CN111505077B - A method for detecting GPC3 based on RGO-Hemin/Au NPs nanocomposites - Google Patents

Abstract

Translated fromChinese

一种基于RGO‑Hemin/Au NPs纳米复合材料检测GPC3的方法，采用电沉积技术以及静电吸附作用将RGO‑Hemin/Au NPs修饰在丝网印刷电极表面。将GPC3‑Apt负载在RGO‑Hemin/Au NPs材料表面，适配体因以单链结构的形式而呈不稳定的空间结构分布在生物传感界面上。在生物传感界面中加入GPC3后，GPC3能够与GPC3‑Apt特异性结合形成蛋白‑适配体复合物而呈稳定的空间结构，从而有序排列在工作电极上。通过DPV方法检测电流值响应值，并描绘出该电流响应值与GPC3浓度的关系曲线，实现对GPC3的定量检测。该方法操作简单、省时、费用低且具有较低的检测限。

A method for detecting GPC3 based on RGO-Hemin/Au NPs nanocomposite materials, using electrodeposition technology and electrostatic adsorption to modify RGO-Hemin/Au NPs on the surface of screen-printed electrodes. GPC3-Apt was loaded on the surface of RGO-Hemin/Au NPs material, and the aptamer was distributed on the biosensing interface in the form of a single-chain structure in an unstable spatial structure. After adding GPC3 to the biosensing interface, GPC3 can specifically bind to GPC3-Apt to form a protein-aptamer complex to form a stable spatial structure, which is arranged on the working electrode in an orderly manner. The response value of the current value was detected by the DPV method, and the relationship between the current response value and the concentration of GPC3 was drawn to realize the quantitative detection of GPC3. The method is simple, time-saving, low-cost and has a low detection limit.

Description

Translated fromChinese

一种基于RGO-Hemin/Au NPs纳米复合材料检测GPC3的方法A method for detecting GPC3 based on RGO-Hemin/Au NPs nanocomposites

技术领域technical field

本发明属于生物检测领域，具体涉及一种基于电化学生物传感器检测磷脂酰肌醇蛋白聚糖-3（GPC3）的方法。The invention belongs to the field of biological detection, in particular to a method for detecting glypican-3 (GPC3) based on an electrochemical biosensor.

背景技术Background technique

磷脂酰肌醇蛋白聚糖-3（GPC3）是在研究过度生长综合征（SGBS）时发现的，是硫酸乙酰肝素糖蛋白中的一种。GPC3在人的正常的肝细胞中无表达，在其他肿瘤组织也低表达或无表达，而在肝癌组织中表达显著高于癌旁组织。GPC3检测方法主要有放射免疫分析法、荧光免疫分析法、酶联免疫吸附法、化学发光免疫分析法、流式免疫分析法、电化学免疫传感器、压电免疫传感器及多肽复合蛋白微阵列等。公开号CN 106636108 A的发明专利，涉及一种靶向GPC3的核酸适配体及其应用，利用结合毛细管电泳的SELEX技术筛选与肝癌标志物GPC3特异性结合的核酸适配体，获得具有特异结合GPC3的适配体序列G625。所述的GPC3核酸适配体可用于设计、制备肝癌血清诊断试剂、肝癌组织切片染色试剂以及肝癌相关的活体成像造影剂。公开号CN 105717104 A的发明专利，涉及一种肝细胞癌患者外周血GPC3检测方法：利用膜过滤装置分离获取无法获得组织标本的肝癌患者外周血中的CTC，运用细胞蜡块技术制作薄层切片，进而检测GPC3表达情况。申请号为CN101290318的发明专利，公开了一种肝癌的检测试剂盒，所述检测试剂盒中含有结合硫酸肝素蛋白多糖3第379-393位中的抗原表位的多克隆抗体，以及结合硫酸肝素蛋白多糖3第379-393位以外抗原表位的单克隆抗体或多克隆抗体，在检测AFP标志物的同时采用本发明的试剂盒检测GPC3标记物可显著提高肝癌的诊断敏感性。这些方法所用仪器昂贵、操作复杂、费时且技术要求高，所以需要建立一种快速、灵敏、操作简便的GPC3检测方法。Glypican-3 (GPC3) was discovered while studying overgrowth syndrome (SGBS) and is one of the heparan sulfate glycoproteins. GPC3 is not expressed in normal human hepatocytes, and also has low or no expression in other tumor tissues, while the expression in liver cancer tissues is significantly higher than that in adjacent tissues. GPC3 detection methods mainly include radioimmunoassay, fluorescence immunoassay, enzyme-linked immunosorbent assay, chemiluminescence immunoassay, flow immunoassay, electrochemical immunosensors, piezoelectric immunosensors and polypeptide complex protein microarrays. The invention patent of publication number CN 106636108 A relates to a nucleic acid aptamer targeting GPC3 and its application. The nucleic acid aptamer that specifically binds to the liver cancer marker GPC3 is screened by using the SELEX technology combined with capillary electrophoresis, and the nucleic acid aptamer that specifically binds to GPC3 is obtained. The aptamer sequence for GPC3 is G625. The GPC3 nucleic acid aptamer can be used for designing and preparing liver cancer serum diagnostic reagents, liver cancer tissue section staining reagents and liver cancer-related in vivo imaging contrast agents. The invention patent with publication number CN 105717104 A relates to a method for detecting GPC3 in peripheral blood of patients with hepatocellular carcinoma: using a membrane filtration device to separate and obtain CTCs in peripheral blood of patients with liver cancer for which tissue samples cannot be obtained, and using cell wax block technology to make thin-layer slices , and then detect the expression of GPC3. The invention patent with the application number of CN101290318 discloses a detection kit for liver cancer. The detection kit contains a polyclonal antibody that binds to the epitopes in positions 379-393 of heparan sulfate proteoglycan 3, and a polyclonal antibody that binds to heparin sulfate. The monoclonal antibody or polyclonal antibody of the epitope other than the 379-393 position of proteoglycan 3 can significantly improve the diagnostic sensitivity of liver cancer by using the kit of the invention to detect the GPC3 marker while detecting the AFP marker. The instruments used in these methods are expensive, complicated, time-consuming and technically demanding, so it is necessary to establish a fast, sensitive and easy-to-operate GPC3 detection method.

发明内容SUMMARY OF THE INVENTION

本发明所要解决的技术问题是提供一种基于还原性氧化石墨烯-血红素/金纳米复合材料（RGO-Hemin/Au NPs）结合适配体来检测GPC3的方法，提高灵敏度、增强特异性。The technical problem to be solved by the present invention is to provide a method for detecting GPC3 based on reducing graphene oxide-hemin/gold nanocomposite material (RGO-Hemin/Au NPs) combined with aptamer, so as to improve the sensitivity and enhance the specificity.

为了解决该技术问题，采用电沉积技术以及静电吸附作用制作了基于RGO-Hemin/Au NPs的GPC3适配体电化学生物传感器。利用石墨烯及纳米金对GPC3适配体（GPC3-Apt）的高负载能力和良好的电子传递效应，以及GPC3-Apt对GPC3的特异性识别作用，采用电化学工作站的微分脉冲伏安法 (DPV)，记录其峰电流。对GPC3的孵育温度、孵育时间、PBS的pH值、GPC3-Apt浓度和复合材料的量进行了优化，绘制了标准曲线，通过与标准工作曲线对比得到准确的GPC3浓度。与现有的方法相比，操作相对简单，特异性高，时间和费用的消耗更少，能达到2.86 ng/mL的检测限。To solve this technical problem, GPC3 aptamer electrochemical biosensors based on RGO-Hemin/Au NPs were fabricated by electrodeposition technology and electrostatic adsorption. Taking advantage of the high loading capacity and good electron transfer effect of graphene and gold nanoparticles on GPC3 aptamer (GPC3-Apt), as well as the specific recognition effect of GPC3-Apt on GPC3, the differential pulse voltammetry of electrochemical workstation ( DPV), and record its peak current. The incubation temperature, incubation time, pH value of PBS, GPC3-Apt concentration and the amount of composite materials were optimized for GPC3, and a standard curve was drawn, and the accurate GPC3 concentration was obtained by comparing with the standard working curve. Compared with the existing methods, the operation is relatively simple, the specificity is high, and the consumption of time and expense is less, and the detection limit of 2.86 ng/mL can be achieved.

本发明的检测原理为：在硫酸活化预处理后的丝网印刷电极上修饰沉积上一层金纳米粒子，利用金的吸附作用将制备好的RGO-Hemin复合材料固定在修饰好的电极上，接着通过分子间作用力将GPC3-Apt固定在RGO-Hemin/Au NPs的表面，构建适配体传感界面，此时适配体因以单链结构的形式而呈不稳定的空间结构分布在生物传感界面上。而后将GPC3蛋白导入到上述生物传感界面，因GPC3蛋白能与GPC3适配体发生特异性结合形成蛋白-适配体复合物而呈稳定的空间结构，从而有序排列在电极表面。通过电化学工作站，使用循环脉冲伏安法（DPV）检测Hemin的电流响应信号变化值。The detection principle of the invention is as follows: a layer of gold nanoparticles is modified and deposited on the screen-printed electrode after sulfuric acid activation and pretreatment, and the prepared RGO-Hemin composite material is fixed on the modified electrode by the adsorption of gold. Then, GPC3-Apt was immobilized on the surface of RGO-Hemin/Au NPs by intermolecular force to construct an aptamer sensing interface. At this time, the aptamer was in the form of a single-chain structure and was distributed in an unstable spatial structure. on the biosensing interface. Then, the GPC3 protein is introduced into the above-mentioned biosensing interface, because the GPC3 protein can specifically bind to the GPC3 aptamer to form a protein-aptamer complex, which forms a stable spatial structure and is arranged on the electrode surface in an orderly manner. Cyclic pulse voltammetry (DPV) was used to detect the current response signal change value of Hemin by an electrochemical workstation.

本发明按照以下步骤进行：The present invention is carried out according to the following steps:

步骤1：RGO-Hemin材料的制备Step 1: Preparation of RGO-Hemin material

将氧化石墨烯（GO）倒入蒸馏水中，使用超声细胞破碎仪超声，使其充分溶解均匀，制成GO水溶液。将血红素（Hemin）加入氨水溶解后加少量蒸馏水，与RGO混合。将水合肼加入RGO和Hemin混合液进行还原，水浴反应后，离心得RGO-Hemin溶液。Graphene oxide (GO) was poured into distilled water and sonicated with an ultrasonic cell disruptor to fully dissolve it and make a GO aqueous solution. Add hemin (Hemin) into ammonia water to dissolve, add a small amount of distilled water, and mix with RGO. Hydrazine hydrate was added to the mixture of RGO and Hemin for reduction, and after reaction in a water bath, the RGO-Hemin solution was obtained by centrifugation.

步骤2：电极的修饰与生物传感界面的构建Step 2: Modification of electrodes and construction of biosensing interface

(1)将丝网印刷电极（SPCE）置于H₂SO₄溶液中，进行循环伏安扫描，得到活化后的丝网印刷电极，用水冲洗干净。(1) The screen-printed electrode (SPCE) was placed in a H₂ SO₄ solution, and cyclic voltammetry was performed to obtain an activated screen-printed electrode, which was rinsed with water.

(2)将活化后的丝网印刷电极置入氯金酸溶液中，进行恒电位沉积，沉积结束后用水将电极冲洗干净，得到Au NPs/SPCE电极。(2) The activated screen-printed electrode was placed in a chloroauric acid solution for potentiostatic deposition. After the deposition, the electrode was rinsed with water to obtain an Au NPs/SPCE electrode.

(3)将Au NPs/SPCE电极用戊二醛浸泡，用PBS洗涤，吹干，而后滴加RGO-Hemin悬浊液孵育一段时间，PBS洗涤，晾干，得到RGO-Hemin/Au NPs/SPCE电极。(3) Soak the Au NPs/SPCE electrode with glutaraldehyde, wash with PBS, blow dry, then dropwise add RGO-Hemin suspension to incubate for a period of time, wash with PBS, and air dry to obtain RGO-Hemin/Au NPs/SPCE electrode.

(4)取氨基化的GPC3-Apt滴加到传感器界面，孵育一段时间，用PBS溶液洗涤未固定到界面的GPC3-Apt，滴加牛血清蛋白（BSA）溶液进行封闭，得到GPC3-Apt /RGO-Hemin/AuNPs/SPCE传感界面，晾干备用。(4) Add the aminated GPC3-Apt dropwise to the sensor interface, incubate for a period of time, wash the GPC3-Apt not immobilized on the interface with PBS solution, and dropwise add bovine serum albumin (BSA) solution for blocking to obtain GPC3-Apt/ The RGO-Hemin/AuNPs/SPCE sensing interface was dried for use.

步骤3：GPC3的标准曲线绘制Step 3: Standard Curve Drawing for GPC3

(1)将标准GPC3溶液滴加到步骤2得到的GPC3-Apt /RGO-Hemin/Au NPs/SPCE传感界面，孵育一段时间，用PBS溶液清洗，得到工作电极，晾干备用。(1) Add the standard GPC3 solution dropwise to the GPC3-Apt/RGO-Hemin/Au NPs/SPCE sensing interface obtained instep 2, incubate for a period of time, wash with PBS solution to obtain a working electrode, and dry it for later use.

(2)将工作电极放入PBS溶液中，采用电化学工作站的DPV扫描，记录其峰电流。(2) Put the working electrode into the PBS solution, use the DPV scan of the electrochemical workstation, and record its peak current.

(3)分别对不同浓度的GPC3进行检测，绘制标准曲线，计算出该方法的最低检测限。(3) Detect different concentrations of GPC3 respectively, draw a standard curve, and calculate the minimum detection limit of the method.

步骤4：实际血清样本中GPC3的检测Step 4: Detection of GPC3 in Actual Serum Samples

(1)在步骤2得到的GPC3-Apt /RGO-Hemin/Au NPs/SPCE传感界面，滴加待测实际血清样本，孵育一段时间，用PBS溶液清洗，得到工作电极，晾干备用。(1) On the GPC3-Apt/RGO-Hemin/Au NPs/SPCE sensing interface obtained instep 2, drop the actual serum sample to be tested, incubate for a period of time, wash with PBS solution to obtain a working electrode, and dry it for use.

(2)将工作电极放入PBS溶液中，采用电化学工作站的DPV扫描，记录其峰电流。(2) Put the working electrode into the PBS solution, use the DPV scan of the electrochemical workstation, and record its peak current.

(3)根据步骤3所述标准曲线，计算得到所述待测实际血清样本中GPC3浓度。(3) Calculate the GPC3 concentration in the actual serum sample to be tested according to the standard curve in step 3.

进一步，所述步骤1中氨水为10 μL。Further, the ammonia water in the step 1 is 10 μL.

进一步，所述步骤1中水合肼为质量分数为80%，用量为8 μL。Further, in the step 1, the mass fraction of hydrazine hydrate is 80%, and the amount is 8 μL.

进一步，所述步骤1中将GO和Hemin材料混合，以水合肼还原，60°C水浴4 h后离心，得到RGO-Hemin悬浊液。Further, in the step 1, the GO and Hemin materials were mixed, reduced with hydrazine hydrate, and centrifuged in a 60°C water bath for 4 h to obtain a RGO-Hemin suspension.

进一步，所述步骤2中H₂SO₄溶液浓度为0.5 mol/L。Further, in thestep 2, the concentration of the H₂ SO₄ solution is 0.5 mol/L.

进一步，所述步骤2中扫描电压为-0.4 V- 1.2 V，扫描段数为20。Further, in thestep 2, the scanning voltage is -0.4 V-1.2 V, and the number of scanning segments is 20.

进一步，所述步骤2中将电极置于H₂SO₄中进行循环伏安扫描后，用纯水冲洗干净后，再将电极置于氯金酸溶液中分别进行循环伏安扫描，最后用纯水冲洗晾干备用。Further, in thestep 2, the electrode is placed in H₂ SO₄ for cyclic voltammetry scanning, and after being rinsed with pure water, the electrode is placed in chloroauric acid solution for cyclic voltammetry scanning respectively, and finally pure water is used for cyclic voltammetry scanning. Rinse with water and dry.

进一步，所述步骤2中，使用的氯金酸浓度为0.01%，沉积条件为-0.5 V，沉积时间120s。Further, in thestep 2, the concentration of chloroauric acid used is 0.01%, the deposition condition is -0.5 V, and the deposition time is 120s.

进一步，所述步骤2中，戊二醛浓度为2.5%。Further, in thestep 2, the concentration of glutaraldehyde is 2.5%.

进一步，所述BSA溶液浓度为0.5%，PBS的浓度为0.2 mol/L，pH值为6.0。Further, the concentration of the BSA solution is 0.5%, the concentration of PBS is 0.2 mol/L, and the pH value is 6.0.

进一步，步骤2中GPC3适配体浓度为0.1 μmol/L。Further, instep 2, the concentration of GPC3 aptamer was 0.1 μmol/L.

进一步，GPC3-Apt在电极的孵育温度为25°C，孵育时间为2小时。Further, the incubation temperature of GPC3-Apt at the electrode was 25°C, and the incubation time was 2 hours.

优选，所述步骤3中GPC3的最佳孵育温度为25℃，最佳孵育时间为30min。Preferably, in the step 3, the optimal incubation temperature of GPC3 is 25° C., and the optimal incubation time is 30 min.

优选，所述步骤3和4中的线性扫描范围-0.4 V-1.2 V，扫描速率为0.01 V/s。Preferably, the linear scanning range insteps 3 and 4 is -0.4 V-1.2 V, and the scanning rate is 0.01 V/s.

其中，步骤1为步骤2提供一种高导电率的包含有电活性物质（Hemin）的纳米复合材料。步骤2构成特异性识别GPC3的生物传感界面，并有利于电子的传递。步骤2中生物传感界面的构建是步骤3和步骤4中GPC3的电化学检测中必不可少的关键步骤。步骤3的GPC3的工作曲线为步骤4的实际样本中GPC3浓度的测定提供计算依据。可见步骤1-4相互支撑，共同作用，才能利用以RGO-Hemin/Au NPs复合材料和GPC3-Apt为识别探针实现GPC3的检测。Wherein, step 1 provides a high-conductivity nanocomposite material containing electroactive material (Hemin) forstep 2.Step 2 constitutes a biosensing interface that specifically recognizes GPC3 and facilitates the transfer of electrons. The construction of the biosensing interface instep 2 is an essential key step in the electrochemical detection of GPC3 insteps 3 and 4. The working curve of GPC3 in step 3 provides a calculation basis for the determination of GPC3 concentration in the actual sample instep 4. It can be seen that steps 1-4 support each other and work together to realize the detection of GPC3 by using the RGO-Hemin/Au NPs composite material and GPC3-Apt as the identification probe.

本发明与现有技术相比具有如下优点：Compared with the prior art, the present invention has the following advantages:

1、RGO-Hemin/Au NPs纳米复合材料具有比表面积大，导电性强，可以有效的提高检测速率；其中，石墨烯复合材料和纳米金的比表面积大、吸附能力强，可以有效地将GPC3-Apt吸附固定于电极的表面，增加传感器的稳定性，提高检测能力；GPC3能够与GPC3-Apt发生特异性结合反应，生成稳定的空间结构。与传统的传感器相比，新型纳米材料传感器不仅体积更小，速度更快，而且精度更高，可靠性更高。1. RGO-Hemin/Au NPs nanocomposites have large specific surface area and strong electrical conductivity, which can effectively improve the detection rate; among them, graphene composites and nano-gold have large specific surface areas and strong adsorption capacity, which can effectively convert GPC3 -Apt is adsorbed and fixed on the surface of the electrode, which increases the stability of the sensor and improves the detection ability; GPC3 can specifically bind to GPC3-Apt to generate a stable spatial structure. Compared with traditional sensors, the new nanomaterial sensor is not only smaller and faster, but also has higher precision and higher reliability.

2、采用以GPC3-Apt为识别探针检测GPC3的背景干扰小，能达到2.86 ng/mL的检测限。适配体和目标物之间的亲和力常常比抗原和抗体之间的亲和力强。此外，适配体比抗体更易被化学方法标记和修饰，这些处理有助于纳米粒子和其表面的功能化。2. Using GPC3-Apt as the identification probe to detect GPC3 has little background interference and can reach the detection limit of 2.86 ng/mL. The affinity between aptamer and target is often stronger than that between antigen and antibody. In addition, aptamers are more easily labeled and modified by chemical methods than antibodies, and these treatments facilitate the functionalization of nanoparticles and their surfaces.

附图说明Description of drawings

图1基于RGO-Hemin/Au NPs的纳米适配体传感器检测GPC3的原理图；Fig. 1 Schematic diagram of the nano-aptamer sensor based on RGO-Hemin/Au NPs to detect GPC3;

图2 RGO（A）和RGO-Hemin（B）的透射电镜图；Fig. 2 TEM images of RGO (A) and RGO-Hemin (B);

图3 电极表面不同修饰过程的扫描电子显微镜表征图；Fig. 3 Scanning electron microscope characterization images of different modification processes on the electrode surface;

图4 基于RGO-Hemin/Au NPs的GPC3纳米适配体传感器的工作曲线；图4A为不同GPC3浓度的DPV曲线，图4B为GPC3适配体传感器的工作曲线。Fig. 4 The working curve of the GPC3 nano-aptamer sensor based on RGO-Hemin/Au NPs; Fig. 4A is the DPV curve of different GPC3 concentrations, and Fig. 4B is the working curve of the GPC3 aptamer sensor.

具体实施方式Detailed ways

下面结合附图和具体实施方式对本发明进行详细说明。The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

一种基于RGO-Hemin/Au NPs纳米复合材料检测GPC3的方法，检测原理见图1。采用电沉积技术以及静电吸附作用将RGO-Hemin/Au NPs修饰在丝网印刷电极表面。通过纳米技术以及分子间作用力将GPC3-Apt负载在RGO-Hemin/Au NPs材料表面，适配体因以单链结构的形式而呈不稳定的空间结构分布在生物传感界面上。在生物传感界面中加入GPC3后，GPC3能够与GPC3-Apt特异性结合形成蛋白-适配体复合物而呈稳定的空间结构，从而有序排列在生物传感界面上。通过循环脉冲伏安法（DPV）检测传感器中Hemin的电化学信号的变化（扫描速率为0.01V/s，扫描的电压区间是 -0.4V-1.2V），并描绘出该电流与GPC3浓度的工作曲线，从而实现对GPC3的检测。A method based on RGO-Hemin/Au NPs nanocomposite to detect GPC3, the detection principle is shown in Figure 1. The RGO-Hemin/Au NPs were modified on the screen-printed electrode surface by electrodeposition technique and electrostatic adsorption. GPC3-Apt was loaded on the surface of RGO-Hemin/Au NPs by nanotechnology and intermolecular forces, and the aptamer was distributed on the biosensing interface in the form of a single-chain structure with an unstable spatial structure. After adding GPC3 to the biosensing interface, GPC3 can specifically bind to GPC3-Apt to form a protein-aptamer complex to form a stable spatial structure, which is arranged in an orderly manner on the biosensing interface. The change of the electrochemical signal of Hemin in the sensor was detected by cyclic pulse voltammetry (DPV) (the scan rate was 0.01V/s, and the scanning voltage range was -0.4V-1.2V), and the current and GPC3 concentration were plotted. working curve, so as to realize the detection of GPC3.

实施步骤如下：The implementation steps are as follows:

1、RGO-Hemin纳米复合材料的制备：1. Preparation of RGO-Hemin nanocomposites:

在电子天平上称取5 mg氧化石墨烯（GO），置于烧杯中，加入50 mL蒸馏水，用超声细胞破碎仪超声氧化石墨烯，使其充分溶解均匀，制成浓度为0.1 mg/mL的GO溶液。再称取10.0 mg血红素于10 μL氨水中溶解，再向其加入10 mL蒸馏水，制成1.0 mg/mL的Hemin溶液。将Hemin溶液与RGO按1：1的比例混合。混合后将8 μL质量分数为80％的水合肼加入RGO和Hemin混合液，涡旋10 min后，水浴4 h，5000 r/min离心去除上清液，洗涤两次。 最后将RGO-Hemin重新溶于超纯水中，得到1.0 mg/mL HGNs溶液。采用透射电镜（TEM）对RGO-Hemin进行表征分析，见如图2所示。图2A为RGO的TEM图，RGO呈片状结构，小点多而分散。图2B为RGO-Hemin的TEM图，RGO-Hemin也呈现片状结构，且两种物质已经凝聚在一起。Weigh 5 mg of graphene oxide (GO) on an electronic balance, put it in a beaker, add 50 mL of distilled water, and ultrasonically sonicate the graphene oxide with an ultrasonic cell disruptor to fully dissolve it and make it into a 0.1 mg/mL concentration of GO. GO solution. Then 10.0 mg of heme was weighed and dissolved in 10 μL of ammonia water, and 10 mL of distilled water was added to it to prepare a 1.0 mg/mL Hemin solution. The Hemin solution was mixed with RGO in a ratio of 1:1. After mixing, 8 μL of 80% hydrazine hydrate was added to the mixture of RGO and Hemin, vortexed for 10 min, water bathed for 4 h, centrifuged at 5000 r/min to remove the supernatant, and washed twice. Finally, RGO-Hemin was redissolved in ultrapure water to obtain a 1.0 mg/mL HGNs solution. The RGO-Hemin was characterized and analyzed by transmission electron microscopy (TEM), as shown in Figure 2. Figure 2A is the TEM image of RGO. RGO has a sheet-like structure with many small dots scattered. Figure 2B is the TEM image of RGO-Hemin. RGO-Hemin also exhibits a sheet-like structure, and the two substances have been condensed together.

2、电极的预处理：2. Pretreatment of electrodes:

基础丝网印刷电极（SPCE）在使用前首先浸泡在0.5 mol/L H₂SO₄ 溶液中进行循环伏安法（CV）扫描，在-0.4V -1.2V的电压范围内扫描20段；扫描完成之后用水洗净，晾干，得到活化的SPCE。The basic screen-printed electrode (SPCE) was first soaked in 0.5 mol/LH₂ SO₄ solution for cyclic voltammetry (CV) scanning before use, and 20 segments were scanned in the voltage range of -0.4V -1.2V; the scanning was completed After that, it is washed with water and air-dried to obtain activated SPCE.

3、电极的修饰与生物传感界面的构建：3. Electrode modification and biosensing interface construction:

将活化后的SPCE电极放入4 mL 0.01%氯金酸溶液，在-0.5 V恒电位沉积120 s，沉积完成后用纯水洗涤3次，吹干得到Au NPs/SPCE电极。将Au NPs/SPCE电极用2.5%戊二醛浸泡15 min，用pH7.0 PBS洗涤3次，吹干，而后滴加5μL的RGO-Hemin悬浊液孵育30 min，PBS洗涤3次，晾干，得RGO-Hemin/Au NPs/SPCE。取2μL氨基化的GPC3适配体（5’-NH₂-TAA CGC TGACCT TAG CTG CAT GGC TTT ACA TGT TCC A-3’）滴加于RGO-Hemin /Au NPs/SPCE传感界面上，孵育2 h，洗涤未能固定到界面的适配体，滴加6μL 0.5%的BSA溶液进行封闭，自然晾干，得到GPC3-Apt /RGO-Hemin/Au NPs/SPCE传感界面。采用扫描电镜（SEM）对电极表面不同修饰过程进行表征，得到各个阶段的SEM表征图见如图3所示。图3A为基础丝网电极，其表面只有一层片状的结构，一片灰暗；图3B为Au NPs/SPCE，表面明显变得亮了，并且有小颗粒附着，说明金纳米粒子修饰电极成功；图3C为RGO-Hemin /Au NPs/SPCE，可以看出覆盖一层黑色物质，表面明显变暗且略显粗糙；图3D为GPC3-Apt /RGO-Hemin/Au NPs/SPCE，电极表面覆盖上了一层絮状般的膜状物质，说明GPC3-Apt已经固定在电极表面。The activated SPCE electrode was put into 4 mL of 0.01% chloroauric acid solution, and deposited at a constant potential of -0.5 V for 120 s. After the deposition was completed, it was washed three times with pure water and dried to obtain the Au NPs/SPCE electrode. The Au NPs/SPCE electrode was soaked in 2.5% glutaraldehyde for 15 min, washed three times with pH 7.0 PBS, air-dried, and then dripped with 5 μL of RGO-Hemin suspension for 30 min, washed three times with PBS, and air-dried. , to obtain RGO-Hemin/Au NPs/SPCE. Take 2 μL of aminated GPC3 aptamer (5'-NH₂ -TAA CGC TGACCT TAG CTG CAT GGC TTT ACA TGT TCC A-3') dropwise on the RGO-Hemin/Au NPs/SPCE sensing interface, and incubate for 2 h, Wash the aptamers that could not be immobilized on the interface, add 6 μL of 0.5% BSA solution dropwise to block, and dry naturally to obtain the GPC3-Apt/RGO-Hemin/Au NPs/SPCE sensing interface. Scanning electron microscopy (SEM) was used to characterize the different modification processes on the electrode surface, and the SEM characterization images of each stage were obtained as shown in Figure 3. Figure 3A shows the basic wire mesh electrode, and its surface has only a sheet-like structure, which is dark; Figure 3B shows the Au NPs/SPCE, the surface becomes brighter obviously, and small particles are attached, indicating that the gold nanoparticles modified the electrode successfully; Figure 3C shows RGO-Hemin/Au NPs/SPCE, it can be seen that it is covered with a layer of black material, and the surface is obviously darkened and slightly rough; Figure 3D shows GPC3-Apt/RGO-Hemin/Au NPs/SPCE, the electrode surface is covered with A layer of flocculent film-like substance was formed, indicating that GPC3-Apt had been fixed on the electrode surface.

4、GPC3标准曲线的绘制：4. Drawing of GPC3 standard curve:

在GPC3-Apt /RGO-Hemin/Au NPs/SPCE传感界面滴加2 µLGPC3溶液，25℃温度下孵育30min，用pH 7.0 PBS溶液和蒸馏水清洗，吹干，得到工作电极。图3E为GPC3吸附在生物传感界面的SEM图，对比图3D，可知GPC3与GPC3适配体特异性结合之后以整齐的结构排列在电极表面，电极表面结构变得平整。然后将上述所得的工作电极放到PBS缓冲液（0.2 mol/L， pH6.0）中，采用电化学工作站的DPV扫描，记录其峰电流。不同GPC3浓度的DPV曲线图见图4A。GPC3浓度在0.001-10 µg/mL范围内时，传感器电流响应值（Y）与GPC3浓度（X）之间的关系呈线性，标准曲线见图4B，其线性回归方程为Y=-5.2446X+66.8748，相关系数为0.9884。将空白对照的三倍标准差定义为检测下限，计算出GPC3的最低检测限为2.86 ng/mL。2 µL of LGPC3 solution was added dropwise to the GPC3-Apt/RGO-Hemin/Au NPs/SPCE sensing interface, incubated at 25 °C for 30 min, washed with pH 7.0 PBS solution and distilled water, and dried to obtain a working electrode. Figure 3E is the SEM image of GPC3 adsorbed on the biosensing interface. Compared with Figure 3D, it can be seen that GPC3 and GPC3 aptamers are specifically combined on the electrode surface in a neat structure, and the electrode surface structure becomes flat. Then, the working electrode obtained above was placed in PBS buffer (0.2 mol/L, pH 6.0), and its peak current was recorded by DPV scanning of electrochemical workstation. The DPV curves of different GPC3 concentrations are shown in Figure 4A. When the GPC3 concentration is in the range of 0.001-10 µg/mL, the relationship between the sensor current response value (Y) and the GPC3 concentration (X) is linear. The standard curve is shown in Figure 4B, and the linear regression equation is Y=-5.2446X+ 66.8748, the correlation coefficient is 0.9884. Three times the standard deviation of the blank control was defined as the lower limit of detection, and the minimum detection limit of GPC3 was calculated to be 2.86 ng/mL.

5、实际血清样本中GPC3的检测：5. Detection of GPC3 in actual serum samples:

分别将已知浓度的GPC3溶液（1 μg/ mL，5 μg/mL，10 μg/mL）与血清按照1:1的比例混合，取2 μL上述混合液滴加在GPC3-Apt/RGO-Hemin/Au NPs/SPCE电极表面，25℃温度下孵育30min，用pH 7.0 PBS溶液和蒸馏水清洗，吹干，得到工作电极。按照步骤4所述，浸没在pH 6.0的PBS缓冲液中进行DPV扫描，平行测定三次，利用加标法检测血清样本的电流峰值。根据步骤4的标准曲线Y=-5.2446X+66.8748，计算可得到对应的实际血清样本中GPC3的浓度，检测结果见表1。GPC3 solutions of known concentrations (1 μg/mL, 5 μg/mL, 10 μg/mL) were mixed with serum in a ratio of 1:1, and 2 μL of the above mixture was added dropwise to GPC3-Apt/RGO-Hemin The surface of the /Au NPs/SPCE electrode was incubated at 25 °C for 30 min, washed with pH 7.0 PBS solution and distilled water, and dried to obtain a working electrode. As described instep 4, DPV scanning was performed by immersing in PBS buffer at pH 6.0, and three parallel measurements were performed, and the current peak value of serum samples was detected by standard addition method. According to the standard curve Y=-5.2446X+66.8748 instep 4, the concentration of GPC3 in the corresponding actual serum samples can be obtained by calculation, and the detection results are shown in Table 1.

表1 实际血清样本中GPC3的检测结果 Table 1 Detection results of GPC3 in actual serum samples

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以上所述仅是对本发明的较佳实施方式而已，并非对本发明作任何形式上的限制，凡是依据本发明的技术实质对以上实施方式所做的任何简单修改，等同变化与修饰，均属于本发明技术方案的范围内。The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention belong to the present invention. within the scope of the technical solution of the invention.

Claims (9)

Translated fromChinese

1.一种基于RGO-Hemin/Au NPs纳米复合材料检测GPC3的方法，按以下步骤进行：1. A method for detecting GPC3 based on RGO-Hemin/Au NPs nanocomposite material, according to the following steps:步骤1：RGO-Hemin材料的制备Step 1: Preparation of RGO-Hemin material将氧化石墨烯GO倒入蒸馏水中，使用超声细胞破碎仪超声，使其充分溶解均匀，制成GO水溶液；将血红素加入氨水溶解后加少量蒸馏水，制成Hemin溶液；将Hemin与GO混合；将水合肼加入GO和Hemin混合液进行还原反应后，离心得RGO-Hemin材料；Pour graphene oxide GO into distilled water, use ultrasonic cell disruptor to sonicate to make it fully dissolved and uniform, and make GO aqueous solution; add heme into ammonia water to dissolve, add a small amount of distilled water to make Hemin solution; mix Hemin and GO; After adding hydrazine hydrate to the mixture of GO and Hemin for reduction reaction, the RGO-Hemin material was obtained by centrifugation;步骤2：电极的修饰与生物传感界面的构建Step 2: Modification of electrodes and construction of biosensing interface（1）将丝网印刷电极SPCE置于H₂SO₄溶液中，进行循环伏安扫描，得到活化后的丝网印刷电极，用水冲洗干净，得到活化的丝网印刷电极；(1) The screen-printed electrode SPCE was placed in a H₂ SO₄ solution, and cyclic voltammetry was performed to obtain an activated screen-printed electrode, which was rinsed with water to obtain an activated screen-printed electrode;（2）将活化后的丝网印刷电极置入氯金酸溶液中，进行恒电位沉积，沉积结束后用水将电极冲洗干净，得到AuNPs/SPCE电极；(2) The activated screen-printed electrode was placed in a chloroauric acid solution for potentiostatic deposition. After the deposition, the electrode was rinsed with water to obtain an AuNPs/SPCE electrode;（3）将AuNPs/SPCE电极用戊二醛浸泡，用PBS洗涤，吹干，而后滴加RGO-Hemin悬浊液孵育一段时间，PBS洗涤，晾干，得到RGO-Hemin/Au NPs/SPCE电极；(3) Soak the AuNPs/SPCE electrode with glutaraldehyde, wash with PBS, blow dry, then dropwise add RGO-Hemin suspension to incubate for a period of time, wash with PBS, and air dry to obtain RGO-Hemin/Au NPs/SPCE electrode ;（4）取氨基化的GPC3适配体滴加到传感器界面，孵育一段时间，用PBS溶液洗涤未固定到界面的GPC3适配体，滴加牛血清蛋白溶液进行封闭，得到GPC3-Apt/RGO-Hemin/Au NPs/SPCE传感界面，晾干备用；(4) Add the aminated GPC3 aptamer to the sensor interface, incubate for a period of time, wash the GPC3 aptamer not immobilized on the interface with PBS solution, and drop the bovine serum albumin solution for blocking to obtain GPC3-Apt/RGO -Hemin/Au NPs/SPCE sensing interface, dry for use;步骤3：GPC3的工作曲线绘制Step 3: Drawing the working curve of GPC3（1）将标准GPC3溶液滴加到步骤2得到的GPC3-Apt/RGO-Hemin/Au NPs/SPCE传感界面，孵育一段时间，用PBS溶液洗涤，得到工作电极，晾干备用；(1) Add the standard GPC3 solution dropwise to the GPC3-Apt/RGO-Hemin/Au NPs/SPCE sensing interface obtained in step 2, incubate for a period of time, wash with PBS solution to obtain a working electrode, and dry it for later use;（2）将工作电极放入PBS溶液中，采用电化学工作站的DPV扫描，记录其峰电流；(2) Put the working electrode into the PBS solution, use the DPV scan of the electrochemical workstation, and record its peak current;（3）分别对不同浓度的GPC3进行检测，绘制标准曲线，计算出该方法的最低检测限；(3) Detect different concentrations of GPC3, draw a standard curve, and calculate the minimum detection limit of the method;步骤4：实际血清样本中GPC3的检测Step 4: Detection of GPC3 in Actual Serum Samples（1）在步骤2得到的GPC3-Apt/RGO-Hemin/Au NPs/SPCE传感界面，滴加待测实际血清样品，孵育一段时间，用PBS溶液洗涤，得到工作电极，晾干备用；(1) On the GPC3-Apt/RGO-Hemin/Au NPs/SPCE sensing interface obtained in step 2, drop the actual serum sample to be tested, incubate for a period of time, wash with PBS solution to obtain a working electrode, and dry it for use;（2）将工作电极放入PBS溶液中，采用电化学工作站的DPV扫描，记录其峰电流；(2) Put the working electrode into the PBS solution, use the DPV scan of the electrochemical workstation, and record its peak current;（3）根据步骤3所述标准曲线，计算得到待测实际血清样本中GPC3的浓度。(3) Calculate the concentration of GPC3 in the actual serum sample to be tested according to the standard curve described in step 3.2.按照权利要求1所述检测GPC3的方法，其特征在于：步骤1中氨水为10μL。2. according to the method for detecting GPC3 described in claim 1, it is characterized in that: in step 1, ammoniacal liquor is 10 μL.3.按照权利要求1所述检测GPC3的方法，其特征在于：步骤1中水合肼的质量分数为80%，用量为8μL。3. according to the method for detecting GPC3 described in claim 1, it is characterized in that: in step 1, the mass fraction of hydrazine hydrate is 80%, and consumption is 8 μL.4.按照权利要求1所述检测GPC3的方法，其特征在于：步骤2中将电极置于0.5mol/LH₂SO₄中进行循环伏安扫描20段，电压范围为-0.4V-1.2V。4. The method for detecting GPC3 according to claim 1, characterized in that: in step 2, the electrodes are placed in 0.5mol/LH₂ SO₄ for 20 cyclic voltammetry scans, and the voltage range is -0.4V-1.2V.5.按照权利要求1所述检测GPC3的方法，其特征在于：步骤2中用于纳米金的沉积溶液为4 mL 0.01%的氯金酸，沉积电压为-0.5 V，沉积时间120 s。5. according to the method for detecting GPC3 according to claim 1, it is characterized in that: in step 2, the deposition solution for nano-gold is 4 mL of 0.01% chloroauric acid, the deposition voltage is-0.5 V, and the deposition time is 120 s.6.按照权利要求1所述检测GPC3的方法，其特征在于：步骤2中BSA溶液为6μL 0.5%的BSA溶液。6. according to the method for detecting GPC3 described in claim 1, it is characterized in that: in step 2, BSA solution is the BSA solution of 6 μL 0.5%.7.按照权利要求1所述检测GPC3的方法，其特征在于：步骤3和步骤4中孵育温度为25°C，孵育时间为30 min。7. according to the method for detecting GPC3 described in claim 1, it is characterized in that: incubation temperature is 25 DEG C in step 3 and step 4, and incubation time is 30 min.8.按照权利要求1所述检测GPC3的方法，其特征在于：步骤3和步骤4中DPV扫描所用的溶液为pH值为6.0的PBS溶液。8. The method for detecting GPC3 according to claim 1, wherein the solution used for DPV scanning in step 3 and step 4 is a PBS solution with a pH value of 6.0.9.按照权利要求1所述检测GPC3的方法，其特征在于：步骤3和步骤4中扫描范围为-0.4V-1.2V，扫描速率为0.01V/s。9. The method for detecting GPC3 according to claim 1, characterized in that: in step 3 and step 4, the scanning range is -0.4V-1.2V, and the scanning rate is 0.01V/s.

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