Preparation and detection method of electrochemical immunosensor based on NiFe-LDO (at) Au / Pd (at) GO bidirectional signal amplification

Abstract

The invention relates to the technical field of immunosensors, and discloses a preparation and detection method of an electrochemical immunosensor based on NiFe-LDO (at) Au / Pd (at) GO bidirectional signal amplification, NiFe-LDO (at) Au and Pd (at) GO are combined to be applied to construction of a double-antibody sandwich type immunosensor, relatively strong electron transfer performance and a unique three-dimensional structure of NiFe-LDO are fully utilized, and the electrochemical immunosensor is prepared. The specific surface area of the electrode is increased, the electron transfer rate is increased, the load rate of Au-coated AB1 is increased, and the capturing capacity of the electrode on ProGRP is improved. Meanwhile, the loading capacity of Pd-Ab2 is promoted, so that a large amount of HPR and Pd are loaded in a detection system, and the catalytic efficiency on H2O2 is improved. The electrochemical immunosensor has the advantages of high sensitivity, wide detection range, good repeatability, good stability and ideal specificity.

Description

Technical Field

[0001] This invention relates to the field of electrochemical immunosensor technology, specifically to the preparation and detection method of an electrochemical immunosensor based on bidirectional signal amplification of NiFe-LDO@Au / Pd@GO. Background Technology

[0002] Progastrin-releasing peptide (ProGRP) is a stable precursor of gastrin-releasing peptide (GRP). It is expressed in very low amounts in normal adult tissues but is specifically highly expressed in small cell lung cancer (SCLC) cells, thus being considered an ideal tumor marker with high specificity for the auxiliary diagnosis of SCLC. In clinical practice, neuron-specific enolase (NSE) is a traditional serum biomarker for SCLC diagnosis, but its application has significant limitations. On the one hand, its concentration is also elevated in some non-small cell lung cancer (NSCLC) patients, leading to insufficient diagnostic specificity. On the other hand, serum NSE levels are highly susceptible to interference from hemolyzed samples, affecting the accuracy of the test results. In contrast, ProGRP exhibits excellent stability in peripheral blood and is unaffected by hemolysis, making it a more promising candidate for SCLC diagnosis, treatment evaluation, recurrence monitoring, and early prediction of NSCLC transformation into SCLC. Therefore, developing efficient and sensitive ProGRP detection technology is crucial for improving the clinical diagnosis and treatment of SCLC.

[0003] However, ProGRP's high-sensitivity detection still faces significant challenges, with existing technologies having obvious shortcomings. The difficulties mainly stem from the following two aspects: 1. The contradiction between extremely low target concentrations in serum and complex matrix interference. ProGRP has extremely low background concentrations in the serum of healthy individuals (typically at the pg / mL level), while it only significantly increases in the serum of SCLC patients. This necessitates detection methods with extremely high sensitivity. Currently, the mainstream detection method is an immunoassay based on antibody-antigen reactions. However, when detecting ultra-low concentrations of biomarkers, these methods are easily interfered with by the non-specific adsorption of a large number of high-concentration matrix components such as miscellaneous proteins in the serum. This leads to a decreased signal-to-noise ratio, increased background signal, and difficulty in accurately distinguishing true positive signals from background noise, thus affecting the accuracy and limit of detection.

[0004] 2. Limitations of traditional detection methods in terms of sensitivity and ease of use. Although some highly sensitive detection techniques have been reported in laboratory studies, these methods are often cumbersome to operate, requiring sophisticated instruments or specialized operators, making them difficult to popularize in routine clinical testing. While existing commercial immunoassay kits are highly automated and relatively easy to operate, their detection sensitivity may have reached its technological limit. Their ability to detect extremely low concentrations of ProGRP released from early, small lesions or residual lesions after treatment is limited, making it difficult to meet the ultra-high sensitivity requirements for early diagnosis and recurrence monitoring. Summary of the Invention

[0005] The present invention aims to provide a method for the preparation and detection of an electrochemical immunosensor based on bidirectional signal amplification of NiFe-LDO@Au / Pd@GO, and to provide a simple, reliable, and highly sensitive electrochemical immunosensor that enables rapid, sensitive, specific, and efficient detection of ProGRP.

[0006] To achieve the above objectives, this invention employs the following technical solution: a method for fabricating an electrochemical immunosensor based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification, resulting in a highly sensitive electrochemical immunosensor for ProGRP detection, comprising the following steps: (1) Preparation of Ab2@Pd@GO 1500 The prepared Pd solution and 500 The prepared GO solution was mixed well to obtain solution A, which was then refrigerated for later use. 100 mg Ab2 was dissolved in 1 mL of deionized water, diluted, and then 15 mg NHS and 5 mg EDC were added. After stirring, the mixture was incubated in a constant temperature incubator for 12 h. Then, 2 mg thionine was added, and the mixture was incubated again for 12 h to obtain solution B. Solution A and solution B were mixed, and 2 mg HPR was added and stirred for 8 h. After aliquoting and centrifugation, the supernatant was discarded, and 500 mg HPR was added to the precipitate. Resuspend in deionized water to obtain the secondary antibody Ab2@Pd@GO solution; (2) Preparation of nano-flower-like NiFe-LDO dispersion Add 2 mg to 4 mg of NiFe-LDO powder to 10 mL of the prepared chitosan solution, stir at room temperature for 4 h, and then refrigerate for later use. (3) Preparation of electrochemical immunosensors After polishing and cleaning, the glassy carbon electrode was subjected to multiple linear scans in 0.1 mM PBS solution to obtain cyclic voltammetry curves ranging from -0.2 to 0.6 V, in order to evaluate the polishing effect. The glassy carbon electrode with good polishing effect was ultrasonically cleaned with deionized water and ethanol and dried with nitrogen gas. 10 NiFe-LDO dispersion was dropped onto the surface of a glassy carbon electrode and allowed to dry at room temperature; then the glassy carbon electrode was placed in HAuCl4 solution for deposition; after washing and drying, ProGRP antibody solution was dropped onto the electrode and dried at 4°C. 10 Bovine serum albumin (BSA) solution was added dropwise to the electrode surface, incubated for 1 hour, rinsed with PBS buffer, and air-dried at 4°C; then 10 Different concentrations of the ProGRP standard to be tested were added to the electrode surface, incubated at room temperature for 30 min, rinsed with PBS buffer, and air-dried at 4°C. 10 The Ab2@Pd@GO solution was added to the electrode surface, incubated at room temperature for 30 min, rinsed with PBS buffer, and dried with nitrogen gas to obtain an electrochemical immunosensor for detecting ProGRP.

[0007] Meanwhile, this solution also provides an electrochemical immunosensor detection method based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification, applied to the above-mentioned electrochemical immunosensor preparation method based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification, for the detection of ProGRP concentration in samples, including the following steps: (1) The glassy carbon electrode modified by the electrochemical immunosensor prepared by the preparation was used as the working electrode, the saturated calomel electrode as the reference electrode, and the platinum wire electrode as the auxiliary electrode; the change of the sensor electrical signal was recorded by differential pulse voltammetry as the detection signal. (2) ProGRP standards with concentrations of 0.1 pg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 320 pg / mL, 1 ng / mL, 2 ng / mL and 5 ng / mL were tested respectively, and working curves were plotted by peak current signal intensity; (3) The ProGRP sample to be tested was replaced with the standard and the differential pulse voltammetry was used for detection. The concentration of ProGRP in the sample was calculated by the working curve.

[0008] The principles and advantages of this scheme are: This method is the first to directly modify an electrode with a nanoflower-like NiFe bimetallic composite oxide (NiFe-LDO), followed by Au deposition via electroplating. Utilizing the unique three-dimensional structure, large specific surface area, and pore volume of NiFe-LDO, the modified electrode can increase the loading of gold nanoparticles (Au), thereby increasing the immobilization capacity of antibody (Ab1), and ultimately improving the electrode's ability to capture trace amounts of ProGRP, thus lowering the detection limit.

[0009] Secondly, Pd@GO was used to label Ab2. Graphene oxide (GO) is both an excellent platform support and a highly efficient signal transduction and amplification medium; Pd (palladium) is a highly efficient electrocatalyst, which can both immobilize the support and act as a powerful signal amplification label. The application of Pd@GO increases the loading of Ab2, improving Ab2's recognition and binding ability to trace amounts of ProGRP. At the same time, because the GO support platform increases the loading of thionine and HPR, it increases the sensor's catalytic efficiency and electron transport efficiency for the substrate H2O2, amplifies the detection signal, and thus achieves higher sensitivity. Attached Figure Description

[0010] Figure 1 This is a flowchart illustrating the fabrication method of the electrochemical immunosensor based on bidirectional signal amplification of NiFe-LDO@Au / Pd@GO according to the present invention. Figure 2 This is a schematic diagram of the fabrication process of the immunosensor in the electrochemical immunosensor fabrication method based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification of the present invention. Detailed Implementation

[0011] The following detailed description illustrates the specific implementation method: This embodiment presents a method for fabricating an electrochemical immunosensor based on bidirectional signal amplification using NiFe-LDO@Au / Pd@GO, which is used for highly sensitive detection of progastrin-releasing peptide (ProGRP). The immunosensor is constructed by immobilizing the secondary antibody-ProGRP and GO via Pd linkage, and then sequentially modifying a glassy carbon electrode with NiFe-LDO@Au to connect the primary antibody-ProGRP. This significantly improves electron transport efficiency, enhances the sensor's detection performance, and gives the immunosensor good specificity, high stability, and reproducibility.

[0012] In this embodiment, as shown in the appendix Figure 1 As shown, the preparation method of the electrochemical immunosensor includes the following steps: (1) Preparation of Ab2@Pd@GO 1500 The prepared Pd solution and 500 The prepared GO solution was mixed and the resulting solution A was refrigerated for later use.

[0013] The Pd solution was prepared by using sodium borohydride as a reducing agent. 35 mg to 65 mg of PVP (polyvinylpyrrolidone) was dissolved in 50 mL of DEPC water. 1 mL to 3 mL of K₂PdCl₄ (0.4 mg / mL) was added to the solution, and the mixture was then stirred at low speed on a magnetic stirrer. In this example, the stirring speed was 300 rpm.

[0014] Then, take another 25 mL beaker and prepare 20 mL of NaBH4 solution with a concentration of 0.5 mg / mL to 1.5 mg / mL. Use 20 The prepared NaBH4 solution was slowly added dropwise to the current solution using a pipette until no more flocculent matter was formed. The mixture was then stirred for 30 minutes to obtain a black, turbid liquid. The liquid was centrifuged and then repeatedly washed with DEPC water and ethanol to remove excess sodium borohydride. The precipitate (Pd) was then dispersed in 5 mL of deionized water to obtain a Pd solution, which was dark black and had a concentration of 0.025 mg / mL to 0.075 mg / mL.

[0015] The preparation process of GO solution is as follows: 10 mg to 20 mg of GO is dispersed in 20 mL of distilled water and ultrasonically stirred for 2 to 4 hours to obtain a dark black GO solution; in this example, the concentration of GO solution is 0.5 mg / mL to 1 mg / mL.

[0016] Then, the Pd solution and GO solution were mixed and stirred at 500 rpm for 12 hours using a magnetic stirrer. Pd adhered to the GO surface through physical adsorption and chemical bonding to form a strong Pd-GO, which allowed Pd and GO to be fully combined, resulting in solution A, which was then stored in a refrigerator for later use.

[0017] 100 mg Ab2 (Mouse anti-Human ProGRP (132-148)) was dissolved in 1 mL of deionized water to prepare 1 mL of antibody solution. Then, 15 mg NHS (N-hydroxysuccinimide) and 5 mg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were added, and the solution was stirred and incubated in a constant temperature incubator for 12 h. In this example, the concentration of the antibody solution after Ab2 dissolution and dilution was 0.1 mg / mL to 0.3 mg / mL. The solution was stirred at 300 rpm for 4 h at 4 °C and then incubated in a constant temperature incubator at 4 °C for 12 h. Subsequently, 2 mg of thionine was added to the antibody solution, and the solution was stirred again at 300 rpm for 4 h at 4 °C and then incubated in a constant temperature incubator for 12 h to obtain solution B.

[0018] Mix solution A and solution B, add 2 mg HPR, and stir at 300 rpm for 8 hours at 4°C. Then, aliquot the mixture into three EP tubes and centrifuge them in a high-speed refrigerated centrifuge at 6000 rpm for 10 minutes at 4°C. After centrifugation, discard the supernatant and add 500 mg HPR to the precipitate. The secondary antibody Ab2@Pd@GO was resuspended in deionized water to obtain a solution with a concentration of 0.775 mg / mL to 1.825 mg / mL.

[0019] (2) Preparation of nano-flower-like NiFe-LDO dispersion In this embodiment, 2 mg to 4 mg of chitosan was first dissolved in 10 mL of DEPC water, and then 100 mg of [unspecified substance] was added dropwise. ~200 Add glacial acetic acid and stir for 10-15 minutes to completely dissolve the chitosan, resulting in a chitosan solution with a concentration of 0.2 mg / mL to 0.4 mg / mL.

[0020] Weigh 2-4 mg of NiFe-LDO powder and add it to 10 mL of the prepared chitosan solution. Stir the mixture at 500 rpm for 4 hours at room temperature using a magnetic stirrer to obtain a light brownish-yellow NiFe-LDO dispersion. Store the dispersion at 4°C for later use. When needed, sonicate at room temperature for 5-10 minutes. The concentration of the NiFe-LDO dispersion is 0.2 mg / mL to 0.4 mg / mL.

[0021] (3) Preparation of electrochemical immunosensors In this embodiment, as shown in the appendix Figure 2 As shown, 0.3 was used respectively. 0.05 Alumina polishing powder slurry was used to polish 4mm diameter glassy carbon electrodes (GCE) for 5-10 minutes, followed by rinsing with ultrapure water. After polishing and cleaning, the glassy carbon electrodes were subjected to multiple linear scans in 0.1mM PBS solution (containing 5 mmol K3Fe(CN)6 and K4Fe(CN)6 (pH 7.4)) to obtain repeatable cyclic voltammetry (CV) curves from -0.2 to 0.6 V, which were used to evaluate the electrode polishing effect. Glassy carbon electrodes with better polishing effects were ultrasonically cleaned with deionized water and ethanol for 1-2 minutes and dried with nitrogen. In this embodiment, the electrode polishing effect was determined by cyclic voltammetry, observing the peak current and peak potential on the CV curve. A good polished surface usually results in a sharper and higher peak current, as well as a more stable peak potential. A good polished surface typically produces a symmetrical and repeatable CV curve. Therefore, the CV curves after different polishing treatments were compared, and the top 3-5 glassy carbon electrodes with better polishing effects were selected.

[0022] 10 A NiFe-LDO dispersion was dropwise added to the surface of a glassy carbon electrode and allowed to dry at room temperature, allowing the nanomaterials to adhere to the electrode surface. After the electrode dried, the glassy carbon electrode was placed in a HAuCl4 solution, and a reference electrode and a platinum wire electrode were connected. An electrochemical workstation ("chi650e") ​​was then turned on to perform deposition. In this example, the HAuCl4 solution was 10 mL with a concentration of 0.2%–0.4%, and the deposition time was 20–40 s, ensuring uniform Au deposition on the NiFe-LDO-modified electrode.

[0023] After washing and drying, add 10 drops to the electrode. The ProGRP antibody solution (Mouse anti-Human ProGRP(79-96)) was prepared and dried at 4°C. The concentration of the ProGRP antibody solution was 5%. / mL~9 / mL.

[0024] 10 Bovine serum albumin (BSA) solution was added dropwise to the electrode surface, with a volume fraction of 1%–2%. Incubation for 1 hour was performed to block non-specific active sites on the electrode surface. The electrode was then rinsed with PBS buffer and air-dried at 4°C. Then, 10… Different concentrations of the ProGRP standard to be tested were added to the electrode surface, incubated at room temperature for 30 min, rinsed with PBS buffer, and air-dried at 4°C. In this example, the pH of the PBS buffer was 7.4.

[0025] 10 The Ab2@Pd@GO solution was dropped onto the electrode surface, incubated at room temperature for 30 min, rinsed with PBS buffer at pH 7.4 and dried with nitrogen gas to obtain an electrochemical immunosensor for detecting ProGRP.

[0026] In this embodiment, an electrochemical immunosensor detection method based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification is also provided. This method is applied to the above-mentioned electrochemical immunosensor preparation method based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification for the detection of ProGRP concentration in samples, and includes the following steps: (1) The three-electrode system of the electrochemical workstation was used for testing. The glassy carbon electrode modified by the electrochemical immunosensor prepared by the preparation was used as the working electrode, the saturated calomel electrode (SCE) was used as the reference electrode, and the platinum wire electrode was used as the auxiliary electrode. The changes in the sensor's electrical signal were recorded by differential pulse voltammetry (DPV) as the detection signal.

[0027] In this embodiment, the differential pulse voltammetry (DPV) experiment was performed in 2 mL of acetate-sodium acetate buffer (pH=5.5), with 2... Hydrogen peroxide was used as the substrate, and the voltage was scanned from -0.4 to 0.2 V at a scan rate of 50 mV / s.

[0028] (2) The DPV method was used to detect ProGRP standards with concentrations of 0.1 pg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 320 pg / mL, 1 ng / mL, 2 ng / mL and 5 ng / mL respectively. The working curve was plotted by the peak current signal intensity, and the calibration curve was obtained.

[0029] (3) The ProGRP sample was used instead of the standard for differential pulse voltammetry detection, and the concentration of ProGRP in the sample was calculated using the working curve. Different concentrations of the analyte were detected by the DPV method, resulting in different current curves and peak currents. The working curve was plotted using the calibrator, and the concentration of ProGRP in the detection solution was determined using the working curve.

[0030] In this embodiment, NiFe-LDO is used as the primary material for electrode modification for the first time. NiFe-LDO is a bimetallic composite oxide with a nano-flower-like three-dimensional structure. Compared with other forms of NiFe composites (such as NiFe-LDH), its structure has better stability, larger specific surface area and pore volume. Modifying the electrode with NiFe-LDO significantly increases the deposition efficiency and loading of Au, thereby effectively increasing the loading of Ab1, and thus greatly improving the sensitivity of the sensor.

[0031] Secondly, Ab2 was labeled with the nanomaterial Pd@GO, with thionine linked as the electron mediator and HPR as the catalytic enzyme. Graphene oxide (GO) is both an excellent platform support and a highly efficient signal transduction and amplification medium. Pd is a highly efficient electrocatalyst, capable of both immobilizing the support and acting as a powerful signal amplification label. The application of Pd@GO increases the loading of Ab2, while simultaneously improving the loading of thionine and HPR, enhancing the capture ability of the analyte, increasing the catalytic efficiency and electron transport efficiency of the sensor for the substrate H2O2, amplifying the detection signal, and thus achieving higher sensitivity.

[0032] Finally, this embodiment is the first to combine NiFe-LDO@Au and Pd@GO in the construction of a dual-antibody sandwich immunosensor, which features bidirectional signal amplification. The sensor constructed based on this method is applied to the clinical detection of ProGRP, offering simple operation, rapid detection, low sensitivity, and good specificity. It can significantly improve the accuracy of detection results, providing reliable technical support for the early diagnosis of small cell lung cancer.

[0033] The following detailed description is provided through specific embodiments.

[0034] Example 1 Preparation of Ab2@Pd@GO and nano-flower-like NiFe-LDO dispersions (1) Preparation of Pd solution Dissolve 35 mg PVP in 50 mL of DEPC water, add 1 mL of K₂PdCl₄ (concentration 0.4 mg / mL), and stir at low speed. Prepare 20 mL of 0.5 mg / mL NaBH₄ solution, adding it dropwise slowly until no more flocculent matter is formed. Continue stirring for 30 min, and the resulting Pd solution concentration is 0.025 mg / mL.

[0035] (2) Preparation of GO solution 10 mg of GO was dispersed in 20 mL of distilled water and ultrasonically stirred for 2 hours to obtain a dark black GO solution with a concentration of 0.5 mg / mL.

[0036] (3) Preparation of Ab2@Pd@GO solution 1500 Pd solution and 500 Mix the GO solution thoroughly and stir with a magnetic stirrer at 500 rpm for 12 hours to obtain solution A, which is then refrigerated for later use. Dissolve 100 mg Ab2 in 1 mL of deionized water to prepare 1 mL of antibody solution with a concentration of 0.1 mg / mL. Add 15 mg NHS and 5 mg EDC, stir at 300 rpm for 4 hours at 4°C, and incubate for 12 hours. Add 2 mg thionine to the antibody solution, stir again at 300 rpm for 4 hours at 4°C, and incubate for 12 hours to obtain solution B.

[0037] Mix solution A and solution B, add 2 mg HPR, and stir at 300 rpm for 8 hours at 4°C. Then, aliquot the mixture into three EP tubes and centrifuge them in a high-speed refrigerated centrifuge at 6000 rpm for 10 minutes at 4°C. After centrifugation, discard the supernatant and add 500 mg HPR to the precipitate. The secondary antibody Ab2@Pd@GO was resuspended in deionized water to obtain a solution with a concentration of 0.775 mg / mL.

[0038] (4) Preparation of nano-flower-like NiFe-LDO dispersion Dissolve 2 mg of chitosan in 10 mL of DEPC water, and add 100 mg of chlorine dioxide dropwise. Add glacial acetic acid and stir for 10 minutes to completely dissolve the chitosan, resulting in a chitosan solution concentration of 0.2 mg / mL. Weigh 2 mg of NiFe-LDO powder and add it to 10 mL of the prepared chitosan solution. Stir at 500 rpm for 4 hours at room temperature using a magnetic stirrer to obtain a light brownish-yellow NiFe-LDO dispersion with a concentration of 0.2 mg / mL. Store the dispersion at 4°C for later use. When ready to use, sonicate at room temperature for 5-10 minutes.

[0039] Example 2 Preparation of Ab2@Pd@GO and nano-flower-like NiFe-LDO dispersions Unlike Example 1, in this example, in step (1), 50 mg of PVP, 2 mL of K2PdCl4, and 1 mg / mL of 20 mL of NaBH4 solution were used, and other conditions remained unchanged, resulting in a Pd solution concentration of 0.05 mg / mL.

[0040] In step (2), 15 mg of GO was dispersed in 20 mL of distilled water and ultrasonically stirred for 3 hours to obtain a light dark black GO solution with a concentration of 0.75 mg / mL.

[0041] In step (3), the antibody solution is diluted to a concentration of 0.2 mg / mL, and other conditions remain unchanged to obtain a secondary antibody Ab2@Pd@GO solution with a concentration of 1.2 mg / mL.

[0042] In step (4), 3 mg of chitosan was weighed and dissolved in 10 mL of DEPC water, and 150 mg of the solution was added dropwise. Glacial acetic acid was added and stirred for 15 minutes to obtain a chitosan solution concentration of 0.3 mg / mL. 3 mg of NiFe-LDO powder was weighed and added to 10 mL of the prepared chitosan solution, with other conditions remaining unchanged, to obtain a light brownish-yellow NiFe-LDO dispersion with a concentration of 0.3 mg / mL.

[0043] Example 3 Preparation of Ab2@Pd@GO and nano-flower-like NiFe-LDO dispersions Unlike Example 1, in this example, in step (1), 65 mg of PVP, 3 mL of K2PdCl4, and 1.5 mg / mL of 20 mL of NaBH4 solution were used, and other conditions remained unchanged, resulting in a Pd solution concentration of 0.075 mg / mL.

[0044] In step (2), 20 mg of GO is dispersed in 20 mL of distilled water and ultrasonically stirred for 4 hours to obtain a light dark black GO solution with a concentration of 1 mg / mL.

[0045] In step (3), the antibody solution was diluted to a concentration of 0.3 mg / mL, and other conditions remained unchanged to obtain a secondary antibody Ab2@Pd@GO solution with a concentration of 1.825 mg / mL.

[0046] In step (4), 4 mg of chitosan was weighed and dissolved in 10 mL of DEPC water, and 200 mg of water was added dropwise. Glacial acetic acid was added and stirred for 20 min to obtain a chitosan solution concentration of 0.3 mg / mL. 4 mg of NiFe-LDO powder was weighed and added to 10 mL of the prepared chitosan solution, with other conditions remaining unchanged, to obtain a light brownish-yellow NiFe-LDO dispersion with a concentration of 0.4 mg / mL.

[0047] Example 4 Fabrication of electrochemical immunosensors (1) Use 0.3 respectively 0.05 The alumina polishing powder slurry was used to polish a glassy carbon electrode (GCE) with a diameter of 4 mm for 5 min to 10 min, and then rinsed with ultrapure water. Multiple linear scans were performed in 0.1 mM PBS solution to obtain repeatable cyclic voltammetry curves of -0.2 to 0.6 V. The electrode was then ultrasonically cleaned with deionized water and ethanol for 1 min to 2 min and dried with nitrogen gas.

[0048] (2) Place 10 A 0.2 mg / mL NiFe-LDO dispersion was dropwise added to the surface of a glassy carbon electrode and allowed to dry at room temperature. The glassy carbon electrode was then placed in 10 mL of a 0.2% HAuCl4 solution, and a reference electrode and a platinum wire electrode were connected, respectively, and deposition was allowed for 20 s.

[0049] (3) After washing and drying, add 10 drops to the electrode. The ProGRP antibody solution has a concentration of 5. / mL, and air dry at 4℃. Then add 10 A 1% (v / v) bovine serum albumin (BSA) solution was added to the electrode surface and incubated for 1 hour. The electrode was then rinsed with PBS buffer (pH 7.4) and air-dried at 4°C.

[0050] (4) Place 10 Different concentrations of the ProGRP standard to be tested were added to the electrode surface and incubated at room temperature for 30 min. The electrode was then rinsed with PBS buffer at pH 7.4 and air-dried at 4°C.

[0051] (5) Place 10 An Ab2@Pd@GO solution with a concentration of 1.2 mg / mL was added to the electrode surface, incubated at room temperature for 30 min, rinsed with PBS buffer at pH 7.4, and dried with nitrogen gas.

[0052] Example 5 Fabrication of electrochemical immunosensors Unlike Example 4, steps (1) and (4) remain unchanged in this example.

[0053] In step (2), the concentration of NiFe-LDO dispersion is 0.3 mg / ml, the concentration of HAuCl4 solution is 0.3%, the deposition time is 30 s, and other conditions remain unchanged.

[0054] In step (3), the concentration of the ProGRP antibody solution is 7. / mL. The volume fraction of bovine serum albumin (BSA) solution was 1.5%, with other conditions remaining unchanged.

[0055] In step (5), the incubation at room temperature is 40 minutes, and other conditions remain unchanged.

[0056] Example 6 Fabrication of electrochemical immunosensors Unlike Example 4, steps (1) and (4) remain unchanged in this example.

[0057] In step (2), the concentration of NiFe-LDO dispersion is 0.4 mg / ml, the concentration of HAuCl4 solution is 0.4%, the deposition time is 40 s, and other conditions remain unchanged.

[0058] In step (3), the concentration of the ProGRP antibody solution is 9. / mL. The volume fraction of bovine serum albumin (BSA) solution was 2%, with other conditions remaining unchanged.

[0059] In step (5), the Ab2@Pd@GO solution concentration is 1.825 mg / mL, and it is incubated at room temperature for 30 min, with other conditions remaining unchanged.

[0060] Example 7 Electrochemical immunosensor for detecting ProGRP concentration in samples (1) The electrochemical workstation was used for testing. The glassy carbon electrode modified with the prepared electrochemical immunosensor was used as the working electrode, the saturated calomel electrode (SCE) as the reference electrode, and the platinum wire electrode as the auxiliary electrode. Differential pulse voltammetry (DPV) was used to record the changes in the sensor's electrical signal as the detection signal. The DPV experiment was carried out in 2 mL of acetate-sodium acetate buffer (pH 5.5), with 2 mL of acetate added. Hydrogen peroxide was used as the substrate, from -0.4 to 0.2 V, at a scan rate of 50 mV / s.

[0061] (2) The prepared sensor was used to detect ProGRP standards with concentrations of 0.1 pg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 320 pg / mL, 1 ng / mL, 2 ng / mL and 5 ng / mL respectively by the DPV method, and the working curve was plotted by the peak current signal intensity.

[0062] (3) Replace the standard with the ProGRP test sample for DPV detection, and calculate the ProGRP concentration in the sample by working curve.

[0063] Detection of ProGRP concentration in serum samples The sensors constructed in Examples 1, 4, and 7 above were used to detect serum samples with known ProGRP concentrations, and the recovery rate was calculated. The results are shown in Table 1 below, which shows the detection of ProGRP in serum samples using the constructed scheme.

[0064] Table 1

[0065] As shown in Table 1, the recovery rate of the immunosensor was 93.59%–105.15%, and the RSD was 1.26%–4.86%. The results indicate that the constructed immunosensor performed well in actual sample detection.

[0066] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing an electrochemical immunosensor based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification, characterized in that, Comprising the following steps: (1) Preparation of Ab2@Pd@GO Prepare 1500 The prepared Pd solution is mixed with 500 The prepared GO solution is mixed and uniformly obtained as solution A for cold storage standby; 100 mg of Ab2 is dissolved in 1 mL of deionized water, diluted, and then 15 mg of NHS and 5 mg of EDC are added, stirred, and then incubated in an incubator for 12 h, followed by the addition of 2 mg of thionine and incubation in an incubator for another 12 h to obtain solution B; solution A and solution B are mixed, 2 mg of HPR is added, stirred for 8 h, centrifuged after aliquotting, the supernatant is discarded, and 500 deionized water is added for resuspension to obtain a secondary antibody Ab2@Pd@GO solution; (2) Preparation of nanoflower-like NiFe-LDO dispersion 2mg~4mg NiFe-LDO powder is added to 10mL prepared chitosan solution, and after stirring at room temperature for 4h, it is refrigerated for standby; (3) Preparation of electrochemical immunosensor After the glassy carbon electrode is polished and cleaned, multiple linear scans are performed in a 0.1mM PBS solution to obtain a cyclic voltammogram of-0.2~0.6V to evaluate the polishing effect of the electrode; the glassy carbon electrode with good polishing treatment effect is ultrasonically cleaned with deionized water and ethanol, and dried with nitrogen; 10 drops of NiFe-LDO dispersion solution were added to the surface of the glassy carbon electrode to dry at room temperature; the glassy carbon electrode was then placed in a HAuCl4 solution for deposition; after washing and drying, a ProGRP antibody solution was added to the electrode and dried at 4°C; 10 Bovine serum albumin (BSA) solution was added dropwise to the electrode surface, incubated for 1 hour, rinsed with PBS buffer, and air-dried at 4°C; then 10 Different concentrations of the ProGRP standard to be tested were added to the electrode surface, incubated at room temperature for 30 min, rinsed with PBS buffer, and dried at 4°C. The 10 Ab2@Pd@GO solution was added to the electrode surface, and after incubation at room temperature for 30 min, the electrode was rinsed with PBS buffer and dried with nitrogen to obtain an electrochemical immunosensor for detecting ProGRP.

2. The preparation method of the electrochemical immunosensor based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification according to claim 1, characterized in that: In step (1), the preparation process of the Pd solution is as follows: 35mg~65mg PVP is dissolved in 50mL DEPC water, 1mL~3mL K2PdCl4 is added, and low-speed stirring is performed; and the prepared NaBH4 solution is slowly added dropwise to the current solution until no flocculation is generated; continue to stir for 30min; the obtained liquid is centrifuged and repeatedly washed with DEPC water and ethanol, and then the precipitate is dispersed in 5mL deionized water; the Pd solution is dark black, and the concentration is 0.025mg / mL~0.075mg / mL.

3. The preparation method of the electrochemical immunosensor based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification according to claim 1, characterized in that: In step (1), the preparation process of the GO solution is as follows: 10mg~20mg GO is dispersed in 20mL distilled water, and ultrasonic stirring is performed for 2~4h to obtain a dark black GO solution; the concentration of the GO solution is 0.5 mg / mL~1mg / mL.

4. The preparation method of the electrochemical immunosensor based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification according to claim 1, characterized in that: In step (1), the concentration of the diluted Ab2 solution is 0.1mg / mL~0.3mg / mL; after stirring at 300rpm at 4℃ for 4h, it is placed in a 4℃ incubator for incubation.

5. The preparation method of the electrochemical immunosensor based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification according to claim 1, characterized in that: In step (1), the centrifugation condition is 6000r at 4℃ for 10min; the concentration of the obtained Ab2@Pd@GO solution is 0.775mg / mL~1.825mg / mL.

6. The preparation method of the electrochemical immunosensor based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification according to claim 1, characterized in that: In step (2), the chitosan solution is 2-4 mg chitosan dissolved in 10 mL DEPC water, and 100 ~200 Glacial acetic acid is added and stirred for 10-15 min to completely dissolve it; the concentration of the chitosan solution is 0.2-0.4 mg / mL; the concentration of the NiFe-LDO dispersion is 0.2-0.4 mg / mL.

7. The preparation method of the electrochemical immunosensor based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification according to claim 1, characterized in that: In step (3), the glassy carbon electrode with a diameter of 4 mm was polished with 0.3 0.05alumina polishing powder slurry for 5 min to 10 min, and then washed with ultrapure water; the HAuCl4 solution was 10 mL with a concentration of 0.2% to 0.4%, and the deposition time was 20 s to 40 s.

8. The preparation method of the electrochemical immunosensor based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification according to claim 1, characterized in that: In step (3), the concentration of the ProGRP antibody solution is 5 / mL ~ 9 / mL; the volume fraction of the BSA solution is 1% ~ 2%; and the pH of the PBS buffer is 7.

4.

9. The electrochemical immunosensor detection method based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification, characterized in that, The electrochemical immunosensor preparation method based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification according to any one of the above claims 1-8 is used for ProGRP concentration detection in a sample, comprising the following steps: (1) The glassy carbon electrode modified by the prepared electrochemical immunosensor is used as a working electrode, a saturated mercury electrode is used as a reference electrode, and a platinum wire electrode is used as an auxiliary electrode; differential pulse voltammetry is used to record the change of the sensor electric signal as a detection signal; (2) 0.1pg / mL, 1pg / mL, 10pg / mL, 100pg / mL, 320pg / mL, 1ng / mL, 2ng / mL, and 5ng / mL ProGRP standard samples are detected respectively, and a working curve is drawn by the peak current signal intensity; (3) The ProGRP sample to be tested is replaced by the standard sample for differential pulse voltammetry detection, and the ProGRP concentration in the sample is calculated by the working curve.

10. The electrochemical immunosensor detection method based on NiFe-LDO@Au / Pd@GO bidirectional signal amplification according to claim 9, characterized in that: The differential pulse voltammetry experiment was carried out in 2 mL of acetic acid-sodium acetate buffer, 2 mM of hydrogen peroxide as substrate, scanning from -0.4 to 0.2 V at a scan rate of 50 mV / s.

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