide array concentration of the polypeptide microarray chip is 0.1-2 mg/mL.
优选的是，将多肽微阵列芯片与激活的血浆在37°C反应40min。Preferably, react the polypeptide microarray chip with the activated plasma at 37°C for 40min.
优选的是，所述亲和素的浓度为3μM以上。Preferably, the concentration of the avidin is above 3 μM.
优选的是，所述多肽修饰的金纳米粒子的浓度为3nM以上。Preferably, the concentration of the polypeptide-modified gold nanoparticles is above 3nM.
优选的是，所述金纳米粒子的平均粒径为25-30nm。Preferably, the average particle diameter of the gold nanoparticles is 25-30nm.
优选的是，所述激活的血浆的体积为30μL。Preferably, the volume of the activated plasma is 30 μL.
优选的是，所述激活的血浆是采用以下方法制备的：向血浆中加入稀释缓冲液、重组人组织因子、磷脂混合物和氯化钙。Preferably, the activated plasma is prepared by the following method: adding dilution buffer, recombinant human tissue factor, phospholipid mixture and calcium chloride to the plasma.
本发明还提供多肽微阵列芯片在血浆中筛选凝血酶抑制剂的方法，包括以下步骤：The present invention also provides a method for screening thrombin inhibitors in blood plasma by a polypeptide microarray chip, comprising the following steps:
(1)将多肽微阵列芯片与加入凝血酶抑制剂的激活的血浆在36-38°C反应40min以上，得到反应后的多肽微阵列芯片；(1) reacting the polypeptide microarray chip with the activated plasma added with a thrombin inhibitor at 36-38° C. for more than 40 minutes to obtain the reacted polypeptide microarray chip;
所述多肽微阵列芯片以生物素修饰的多肽为底物，所述多肽的氨基酸序列为Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys，Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys，Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys，Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys，Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys，Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys，Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys或者Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys，其中，生物素均修饰在末端赖氨酸上，D-Phe为D型苯丙氨酸，其它氨基酸均为L型氨基酸；The polypeptide microarray chip uses a biotin-modified polypeptide as a substrate, and the amino acid sequence of the polypeptide is Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys , Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg -Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg -Ser-Phe-Lys-Val-Val-Lys, Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys, Cys-Ala-Glu-Gly-Gly -D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys or Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala -Leu-Phe-Asp-Lys, wherein biotin is modified on the terminal lysine, D-Phe is D-type phenylalanine, and other amino acids are L-type amino acids;
(2)将反应后的多肽微阵列芯片与亲和素反应1h以上，再与多肽修饰的金纳米粒子反应1h以上，得到金纳米粒子标记的多肽微阵列芯片；(2) Reacting the reacted polypeptide microarray chip with avidin for more than 1 hour, and then reacting with polypeptide-modified gold nanoparticles for more than 1 hour to obtain a gold nanoparticle-labeled polypeptide microarray chip;
所述多肽修饰的金纳米粒子为Cys-Ala-Leu-Asn-Asn与Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly修饰的金纳米粒子，其中，Lys上修饰有生物素；The polypeptide-modified gold nanoparticles are Cys-Ala-Leu-Asn-Asn and Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly modified gold nanoparticles, wherein Lys is modified with biotin;
(3)检测金纳米粒子标记的多肽微阵列芯片上金纳米粒子的共振光散射信号，实现对凝血酶抑制剂的筛选。(3) detecting the resonant light scattering signal of the gold nanoparticle on the polypeptide microarray chip labeled with the gold nanoparticle, so as to realize the screening of thrombin inhibitors.
优选的，所述的凝血酶抑制剂为阿加曲班、抗凝血酶III或者4-(2-氨乙基)苯磺酰氟盐酸盐(AEBSF)。Preferably, the thrombin inhibitor is argatroban, antithrombin III or 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF).
本发明的有益效果是：The beneficial effects of the present invention are:
(1)本发明以生物素修饰的多肽为底物制备多肽微阵列芯片，在血浆中检测凝血酶活性及筛选凝血酶抑制剂，首先激活的血浆中活化的凝血酶作用于芯片上多肽底物的凝血酶作用位点，使带生物素的多肽片段水解下来，芯片上存留不带生物素的多肽片段，然后利用亲和素-生物素反应，先采用亲和素标记多肽微阵列芯片，再以Cys-Ala-Leu-Asn-Asn和Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly修饰的金纳米粒子标记芯片，检测金纳米粒子标记的芯片上金纳米粒子的共振光散射信号(Resonance Light Scattering，RLS)，芯片与凝血酶作用后，部分带生物素的多肽片段从芯片上解离，多肽点阵的共振光散射信号随之减弱，通过检测共振光散射信号，实现对凝血酶活性的检测及凝血酶抑制剂的筛选；(1) The present invention uses biotin-modified polypeptides as substrates to prepare polypeptide microarray chips, detects thrombin activity and screens thrombin inhibitors in plasma, and first activates activated thrombin in plasma to act on polypeptide substrates on the chip The site of thrombin to hydrolyze the peptide fragments with biotin, the peptide fragments without biotin remain on the chip, and then use avidin-biotin reaction, first use avidin to label the peptide microarray chip, and then Gold nanoparticles labeled chip with Cys-Ala-Leu-Asn-Asn and Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly, detection of resonance light scattering signal of gold nanoparticles on the chip labeled with gold nanoparticles (Resonance Light Scattering, RLS), after the chip interacts with thrombin, some peptide fragments with biotin are dissociated from the chip, and the resonance light scattering signal of the polypeptide lattice is weakened accordingly. By detecting the resonance light scattering signal, the blood coagulation Detection of enzyme activity and screening of thrombin inhibitors;
(2)本发明的方法可用于血浆中凝血酶活性的直接检测，并采用从血液凝结级联反应的上游激活凝血途径，初步区分普通血浆、高凝血浆和低凝血浆；(2) The method of the present invention can be used for direct detection of thrombin activity in plasma, and adopts the upstream activation of coagulation pathway from the blood coagulation cascade reaction to initially distinguish normal plasma, hypercoagulable plasma and hypocoagulable plasma;
(3)本发明能够在血浆中筛选凝血酶的抑制剂，可以为抑制剂的筛选提供一个接近真实的血浆环境，为预测实际应用在人体环境中其他因子对抑制剂的影响做出初步判断，更准确地判断抑制剂的抑制效果，实现高通量的、微量的、快速的、灵敏的凝血酶抑制剂的筛选，为凝血酶抑制剂类药物的研究与应用做基础。(3) The present invention can screen the inhibitor of thrombin in blood plasma, can provide a close to real plasma environment for the screening of inhibitor, make preliminary judgment for predicting the influence of other factors on inhibitor in the human body environment of actual application, More accurately judge the inhibitory effect, realize high-throughput, trace, rapid and sensitive screening of thrombin inhibitors, and lay the foundation for the research and application of thrombin inhibitors.
附图说明Description of drawings
图1为本发明不同浓度多肽点阵的多肽微阵列芯片的点阵光学照片；Fig. 1 is the lattice optical photograph of the polypeptide microarray chip of different concentration polypeptide lattices of the present invention;
图2为本发明实施例1不同多肽底物的多肽微阵列芯片与凝血酶作用后的共振光散射信号强度变化；Fig. 2 is the variation of resonance light scattering signal intensity after the interaction between the polypeptide microarray chip and thrombin of different polypeptide substrates in Example 1 of the present invention;
图3为本发明实施例2和实施例3的多肽微阵列芯片的多肽点阵浓度与凝血酶催化反应速度的双倒数曲线；Fig. 3 is the double reciprocal curve of the polypeptide lattice concentration and thrombin catalytic reaction speed of the polypeptide microarray chip of the
图4为本发明实施例4的多肽微阵列芯片检测凝血酶的标准曲线图；Fig. 4 is the standard curve diagram of detecting thrombin by the polypeptide microarray chip of
图5为本发明实施例5的多肽微阵列芯片与激活的普通血浆、高凝血浆和低凝血浆作用后的共振光散射信号强度变化；Fig. 5 is the change of resonance light scattering signal intensity after the polypeptide microarray chip of Example 5 of the present invention interacts with activated normal plasma, hypercoagulable plasma and hypocoagulable plasma;
图6为本发明实施例6的多肽微阵列芯片与加入凝血酶抑制剂的激活的血浆作用后的共振光散射信号；Fig. 6 is the resonance light scattering signal after the polypeptide microarray chip of Example 6 of the present invention interacts with activated plasma added with a thrombin inhibitor;
图7为本发明对比例1的流程图；Fig. 7 is the flowchart of comparative example 1 of the present invention;
图8为本发明对比例2的流程图；Fig. 8 is the flowchart of comparative example 2 of the present invention;
图9为本发明对比例3的流程图；Fig. 9 is the flowchart of comparative example 3 of the present invention;
图10为本发明实施例1-4多肽微阵列芯片检测凝血酶的流程图；Fig. 10 is a flowchart of detection of thrombin by a polypeptide microarray chip in Example 1-4 of the present invention;
图11为本发明实施例5多肽微阵列芯片检测血浆样品的流程图；Fig. 11 is a flowchart of the detection of plasma samples by the polypeptide microarray chip in Example 5 of the present invention;
图12为本发明实施例6多肽微阵列芯片在血浆中筛选凝血酶抑制剂的流程图。Fig. 12 is a flow chart of the screening of thrombin inhibitors in plasma by polypeptide microarray chip according to Example 6 of the present invention.
图中，
具体实施方式Detailed ways
多肽微阵列芯片在血浆中检测凝血酶活性的方法，包括以下步骤：A method for detecting thrombin activity in blood plasma with a polypeptide microarray chip, comprising the following steps:
(1)将多肽微阵列芯片与激活的血浆在36-38°C反应40min以上，激活的血浆中活化的凝血酶作用于芯片上多肽底物的凝血酶作用位点，使带生物素的多肽片段水解下来，芯片上存留不带生物素的多肽片段，得到反应后的多肽微阵列芯片；(1) React the polypeptide microarray chip with the activated plasma at 36-38°C for more than 40 minutes, and the activated thrombin in the activated plasma acts on the thrombin action site of the polypeptide substrate on the chip, so that the biotin-containing polypeptide After the fragments are hydrolyzed, the peptide fragments without biotin remain on the chip, and the reacted peptide microarray chip is obtained;
所述多肽微阵列芯片以生物素修饰的多肽为底物，所述多肽的氨基酸序列为Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys，Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys，Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys，Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys，Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys，Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys，Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys或者Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys，其中，生物素均修饰在末端赖氨酸上，D-Phe为D型苯丙氨酸，其它氨基酸均为L型氨基酸；The polypeptide microarray chip uses a biotin-modified polypeptide as a substrate, and the amino acid sequence of the polypeptide is Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys , Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg -Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg -Ser-Phe-Lys-Val-Val-Lys, Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys, Cys-Ala-Glu-Gly-Gly -D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys or Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala -Leu-Phe-Asp-Lys, wherein biotin is modified on the terminal lysine, D-Phe is D-type phenylalanine, and other amino acids are L-type amino acids;
(2)将反应后的多肽微阵列芯片与亲和素反应1h以上，再与多肽修饰的金纳米粒子反应1h以上，得到金纳米粒子标记的多肽微阵列芯片；(2) Reacting the reacted polypeptide microarray chip with avidin for more than 1 hour, and then reacting with polypeptide-modified gold nanoparticles for more than 1 hour to obtain a gold nanoparticle-labeled polypeptide microarray chip;
所述多肽修饰的金纳米粒子为Cys-Ala-Leu-Asn-Asn与Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly修饰的金纳米粒子，其中，Lys上修饰有生物素；The polypeptide-modified gold nanoparticles are Cys-Ala-Leu-Asn-Asn and Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly modified gold nanoparticles, wherein Lys is modified with biotin;
(3)检测金纳米粒子标记的多肽微阵列芯片上金纳米粒子的共振光散射信号，步骤(1)的多肽微阵列芯片与凝血酶作用后，部分带生物素的多肽片段从芯片上解离，点阵的共振光散射信号随之减弱，通过共振光散射信号的测定实现对凝血酶活性的检测。(3) Detect the resonance light scattering signal of the gold nanoparticles on the peptide microarray chip labeled with gold nanoparticles. After the peptide microarray chip in step (1) interacts with thrombin, part of the peptide fragments with biotin are dissociated from the chip. , the resonance light scattering signal of the lattice weakens accordingly, and the detection of thrombin activity is realized through the measurement of the resonance light scattering signal.
本发明中，以Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)为多肽底物的多肽微阵列芯片和凝血酶的亲和性更高，其检测的灵敏性更好，所以，本发明优选Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)为多肽底物的多肽微阵列芯片在血浆中检测凝血酶的活性。In the present invention, take Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin) as polypeptide microarray chip of polypeptide substrate and thrombin Affinity is higher, and the sensitivity of its detection is better, so, the present invention preferably Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin ) is a polypeptide microarray chip which is a polypeptide substrate to detect the activity of thrombin in blood plasma.
本发明中，在知道多肽的氨基酸序列的情况下，多肽底物的获得为本领域人员公知技术，本实施方式的多肽底物由上海强耀生物科技有限公司合成。In the present invention, when the amino acid sequence of the polypeptide is known, the acquisition of the polypeptide substrate is a well-known technology for those skilled in the art. The polypeptide substrate of this embodiment is synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.
本发明中，所使用的芯片没有特殊要求，商购即可，本实施方式采用北京博奥生物有限公司的
本发明多肽微阵列芯片的制备方法为本领域人员公知技术，如接触法，本实施方式提供一种其制备过程，但本发明不限于此，具体步骤为：The preparation method of the polypeptide microarray chip of the present invention is a well-known technology for those skilled in the art, such as the contact method. This embodiment provides a preparation process thereof, but the present invention is not limited thereto. The specific steps are:
(1)使用北京博奥生物有限公司生产的
(2)将上述芯片置于真空干燥箱中抽真空，30°C过夜反应，得到反应后的芯片；(2) Place the above-mentioned chip in a vacuum drying oven to evacuate, and react overnight at 30° C. to obtain the reacted chip;
(3)用1%(w/v)牛血清白蛋白封闭反应后的芯片，制成多肽微阵列芯片。(3) Block the reacted chip with 1% (w/v) bovine serum albumin to make a polypeptide microarray chip.
本发明中，多肽微阵列芯片的多肽点阵浓度一般采用0.1-2mg/mL，但不限于此，多肽微阵列芯片中多肽点阵的浓度越大，共振光散射信号越强，如图1所示，采用Arrayit微阵列扫描仪测定的多肽底物为Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)的多肽微阵列芯片的点阵光学照片，从左到右多肽点阵浓度依次为0.2、0.3、0.35、0.5、1、1.5mg/mL。In the present invention, the polypeptide lattice concentration of the polypeptide microarray chip is generally 0.1-2mg/mL, but not limited thereto, the greater the concentration of the polypeptide lattice in the polypeptide microarray chip, the stronger the resonance light scattering signal, as shown in Figure 1 shows that the polypeptide substrate determined by the Arrayit microarray scanner is a polypeptide microarray of Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin) The dot matrix optical photo of the chip, from left to right, the peptide dot matrix concentrations are 0.2, 0.3, 0.35, 0.5, 1, 1.5mg/mL.
本发明中，亲和素的浓度为3μM。In the present invention, the concentration of avidin is 3 μM.
本发明中，多肽修饰的金纳米粒子的浓度为3nM，平均粒径为25-30nm，优选30nm(紫外最大吸收峰为528nm)。In the present invention, the concentration of the polypeptide-modified gold nanoparticles is 3nM, and the average particle size is 25-30nm, preferably 30nm (the maximum ultraviolet absorption peak is 528nm).
本发明中，激活的血浆的体积由反应容器的大小而定，本实施方式中，与多肽微阵列芯片作用的激活的血浆的体积为30μL，可以采用以下方法制备：向血浆(40μL)中加入稀释缓冲液(40μL)、重组人组织因子、磷脂混合物和氯化钙，得到激活的血浆，激活的血浆中重组人组织因子的浓度为5pM，磷脂混合物的浓度为4μM和氯化钙的浓度为17mM，所述稀释缓冲液为含有60mg/mL牛血清白蛋白和21.7mM柠檬酸三钠的4-羟乙基哌嗪乙磺酸(HEPES，20mM)溶液，所述磷脂混合物为60mol%的二油酰磷酸胆碱、20mol%的二油酰磷酸乙醇胺和20mol%的二油酰磷酸丝氨酸。In the present invention, the volume of activated plasma depends on the size of the reaction container. In this embodiment, the volume of activated plasma interacting with the polypeptide microarray chip is 30 μL, which can be prepared by the following method: add Dilution buffer (40 μL), recombinant human tissue factor, phospholipid mixture and calcium chloride to obtain activated plasma, the concentration of recombinant human tissue factor in the activated plasma was 5 pM, the concentration of phospholipid mixture was 4 μM and the concentration of calcium chloride was 17mM, the dilution buffer is 4-hydroxyethylpiperazineethanesulfonic acid (HEPES, 20mM) solution containing 60mg/mL bovine serum albumin and 21.7mM trisodium citrate, and the phospholipid mixture is 60mol% di Oleoylphosphorylcholine, 20mol% dioleoylphosphoethanolamine and 20mol% dioleoylphosphoserine.
利用本发明的多肽微阵列芯片在血浆中检测凝血酶时，凝血酶的线性检测范围为2.7×10-2-810nM，检测限为19.7pM。When the polypeptide microarray chip of the present invention is used to detect thrombin in plasma, the linear detection range of thrombin is 2.7×10 -2 -810nM, and the detection limit is 19.7pM.
本发明的多肽微阵列芯片在血浆中检测凝血酶活性的方法能够对普通血浆、高凝血浆和低凝血浆进行初步区分。The method for detecting thrombin activity in plasma by the polypeptide microarray chip of the present invention can initially distinguish normal plasma, hypercoagulable plasma and hypocoagulable plasma.
多肽微阵列芯片在血浆中筛选凝血酶抑制剂的方法，包括以下步骤：A method for screening thrombin inhibitors in blood plasma with a polypeptide microarray chip, comprising the following steps:
(1)将多肽微阵列芯片与加入凝血酶抑制剂的激活的血浆在36-38°C反应40min以上，得到反应后的多肽微阵列芯片；(1) reacting the polypeptide microarray chip with the activated plasma added with a thrombin inhibitor at 36-38° C. for more than 40 minutes to obtain the reacted polypeptide microarray chip;
所述多肽微阵列芯片以生物素修饰的多肽为底物，所述多肽的氨基酸序列为Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys，Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys，Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys，Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys，Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys，Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys，Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys或者Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys，其中，生物素均修饰在末端赖氨酸上，D-Phe为D型苯丙氨酸，其它氨基酸均为L型氨基酸；The polypeptide microarray chip uses a biotin-modified polypeptide as a substrate, and the amino acid sequence of the polypeptide is Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys , Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg -Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys, Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg -Ser-Phe-Lys-Val-Val-Lys, Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys, Cys-Ala-Glu-Gly-Gly -D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys or Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala -Leu-Phe-Asp-Lys, wherein biotin is modified on the terminal lysine, D-Phe is D-type phenylalanine, and other amino acids are L-type amino acids;
(2)将反应后的多肽微阵列芯片与亲和素反应1h以上，再与多肽修饰的金纳米粒子反应1h以上，得到金纳米粒子标记的多肽微阵列芯片；(2) Reacting the reacted polypeptide microarray chip with avidin for more than 1 hour, and then reacting with polypeptide-modified gold nanoparticles for more than 1 hour to obtain a gold nanoparticle-labeled polypeptide microarray chip;
所述多肽修饰的金纳米粒子为Cys-Ala-Leu-Asn-Asn与Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly修饰的金纳米粒子，其中，Lys上修饰有生物素；The polypeptide-modified gold nanoparticles are Cys-Ala-Leu-Asn-Asn and Cys-Ala-Leu-Asn-Asn-Gly-Lys-Gly modified gold nanoparticles, wherein Lys is modified with biotin;
(3)检测金纳米粒子标记的多肽微阵列芯片上金纳米粒子的共振光散射信号，实现对凝血酶抑制剂的筛选。(3) detecting the resonant light scattering signal of the gold nanoparticle on the polypeptide microarray chip labeled with the gold nanoparticle, so as to realize the screening of thrombin inhibitors.
本发明中，凝血酶抑制剂没有特殊限定，一般为阿加曲班、抗凝血酶III或者AEBSF，凝血酶抑制剂在血浆中的浓度为7.5μM。In the present invention, the thrombin inhibitor is not particularly limited, generally it is argatroban, antithrombin III or AEBSF, and the concentration of the thrombin inhibitor in plasma is 7.5 μM.
本发明中，以Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)为多肽底物的多肽微阵列芯片和凝血酶的亲和性更高，其检测的灵敏性更好，所以，本发明优选Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)为多肽底物的多肽微阵列芯片在血浆中筛选凝血酶抑制剂。In the present invention, take Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin) as polypeptide microarray chip of polypeptide substrate and thrombin Affinity is higher, and the sensitivity of its detection is better, so, the present invention preferably Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin ) is a polypeptide microarray chip for screening thrombin inhibitors in plasma.
本发明中，在知道多肽的氨基酸序列的情况下，多肽底物的获得为本领域人员公知技术，本实施方式的多肽底物由上海强耀生物科技有限公司合成。In the present invention, when the amino acid sequence of the polypeptide is known, the acquisition of the polypeptide substrate is a well-known technology for those skilled in the art. The polypeptide substrate of this embodiment is synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.
本发明中，所使用的芯片没有特殊要求，商购即可，本实施方式采用北京博奥生物有限公司的
本发明多肽微阵列芯片的制备方法为本领域人员公知技术，如接触法，本实施方式提供一种其制备过程，但本发明不限于此，具体步骤为：The preparation method of the polypeptide microarray chip of the present invention is a well-known technology for those skilled in the art, such as the contact method. This embodiment provides a preparation process thereof, but the present invention is not limited thereto. The specific steps are:
(1)使用北京博奥生物有限公司生产的
(2)将上述芯片置于真空干燥箱中抽真空，30°C过夜反应，得到反应后的芯片；(2) Place the above-mentioned chip in a vacuum drying oven to evacuate, and react overnight at 30° C. to obtain the reacted chip;
(3)用1%(w/v)牛血清白蛋白封闭反应后的芯片，制成多肽微阵列芯片。(3) Block the reacted chip with 1% (w/v) bovine serum albumin to make a polypeptide microarray chip.
本发明中，多肽微阵列芯片的多肽点阵浓度一般采用0.1-2mg/mL，但不限于此，多肽微阵列芯片中多肽点阵的浓度越大，共振光散射信号越强，如图1所示，采用Arrayit微阵列扫描仪测定的多肽底物为Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)的多肽微阵列芯片的点阵光学照片，从左到右多肽点阵浓度依次为0.2、0.3、0.35、0.5、1、1.5mg/mL。In the present invention, the polypeptide lattice concentration of the polypeptide microarray chip is generally 0.1-2mg/mL, but not limited thereto, the greater the concentration of the polypeptide lattice in the polypeptide microarray chip, the stronger the resonance light scattering signal, as shown in Figure 1 shows that the polypeptide substrate determined by the Arrayit microarray scanner is a polypeptide microarray of Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin) The dot matrix optical photo of the chip, from left to right, the peptide dot matrix concentrations are 0.2, 0.3, 0.35, 0.5, 1, 1.5mg/mL.
本发明中，亲和素的浓度为3μM。In the present invention, the concentration of avidin is 3 μM.
本发明中，多肽修饰的金纳米粒子的浓度为3nM，平均粒径为25-30nm，优选30nm(紫外最大吸收峰为528nm)。In the present invention, the concentration of the polypeptide-modified gold nanoparticles is 3nM, and the average particle size is 25-30nm, preferably 30nm (the maximum ultraviolet absorption peak is 528nm).
对比例1Comparative example 1
结合图7说明对比例1Comparative example 1 is illustrated in conjunction with Fig. 7
(1)将多肽点阵浓度为0.5mg/mL，多肽底物分别为：(1) The peptide array concentration is 0.5mg/mL, and the peptide substrates are:
Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys(biotin)；Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys (biotin);
Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys(biotin)；Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys (biotin);
Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)；Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin);
Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)；Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin);
Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys(biotin)；Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys (biotin);
Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys(biotin)；Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys (biotin);
Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)；Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin);
Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys(biotin)的多肽微阵列芯片，分别与不含凝血酶的缓冲液反应1h，得到反应后的多肽微阵列芯片，所述缓冲液的pH值为7.35，含有20mM4-羟乙基哌嗪乙磺酸，0.14mM氯化钠和2mM氯化钙；The polypeptide microarray chip of Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys (biotin), respectively with and without The buffer solution of thrombin was reacted for 1 hour to obtain the reacted polypeptide microarray chip, the pH value of the buffer solution was 7.35, containing 20mM 4-hydroxyethylpiperazineethanesulfonic acid, 0.14mM sodium chloride and 2mM calcium chloride;
(2)将反应后的的多肽微阵列芯片分别依次与3μM亲和素，3nM多肽修饰的金纳米粒子各反应1h，得到金纳米粒子标记的多肽微阵列芯片；(2) Reacting the reacted polypeptide microarray chip with 3 μM avidin and 3 nM polypeptide-modified gold nanoparticles for 1 hour respectively to obtain a gold nanoparticle-labeled polypeptide microarray chip;
(3)将金纳米粒子标记的多肽微阵列芯片用Arrayit微阵列扫描仪测定金纳米粒子共振光散射信号。(3) The gold nanoparticle-labeled polypeptide microarray chip was used to measure the gold nanoparticle resonant light scattering signal with an Arrayit microarray scanner.
结果表明，对比例1金纳米粒子标记的多肽微阵列芯片的共振光散射信号强度不变，图7为本发明对比例1的流程图。The results show that the resonance light scattering signal intensity of the polypeptide microarray chip labeled with gold nanoparticles in Comparative Example 1 remains unchanged, and FIG. 7 is a flow chart of Comparative Example 1 of the present invention.
对比例2Comparative example 2
结合图8说明对比例2Illustrate Comparative Example 2 in conjunction with Fig. 8
(1)将多肽点阵浓度为0.5mg/mL，多肽底物为Cys-Ala-Leu-Asn-Asn的多肽微阵列芯片，与27nM的凝血酶反应1h，得到反应后的多肽微阵列芯片；(1) React the peptide microarray chip with a peptide array concentration of 0.5 mg/mL and a peptide substrate of Cys-Ala-Leu-Asn-Asn with 27 nM thrombin for 1 hour to obtain a reacted peptide microarray chip;
(2)将反应后的的多肽微阵列芯片依次与3μM亲和素，3nM多肽修饰的金纳米粒子各反应1h，得到金纳米粒子标记的多肽微阵列芯片；(2) Reacting the reacted polypeptide microarray chip with 3 μM avidin and 3 nM polypeptide-modified gold nanoparticles for 1 hour respectively to obtain a gold nanoparticle-labeled polypeptide microarray chip;
(3)将金纳米粒子标记的多肽微阵列芯片用Arrayit微阵列扫描仪测定金纳米粒子共振光散射信号。(3) The gold nanoparticle-labeled polypeptide microarray chip was used to measure the gold nanoparticle resonant light scattering signal with an Arrayit microarray scanner.
结果表明，对比例2的金纳米粒子标记的多肽微阵列芯片不会产生共振光散射信号，图8为本发明对比例2的流程图。The results show that the peptide microarray chip labeled with gold nanoparticles in Comparative Example 2 does not generate resonance light scattering signals, and FIG. 8 is a flow chart of Comparative Example 2 of the present invention.
结合对比例1和对比例2说明本发明中共振光散射信号的产生、变化以及试验结果的产生，都是由凝血酶的作用造成的，排除其它因素产生信号变化及试验结果的可能。Combined with Comparative Example 1 and Comparative Example 2, it is illustrated that the generation, change and test results of the resonant light scattering signal in the present invention are all caused by the action of thrombin, and the possibility of signal changes and test results caused by other factors is excluded.
对比例3Comparative example 3
结合图9说明对比例3Comparative example 3 is illustrated in conjunction with Fig. 9
(1)将多肽点阵浓度为0.5mg/mL，多肽底物为Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)的多肽微阵列芯片，分别与与30μL未激活的普通血浆，30μL未激活的高凝血浆，30uL未激活的低凝血浆在37°C反应40min,得到反应后的多肽微阵列芯片；(1) The peptide matrix concentration is 0.5mg/mL, and the peptide substrate is Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin) The polypeptide microarray chip was reacted with 30 μL of unactivated normal plasma, 30 μL of unactivated hypercoagulable plasma, and 30 μL of unactivated hypocoagulable plasma at 37°C for 40 minutes to obtain the reacted polypeptide microarray chip;
所述普通血浆为健康人的新鲜冰冻血浆，所述高凝血浆为未经治疗的自发性深静脉血栓(Deep vein thrombosis，DVT)或肺栓塞(PuLmonary embolism，PE)病人的新鲜冰冻血浆，所述低凝血浆为加入7.5μM凝血酶抑制剂(抗凝血酶III)的健康人的新鲜冰冻血浆；The normal plasma is the fresh frozen plasma of healthy people, and the hypercoagulable plasma is the fresh frozen plasma of untreated patients with spontaneous deep vein thrombosis (Deep vein thrombosis, DVT) or pulmonary embolism (PuLmonary embolism, PE). The low coagulation plasma is the fresh frozen plasma of healthy people who added 7.5 μM thrombin inhibitor (antithrombin III);
(2)将反应后的的多肽微阵列芯片分别依次与3μM亲和素，3nM多肽修饰的金纳米粒子各反应1h，得到金纳米粒子标记的多肽微阵列芯片；(2) Reacting the reacted polypeptide microarray chip with 3 μM avidin and 3 nM polypeptide-modified gold nanoparticles for 1 hour respectively to obtain a gold nanoparticle-labeled polypeptide microarray chip;
(3)将金纳米粒子标记的多肽微阵列芯片用Arrayit微阵列扫描仪测定金纳米粒子共振光散射信号。(3) The gold nanoparticle-labeled polypeptide microarray chip was used to measure the gold nanoparticle resonant light scattering signal with an Arrayit microarray scanner.
图9为本发明对比例3的流程图。FIG. 9 is a flowchart of Comparative Example 3 of the present invention.
实施例1Example 1
结合图2和图10说明实施例1
多肽微阵列芯片检测凝血酶活性：Peptide microarray chip detection of thrombin activity:
(1)将8种多肽底物的多肽微阵列芯片，多肽点阵浓度为0.5mg/mL，8种多肽底物分别为：(1) The polypeptide microarray chip with 8 kinds of polypeptide substrates, the concentration of the polypeptide array is 0.5mg/mL, and the 8 kinds of polypeptide substrates are respectively:
Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys(biotin)；Cys-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Lys (biotin);
Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys(biotin),；Cys-Glu-Gly-Phe-Phe-Ser-Ala-Arg-Gly-His-Arg-Pro-Leu-Lys(biotin);
Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)；Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin);
Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)；Cys-Ala-Glu-Gly-Gly-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin);
Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys(biotin)；Cys-Ala-Glu-Gly-Gly-Val-Pro-Arg-Ser-Phe-Lys-Val-Val-Lys (biotin);
Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys(biotin)；Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys (biotin);
Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)；Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin);
Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys(biotin)的多肽微阵列芯片分别与27nM凝血酶反应1h，得到反应后的多肽微阵列芯片；The polypeptide microarray chip of Cys-Gly-Gly-Gly-Val-Arg-Pro-Gly-Arg-Val-Gly-Gly-Gly-Glu-Ala-Leu-Phe-Asp-Lys (biotin) was mixed with 27nM thrombin React for 1 hour to obtain the reacted polypeptide microarray chip;
(2)将反应后的多肽微阵列芯片分别依次与3μM亲和素，3nM多肽修饰的金纳米粒子各反应1h，得到金纳米粒子标记的多肽微阵列芯片；(2) respectively reacting the reacted polypeptide microarray chip with 3 μM avidin and 3 nM polypeptide-modified gold nanoparticles for 1 hour respectively to obtain a gold nanoparticle-labeled polypeptide microarray chip;
(3)将金纳米粒子标记的多肽微阵列芯片用Arrayit微阵列扫描仪测定金纳米粒子共振光散射信号。(3) The gold nanoparticle-labeled polypeptide microarray chip was used to measure the gold nanoparticle resonant light scattering signal with an Arrayit microarray scanner.
将实施例2得到的8种多肽点阵的金纳米粒子共振光散射信号分别与对比例1的同种多肽点阵的金纳米粒子共振光散射信号比较，共振光散射信号强度变化结果分别记作S01，S02，S03，S04，S05，S06，S07，S08，作出不同多肽底物的多肽微阵列芯片与凝血酶作用后的共振光散射信号强度变化，如图2所示，从图2可以看出，本发明的8种多肽微阵列芯片都能与凝血酶作用，并通过共振光散射信号强度的变化显示，其中，凝血酶对S07号多肽的作用效率最大，所以底物为Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)的芯片与凝血酶的亲和力更高。The gold nanoparticle resonance light scattering signals of the 8 polypeptide lattices obtained in Example 2 were compared with the gold nanoparticle resonance light scattering signals of the same polypeptide lattice in Comparative Example 1, and the resonance light scattering signal intensity changes were recorded as S01, S02, S03, S04, S05, S06, S07, S08, the changes in the resonance light scattering signal intensity after the polypeptide microarray chip with different polypeptide substrates interacted with thrombin, as shown in Figure 2, can be seen from Figure 2 It can be seen that 8 kinds of polypeptide microarray chips of the present invention can interact with thrombin, and it is shown by the change of resonance light scattering signal intensity, wherein, thrombin has the greatest efficiency of action on S07 polypeptide, so the substrate is Cys-Ala- Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys (biotin) chips have a higher affinity to thrombin.
实施例2Example 2
结合图3和图10说明实施例2
多肽微阵列芯片检测凝血酶活性：Peptide microarray chip detection of thrombin activity:
(1)将多肽点阵浓度分别为0.2、0.3、0.35、0.5、1、1.5mg/mL，多肽底物为Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)的多肽微阵列芯片分别与810nM凝血酶反应1h，得到反应后的多肽微阵列芯片；(1) The concentration of the peptide matrix is 0.2, 0.3, 0.35, 0.5, 1, 1.5 mg/mL, and the peptide substrate is Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe -Arg-Val-Val-Lys (biotin) polypeptide microarray chip was reacted with 810nM thrombin for 1h to obtain the reacted polypeptide microarray chip;
(2)将反应后的多肽微阵列芯片分别依次与3μM亲和素，3nM多肽修饰的金纳米粒子各反应1h，得到金纳米粒子标记的多肽微阵列芯片；(2) respectively reacting the reacted polypeptide microarray chip with 3 μM avidin and 3 nM polypeptide-modified gold nanoparticles for 1 hour respectively to obtain a gold nanoparticle-labeled polypeptide microarray chip;
(3)将金纳米粒子标记的多肽微阵列芯片用Arrayit微阵列扫描仪测定金纳米粒子共振光散射信号。(3) The gold nanoparticle-labeled polypeptide microarray chip was used to measure the gold nanoparticle resonant light scattering signal with an Arrayit microarray scanner.
实施例3Example 3
结合图3和图10说明实施例3Embodiment 3 is illustrated in conjunction with Fig. 3 and Fig. 10
多肽微阵列芯片检测凝血酶活性：Peptide microarray chip detection of thrombin activity:
(1)将多肽点阵浓度分别为0.1、0.15、0.2、0.3、1、2mg/mL，多肽底物为Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu-Val-Gly-Lys(biotin)的多肽微阵列芯片分别与810nM凝血酶反应1h，得到反应后的多肽微阵列芯片；(1) The concentration of the peptide matrix is 0.1, 0.15, 0.2, 0.3, 1, 2 mg/mL, and the peptide substrate is Cys-Gly-Gly-Gly-Ala-Arg-Pro-Arg-Ser-Leu-Leu- The polypeptide microarray chip of Val-Gly-Lys (biotin) was reacted with 810nM thrombin for 1 hour to obtain the reacted polypeptide microarray chip;
(2)将反应后的多肽微阵列芯片分别依次与3μM亲和素，3nM多肽修饰的金纳米粒子各反应1h，得到金纳米粒子标记的多肽微阵列芯片；(2) respectively reacting the reacted polypeptide microarray chip with 3 μM avidin and 3 nM polypeptide-modified gold nanoparticles for 1 hour respectively to obtain a gold nanoparticle-labeled polypeptide microarray chip;
(3)将金纳米粒子标记的多肽微阵列芯片用Arrayit微阵列扫描仪测定金纳米粒子共振光散射信号。(3) The gold nanoparticle-labeled polypeptide microarray chip was used to measure the gold nanoparticle resonant light scattering signal with an Arrayit microarray scanner.
图3a)为实施例2的多肽微阵列芯片的多肽点阵浓度与凝血酶催化反应速度的双倒数曲线，图3b)为实施例3以蛋白酶数据库(MEROPS)提供的多肽底物序列为底物的芯片的多肽点阵浓度与凝血酶催化反应速度的双倒数曲线，从图3可以看出，实施例2的多肽与实施例3的多肽与凝血酶的亲和力较好。Fig. 3 a) is the double reciprocal curve of the polypeptide matrix concentration and thrombin catalytic reaction speed of the polypeptide microarray chip of
实施例4Example 4
结合图4和图10说明实施例4
多肽微阵列芯片检测凝血酶活性：Peptide microarray chip detection of thrombin activity:
(1)将多肽点阵浓度为0.5mg/mL，多肽底物为Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)的多肽微阵列芯片分别与浓度为0.0135、0.027、0.135、1.35、13.5、135、405、810、1350nM的凝血酶反应1h，得到反应后的多肽微阵列芯片；(1) The peptide matrix concentration is 0.5mg/mL, and the peptide substrate is Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin) The polypeptide microarray chip was reacted with thrombin at a concentration of 0.0135, 0.027, 0.135, 1.35, 13.5, 135, 405, 810, and 1350 nM for 1 hour to obtain the reacted polypeptide microarray chip;
(2)将反应后的多肽微阵列芯片分别依次与3μM亲和素，3nM多肽修饰的金纳米粒子各反应1h，得到金纳米粒子标记的多肽微阵列芯片；(2) respectively reacting the reacted polypeptide microarray chip with 3 μM avidin and 3 nM polypeptide-modified gold nanoparticles for 1 hour respectively to obtain a gold nanoparticle-labeled polypeptide microarray chip;
(3)将金纳米粒子标记的多肽微阵列芯片用Arrayit微阵列扫描仪测定金纳米粒子共振光散射信号。(3) The gold nanoparticle-labeled polypeptide microarray chip was used to measure the gold nanoparticle resonant light scattering signal with an Arrayit microarray scanner.
图4为本发明实施例4多肽微阵列芯片检测凝血酶的标准曲线图；从图4可以看出，本发明凝血酶检测的线性范围为2.7×10-2-810nM，检测限为19.7pM。Fig. 4 is a standard curve diagram of thrombin detection by the polypeptide microarray chip in Example 4 of the present invention; it can be seen from Fig. 4 that the linear range of thrombin detection in the present invention is 2.7×10 -2 -810nM, and the detection limit is 19.7pM.
图10为本发明实施例1-4多肽微阵列芯片检测凝血酶的流程图。Fig. 10 is a flowchart of the detection of thrombin by the polypeptide microarray chip in Example 1-4 of the present invention.
实施例5Example 5
结合图5和图11说明实施例5Embodiment 5 is illustrated in conjunction with Fig. 5 and Fig. 11
多肽微阵列芯片在血浆中检测凝血酶活性：Peptide microarray chip detects thrombin activity in plasma:
(1)将多肽点阵浓度为0.5mg/mL，多肽底物为Cys-Ala-Glu-Gly-Gly-D-Phe-ProArg-Ser-Phe-Arg-Val-Val-Lys(biotin)的多肽微阵列芯片分别与13组30uL激活的普通血浆、4组30uL激活的高凝血浆和7组30uL激活的低凝血浆在37°C反应40min，得到反应后的多肽微阵列芯片；(1) The concentration of the peptide matrix is 0.5mg/mL, and the peptide substrate is the peptide of Cys-Ala-Glu-Gly-Gly-D-Phe-ProArg-Ser-Phe-Arg-Val-Val-Lys (biotin) The microarray chip was reacted with 13 groups of 30uL activated normal plasma, 4 groups of 30uL activated hypercoagulable plasma and 7 groups of 30uL activated hypocoagulable plasma at 37°C for 40min to obtain the reacted polypeptide microarray chip;
所述普通血浆为健康人的新鲜冰冻血浆，所述高凝血浆为未经治疗的自发性深静脉血栓(Deep vein thrombosis，DVT)或肺栓塞(PuLmonary embolism，PE)病人的新鲜冰冻血浆，所述低凝血浆为加入7.5μM凝血酶抑制剂(抗凝血酶III)的健康人的新鲜冰冻血浆；The normal plasma is the fresh frozen plasma of healthy people, and the hypercoagulable plasma is the fresh frozen plasma of untreated patients with spontaneous deep vein thrombosis (Deep vein thrombosis, DVT) or pulmonary embolism (PuLmonary embolism, PE). The low coagulation plasma is the fresh frozen plasma of healthy people who added 7.5 μM thrombin inhibitor (antithrombin III);
(2)将反应后的多肽微阵列芯片分别依次与3μM亲和素，3nM的多肽修饰的金纳米粒子反应1h，得到金纳米粒子标记的多肽微阵列芯片；(2) Reacting the reacted polypeptide microarray chip with 3 μM avidin and 3 nM polypeptide-modified gold nanoparticles for 1 hour to obtain a gold nanoparticle-labeled polypeptide microarray chip;
(3)将金纳米粒子标记的多肽微阵列芯片用Arrayit微阵列扫描仪测定金纳米粒子共振光散射信号。(3) The gold nanoparticle-labeled polypeptide microarray chip was used to measure the gold nanoparticle resonant light scattering signal with an Arrayit microarray scanner.
图5为本发明实施例5制备的多肽微阵列芯片检测激活的普通血浆、高凝血浆和低凝血浆中的凝血酶活性图，从图5可以看出，当金纳米粒子共振光散射信号变化量在50.2%以下时，血液为低凝血浆，当金纳米粒子共振光散射信号变化量在73.2%以上时，血液为高凝血浆。Fig. 5 is the figure of thrombin activity in normal plasma, hypercoagulable plasma and hypocoagulable plasma detected by the polypeptide microarray chip prepared in Example 5 of the present invention. As can be seen from Fig. 5, when the gold nanoparticle resonance light scattering signal changes When the gold nanoparticle resonance light scattering signal variation is above 73.2%, the blood is hypercoagulable plasma when the amount is below 50.2%.
图11为本发明实施例5多肽微阵列芯片在血浆中检测凝血酶活性的流程图。Fig. 11 is a flow chart of the detection of thrombin activity in plasma by the polypeptide microarray chip in Example 5 of the present invention.
实施例6Example 6
结合图6和图12说明实施例6
多肽微阵列芯片在血浆中筛选凝血酶抑制剂：Screening of Thrombin Inhibitors in Plasma by Peptide Microarray Chip:
(1)将多肽点阵浓度为0.5mg/mL，多肽底物为Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin)的多肽微阵列芯片分别与激活的普通血浆、加入阿加曲班的激活的普通血浆，加入抗凝血酶III的激活的普通血浆，加入AEBSF的激活的普通血浆，和未激活的普通血浆反应40min，得到反应后的多肽微阵列芯片，阿加曲班、抗凝血酶III和AEBSF的终浓度均为7.5μM；(1) The peptide matrix concentration is 0.5mg/mL, and the peptide substrate is Cys-Ala-Glu-Gly-Gly-D-Phe-Pro-Arg-Ser-Phe-Arg-Val-Val-Lys(biotin) The peptide microarray chip reacted with activated normal plasma, activated normal plasma with added argatroban, activated normal plasma with added antithrombin III, activated normal plasma with added AEBSF, and unactivated normal plasma After 40 minutes, the reacted polypeptide microarray chip was obtained, and the final concentrations of argatroban, antithrombin III and AEBSF were all 7.5 μM;
(2)将反应后的多肽微阵列芯片分别依次与3μM亲和素，3nM多肽修饰的金纳米粒子各反应1h，得到金纳米粒子标记的多肽微阵列芯片；(2) respectively reacting the reacted polypeptide microarray chip with 3 μM avidin and 3 nM polypeptide-modified gold nanoparticles for 1 hour respectively to obtain a gold nanoparticle-labeled polypeptide microarray chip;
(3)将金纳米粒子标记的多肽微阵列芯片用Arrayit微阵列扫描仪测定金纳米粒子共振光散射信号。(3) The gold nanoparticle-labeled polypeptide microarray chip was used to measure the gold nanoparticle resonant light scattering signal with an Arrayit microarray scanner.
图6为本发明实施例6的多肽微阵列芯片与加入凝血酶抑制剂的激活的血浆作用后的共振光散射信号；从图6可以看出，阿加曲班和抗凝血酶III具有较高的抑制凝血酶的能力，本发明可以在血浆中直接筛选凝血酶的抑制剂。Fig. 6 is the resonant light scattering signal after the polypeptide microarray chip of the embodiment of the
图12为本发明实施例6多肽微阵列芯片在血浆中筛选凝血酶抑制剂的流程图。Fig. 12 is a flow chart of the screening of thrombin inhibitors in plasma by polypeptide microarray chip according to Example 6 of the present invention.
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。The descriptions of the above embodiments are only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105884888A (en) * | 2016-04-14 | 2016-08-24 | 上海大学 | Artificial antibody construction method based on gold nanoparticles |
| CN114231594A (en) * | 2021-09-29 | 2022-03-25 | 深圳市赛尔生物技术有限公司 | Method for detecting thrombin activity in plasma and screening thrombin inhibitor by peptide microarray chip |
-
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Non-Patent Citations (3)
| Title |
|---|
| GAO JQ ET AL: "Microarray-Based Study of Carbohydrate-Protein Binding by Gold Nanoparticle Probes", 《ANAL CHEM》 * |
| GAO JQ ET AL: "Screening Lectin-Binding Specificity of Bacterium by Lectin Microarray with Gold Nanoparticle Probes", 《ANAL CHEM》 * |
| LI T ET AL: "Screening Kinase Inhibitors with a Microarray-Based Fluorescent and Resonance Light Scattering Assay", 《ANAL CHEM》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105884888A (en) * | 2016-04-14 | 2016-08-24 | 上海大学 | Artificial antibody construction method based on gold nanoparticles |
| CN114231594A (en) * | 2021-09-29 | 2022-03-25 | 深圳市赛尔生物技术有限公司 | Method for detecting thrombin activity in plasma and screening thrombin inhibitor by peptide microarray chip |
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