Ratio-based electrochemical lateral flow immunochromatography test strip as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological analysis and relates to a ratio-based electrochemical lateral flow immunochromatography test strip as well as a preparation method and application thereof. The preparation method comprises the following steps: step 1, dissolving ferrocenecarboxylic acid (FCA) in dimethyl sulfoxide, and adding graphene ink into the solution; screening the dual-working electrode onto a polyvinyl chloride plastic sheet by using FCA ink, drying, screening a counter electrode and a reference electrode by using graphene and silver ink respectively, and drying to obtain a dual-working screen-printed electrode; step 2, preparation of HCG and MIPs; and 3, preparing the dual-working electrochemical immunochromatography test strip. According to the invention, the detection specificity and sensitivity can be effectively improved, the repeatability and stability of the test strip are enhanced, and the detection reliability and accuracy are improved; the cost is reduced, the operation is simple, the influence of various environmental factors is avoided, the large-scale production is easy, and the application prospect is wide.

Description

Technical Field

[0001] The present invention belongs to the field of bioanalysis technology and relates to a ratiometric electrochemical lateral flow immunochromatography test strip, a preparation method and an application thereof. Background Art

[0002] Immunochromatography assays (ICA) combine immunoassays with novel labeling materials and technologies, utilizing the specific reaction between antigens and antibodies to detect target analytes. They are a key tool for point-of-care (POCT) testing. Lateral flow immunoassays offer ease of operation, significantly shortened testing times, portability, widespread application, strong versatility, and mature technology. However, traditional colloidal gold test strips suffer from drawbacks such as false positives, missed detections, and low sensitivity, and are limited to qualitative or semi-quantitative analysis. This significantly limits their development, necessitating the development of highly specific recognition probes to achieve quantitative detection and enhance sensitivity.

[0003] Currently, common methods include adding a "stack pad" between the binding pad and the test pad of a traditional test strip or introducing a chemiluminescent substance to capture the luminescent signal. However, these technologies are very complex, require a high experimental environment, and have complicated and tedious operation steps.

[0004] Existing test strips, in the application publication number "CN 115327101 A", disclose "An electrochemiluminescent test strip for gamma interferon and its application". The electrochemiluminescent test strip for gamma interferon of the present invention comprises a paper-based channel layer and an electrode layer; the paper-based channel layer comprises a sample loading pad, a conjugation pad, a detection pad and a water-absorbing pad which are arranged in a sequentially overlapping manner; the sample loading pad is fixed with a sample buffer and a heterophilic antibody blocker; the conjugation pad is coated with a ruthenium-coupled labeled antibody; the detection pad is coated with a microsphere-coupled capture antibody and an electrochemiluminescent co-reactant buffer; the electrode layer is provided with a plurality of electrodes, one end of each electrode being connected to the detection pad. Problems exist: the structure is complex, processing difficulty is increased, and production and use costs are increased.

[0005] Existing electrode preparation methods, such as those described in application publication number "CN 107607597 B," disclose "A method for detecting hydrogen peroxide using a screen-printed electrode modified with a persimmon tannin-graphene-Pt composite material." First, using persimmon tannin as the raw material, a persimmon tannin-graphene-Pt nanocomposite material is directly prepared via a one-step reduction method. Gold nanoparticles are then deposited onto the surface of a screen-printed electrode using electrodeposition. The persimmon tannin-graphene-Pt composite material, dropped onto the electrode surface, is then adsorbed to the screen-printed electrode via electrostatic adsorption to produce the electrode. Because detection using a single working electrode results in poor repeatability, there is a need to improve the signal stability and repeatability of conventional electrochemical detectors to achieve accurate detection of target molecules. Summary of the Invention

[0006] The present invention provides a ratiometric electrochemical lateral flow immunochromatographic test strip, a preparation method and an application thereof to solve the problems existing in the prior art such as complex structure, increased processing difficulty, and increased production and use costs. Since detection is performed using a single working electrode, the repeatability is poor. Therefore, it is necessary to improve the signal stability and repeatability of traditional electrochemical detectors to achieve the problem of detecting target molecules.

[0007] In order to achieve the above object, the technical solution of the present invention is: a method for preparing a ratiometric electrochemical lateral flow immunochromatographic test strip, comprising the following steps:

[0008] Step 1: Preparation of screen-printed electrodes

[0009] Ferrocenecarboxylic acid (FCA) is dissolved in dimethyl sulfoxide (DMSO), and graphene ink is added to the solution, which is then stirred evenly to obtain FCA ink. The FCA ink is then used to screen a dual working electrode onto a polyvinyl chloride (PVC) plastic sheet. The electrodes are then dried, and the counter and reference electrodes are screened using graphene and silver inks, respectively, and dried to obtain a dual working screen-printed electrode.

[0010] Step 2: Preparation of HCG@MIPs

[0011] HCG@MIPs surface protein-imprinted polymers were prepared using AuNPs as carriers, HCG as template protein, and dopamine as functional monomer and cross-linker.

[0012] Step 3: Preparation of dual-working electrochemical immunochromatographic test strips

[0013] First, a backing plate with slots is prepared, and the dual working screen-printed electrodes are placed in the slots of the backing plate;

[0014] The NC film is then stacked on the electrode;

[0015] Then, tape the sample pad, conjugate pad, and absorbent pad to the backing card in that order, with each piece of paper overlapping the backing card;

[0016] Finally, the immune probe was fixed on the conjugate pad, and the capture antibody α-HCG was immobilized on the NC membrane corresponding to the working electrode W2 of the dual working screen-printed electrode.

[0017] Furthermore, in the above step 1, the feeding amounts of the dimethyl sulfoxide and ferrocenecarboxylic acid are 50-150 μL: 5-20 mg, and the feeding amounts of the graphene ink and silver ink are 0.5-2.0 g: 0.5-1.0 g.

[0018] Furthermore, the specific steps of the above-mentioned step 2 are as follows: first, take the AuNPs solution, adjust the pH value to 7-9, add HCG protein, first vibrate in a vortex mixer, and then pre-assemble in a constant temperature shaker; then dissolve the AuNPs nanoparticles pre-assembled with the template protein in a TrisHCl buffer solution containing dopamine and incubate; finally, centrifuge and elute to obtain HCG surface-imprinted AuNPs (HCG-AuNPs@MIPs).

[0019] Furthermore, in the above step 2, the coupling amount of the template protein HCG is 0.5-50 μL, the concentration is 50-100 mIU / mL, and the feeding amount of dopamine is 0.01-0.5 g.

[0020] Furthermore, the above-mentioned method for preparing a ratio electrochemical lateral flow immunochromatographic test strip obtains a ratio electrochemical lateral flow immunochromatographic test strip.

[0021] Furthermore, the above-mentioned method for preparing a ratiometric electrochemical lateral flow immunochromatographic test strip obtains the application of the ratiometric electrochemical lateral flow immunochromatographic test strip in the quantitative detection and analysis of proteins.

[0022] Compared with the prior art, the advantages of the present invention are:

[0023] 1. Using HCG as the template protein, AuNPs as the carrier, and DA as the functional monomer and cross-linker, the HCG@MIPs surface protein-imprinted polymer was prepared. This polymer was then immobilized on a conjugate pad as an immunoprobe to detect HCG in a test solution, achieving specific recognition of the HCG protein. This innovative method immobilizes the prepared protein-imprinted nanoparticles as immunoprobes on the conjugate pad of a test strip. Compared to traditional probes, using the imprinted polymer instead of the probe effectively improves detection specificity and sensitivity.

[0024] 2. The present invention introduces a dual-working screen-printed electrode. Compared with the electrodes in the prior art, the dual-working screen-printed electrode differs in that ferrocenecarboxylic acid is used in the present invention, which acts as an electroactive substance. After combining with the ink, it enhances conductivity and is prepared into a dual-working screen-printed electrode. Therefore, it can reduce the interference of background current. After adding the test strip, it can effectively enhance the repeatability stability of the test strip, thereby improving the detection sensitivity. The screen-printed electrode constitutes a dual-signal detection system. Combined with the highly specific recognition ability of the protein imprinted polymer, a highly sensitive electrochemical immunoassay lateral flow test strip suitable for the detection of HCG protein was successfully prepared.

[0025] 3. The present invention connects the electrodes in the dual-working electrochemical immunochromatographic test strip to the electrochemical workstation and uses DPV for detection. The HCG protein in the sample will specifically bind to the immune probe HCG-AuNPs@MIPs on the binding pad to form an immune complex, which will then bind to the α-HCG on W2 to generate current at a specific potential of 0.5V. W1, as a background current, will also generate a response signal. By using the ratio W1 / W2, a dual-working electrochemical lateral flow immune system is constructed, which improves the reliability and accuracy of the detection. The present invention uses protein blotting technology to prepare immune probes instead of traditional antibodies. This method not only reduces the experimental cost, but also is simple to operate, is not affected by many environmental factors, and is easy to mass produce.

[0026] 4. Broad Application Prospects: The dual-action electrochemical immunochromatographic test strip prepared by this invention is not only suitable for the detection of HCG protein, but can also be used for the quantitative detection and analysis of other proteins. Due to its advantages such as high sensitivity, specificity, repeatability, and low cost, it has broad prospects in the fields of pesticide residues, biological analysis, environmental monitoring, and food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 SEM images of electrodes, including (a) bare electrode and (b) FCA modified electrode;

[0028] Figure 2 FTIR spectra of electrodes modified with different materials;

[0029] Figure 3 (a) EIS and (b) CV curve analysis of bare electrode, in the presence of Abα-HCG, and at low concentrations of 450mIU / mL and high concentrations of 800mIU / mL of HCG. DETAILED DESCRIPTION

[0030] The method of the present invention is described below with reference to specific implementation examples and accompanying drawings, but the present invention is not limited thereto.

[0031] The experimental methods described in the following examples are considered conventional methods unless otherwise specified. The reagents and materials described are commercially available unless otherwise specified.

[0032] Example 1: A method for preparing a ratiometric electrochemical lateral flow immunochromatographic test strip, comprising the following steps:

[0033] Step 1: Preparation of screen-printed electrodes

[0034] 5.0 mg of the redox substance ferrocenecarboxylic acid (FCA) was dissolved in 50 μL of dimethyl sulfoxide (DMSO). 0.5 g of conductive graphene ink was added to the solution, and the solution was stirred evenly with a homogenizer for 30 minutes to obtain FCA ink. The dual working electrodes were then printed onto polyvinyl chloride (PVC) plastic sheets using the FCA ink, and the electrodes were dried in a 50°C oven for 3 hours. The counter and reference electrodes were then printed using 1.0 g of graphene and 0.5 g of silver ink, respectively, and dried in a 50°C oven for 3 hours to obtain the dual working screen-printed electrodes.

[0035] Step 2: Preparation of HCG-AuNPs@MIPs immunoprobe

[0036] Using 0.5 mL of AuNPs at pH 7 as the carrier and 5 μL of 80 mIU / mL HCG as the template protein, the mixture was pre-assembled on a constant temperature shaker after vortex mixing. 0.01 g of polymerization-regulated dopamine was used as the functional monomer and cross-linker to prepare AuNP-surface protein-imprinted polymers (HCG@MIPs). The desired AuNP-surface imprinted polymers were obtained after centrifugation and elution.

[0037] Step 3: Preparation of dual-working electrochemical immunochromatographic test strips

[0038] First, a backing plate with slots is prepared, and the dual working screen-printed electrodes are placed in the slots of the backing plate;

[0039] The NC film is then stacked on the electrode;

[0040] Then, stick the sample pad, conjugate pad, and absorbent pad onto the backing card in that order, with each piece of paper overlapping the backing card by 3 mm;

[0041] Finally, the immune probe was fixed on the conjugate pad, and the capture antibody α-HCG was fixed on the NC membrane corresponding to the working electrode W2 of the screen-printed electrode to obtain a ratiometric electrochemical lateral flow immunochromatography test strip.

[0042] Example 2: A method for preparing a ratiometric electrochemical lateral flow immunochromatographic test strip, comprising the following steps:

[0043] Step 1: Preparation of screen-printed electrodes

[0044] 15.0 mg of the redox substance ferrocenecarboxylic acid (FCA) was dissolved in 150 μL of dimethyl sulfoxide (DMSO). 1.5 g of conductive graphene ink was added to the solution, and the solution was stirred evenly for 60 minutes using a homogenizer to obtain FCA ink. The FCA ink was then used to print a dual working electrode onto a polyvinyl chloride (PVC) plastic sheet. The electrode was then dried in a 60°C oven for 1 hour. The counter and reference electrodes were then printed using 1.0 g of graphene and 1.5 g of silver ink, respectively. The electrodes were then dried in a 60°C oven for 1 hour to obtain the dual working screen-printed electrodes.

[0045] Step 2: Preparation of HCG-AuNPs@MIPs immunoprobe

[0046] Using 0.7 mL of AuNPs at pH 8 as the carrier and 7 μL of 90 mIU / mL HCG as the template protein, the mixture was pre-assembled on a constant temperature shaker after vortex mixing. 0.02 g of polymerization-regulated dopamine was used as the functional monomer and cross-linker to prepare AuNP-surface protein-imprinted polymers (HCG@MIPs). The desired AuNP-surface imprinted polymers were obtained after centrifugation and elution.

[0047] Step 3: Different from Example 1, each piece of paper overlaps the backing card by 4 mm.

[0048] Example 3: A method for preparing a ratiometric electrochemical lateral flow immunochromatographic test strip, comprising the following steps:

[0049] Step 1: Preparation of screen-printed electrodes

[0050] 10.0 mg of the redox substance ferrocenecarboxylic acid (FCA) was dissolved in 100 μL of dimethyl sulfoxide (DMSO). 1.0 g of conductive graphene ink was added to the solution, and the solution was stirred evenly with a homogenizer for 40 minutes to obtain FCA ink. The dual working electrodes were then printed onto polyvinyl chloride (PVC) plastic sheets using the FCA ink, and the electrodes were dried in a 55°C oven for 2 hours. The counter and reference electrodes were then printed using 1.5 g of graphene and 1.0 g of silver ink, respectively, and dried in a 55°C oven for 2 hours to obtain the dual working screen-printed electrodes.

[0051] Step 2: Preparation of HCG-AuNPs@MIPs immunoprobe

[0052] Using 2.0 mL of AuNPs at pH 9 as the carrier and 9 μL of 80 mIU / mL HCG as the template protein, the mixture was pre-assembled on a constant temperature shaker after vortex mixing. 0.05 g of polymerization-regulated dopamine was used as the functional monomer and cross-linker to prepare HCG-surface-imprinted AuNP probes (HCG-AuNPs@MIPs). After centrifugation and elution, the desired AuNP-surface protein-imprinted polymer was obtained.

[0053] Step 3: Preparation of dual-working electrochemical immunochromatographic test strips

[0054] Unlike Example 1, each sheet of paper overlapped the backing card by 4 mm.

[0055] The prepared ratio electrochemical lateral flow immunochromatography test strip can be verified by various testing methods such as differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). This embodiment uses an electrochemical method to characterize the ratio electrochemical lateral flow immunochromatography test strip prepared in Example 1: the CV test potential range is -0.2–0.4V, the DPV test range is -0.2-0.4V, the EIS test frequency is 0.1Hz-100KHz, and the amplitude is 5mV. Due to capillary action, it will chromatograph to the conjugate pad and specifically bind to the HCG@MIPs immune probe on the conjugate pad. Continue to chromatograph to the NC membrane and bind to the capture antibody α-HCG above the working electrode W2 to generate a corresponding current signal. W1 is a background current and does not have a fixed antibody, but it also generates a current signal. Different concentrations of HCG protein are detected by the W2 / W1 method.

[0056] The above embodiments take embodiment 1 as the best embodiment. The following are the test results of the ratio electrochemical lateral flow immunochromatographic test strip prepared in embodiment 1.

[0057] See also Figure 1 , the bare, unmodified electrode surface exhibits distinct graphene flakes, while the electrode surface, modified with FCA, is relatively smooth. This is due to the uniform distribution of the added electroactive material on the electrode surface. Surface elemental analysis of the modified electrode using energy dispersive spectroscopy (EDS) revealed the presence of carbon (C), oxygen (O), and iron (Fe), confirming the successful FCA modification.

[0058] See also Figure 2 , we can see that the bare SPGE and FCA-SPGE were compared by infrared spectroscopy. Since only carbon materials were used in the electrode production, it can be observed that there is almost no peak on the bare electrode; while the SPGE modified by FCA has a peak at 3000cm -1 and 950cm -1There are OH stretching and bending vibrations of the carboxyl group on FCA, 1680 cm -1 The bending vibration of C=O bond is 1471cm -1 The stretching vibration of aromatic C=C bond is at 1290cm -1 These peaks are unique to FCA, indicating the stretching vibration of the CO bond. This demonstrates that FCA has been successfully modified on the electrode surface.

[0059] See also Figure 3 , we can see that electron transfer is unimpeded on the bare electrode. However, when Ab α-HCG When fixed above W2, the current decreases due to protein-induced electron transfer hindrance. Similarly, when the HCG target protein is introduced to the device, the current response decreases with increasing HCG concentration. This is due to the formation of immune complexes in the sensing area, which hinder electron transfer and cause the current to decrease with increasing HCG concentration.

[0060] The above description is an explanation of the specific implementation of the present invention, rather than a limitation of the present invention. Those skilled in the relevant technical field can also make various equivalent technical solutions without departing from the scope of the present invention, so all equivalent technical solutions should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a ratiometric electrochemical lateral flow immunochromatographic test strip, characterized in that: The following steps are involved: Step 1: Preparation of screen-printed electrodes Ferrocenecarboxylic acid (FCA) is dissolved in dimethyl sulfoxide (DMSO), and graphene ink is added to the solution, which is then stirred evenly to obtain FCA ink. The FCA ink is then used to screen a dual working electrode onto a polyvinyl chloride (PVC) plastic sheet. The electrodes are then dried, and the counter and reference electrodes are screened using graphene and silver inks, respectively, and dried to obtain a dual working screen-printed electrode. Step 2: Preparation of HCG@MIPs HCG@MIPs surface protein-imprinted polymers were prepared using AuNPs as carriers, HCG as template protein, and dopamine as functional monomer and cross-linker. Step 3: Preparation of dual-working electrochemical immunochromatographic test strips First, a backing plate with slots is prepared, and the dual working screen-printed electrodes are placed in the slots of the backing plate; The NC film is then stacked on the electrode; Then, tape the sample pad, conjugate pad, and absorbent pad to the backing card in that order, with each piece of paper overlapping the backing card; Finally, the immune probe was fixed on the conjugate pad, and the capture antibody α-HCG was immobilized on the NC membrane corresponding to the working electrode W2 of the dual working screen-printed electrode.

2. The method for preparing a ratiometric electrochemical lateral flow immunochromatographic test strip according to claim 1, wherein: In the step 1, the feeding amounts of dimethyl sulfoxide and ferrocenecarboxylic acid are 50-1500 μL: 5-20 mg, and the feeding amounts of the graphene ink and silver ink are 0.5-2.0 g: 0.5-1.0 g.

3. The method for preparing a ratiometric electrochemical lateral flow immunochromatographic test strip according to claim 2, wherein: The specific steps of step 2 are: first, take the AuNPs solution, adjust the pH value to 7-9, add HCG protein, first vibrate in a vortex mixer, and then pre-assemble in a constant temperature shaker; then dissolve the AuNPs nanoparticles pre-assembled with the template protein in a TrisHCl buffer solution containing dopamine and incubate; finally, centrifuge and elute to obtain HCG surface-imprinted AuNPs.

4. The method for preparing a ratiometric electrochemical lateral flow immunochromatographic test strip according to claim 3, wherein: In the step 2, the coupling amount of the template protein HCG is 0.5-50 μL, the concentration is 50-100 mIU / mL, and the feeding amount of dopamine is 0.01-0.5 g.

5. A method for preparing a ratio electrochemical lateral flow immunochromatography test strip according to claim 1 to obtain a ratio electrochemical lateral flow immunochromatography test strip.

6. The method for preparing a ratiometric electrochemical lateral flow immunochromatographic test strip according to claim 1 obtains an application of the ratiometric electrochemical lateral flow immunochromatographic test strip in the quantitative detection and analysis of proteins.

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