Colorimetric / electrochemical dual-mode test strip for detecting OA and preparation method of colorimetric / electrochemical dual-mode test strip

Abstract

The invention provides a colorimetric / electrochemical dual-mode test strip for detecting OA and a preparation method thereof, and belongs to the technical field of biological detection. The test paper is composed of a bifunctional signal probe, a chromatography matrix and an integrated detection module. Cellulose paper is adopted to replace a traditional nitrocellulose membrane to serve as a chromatography substrate, and a screen-printed electrode is integrated in a detection area, so that colorimetric and electrochemical dual-mode signal output can be synchronously triggered by loading a single sample. Wherein the hollow MOF structure realizes physical isolation of two signal carriers AuNPs and Fc-NH2 through a space confinement effect, and signal cross interference is effectively eliminated; the colorimetric module supports on-site rapid screening, and the electrochemical module realizes high-sensitivity quantitative detection. According to the method, the reliability of a detection result is remarkably improved through mutual verification of double signals, the functions of visual interpretation and accurate analysis are achieved, operation is easy and convenient, additional auxiliary steps are not needed, and the method is particularly suitable for rapid detection of trace target objects in complex samples.

Description

Technical Field

[0001] This invention provides a colorimetric / electrochemical dual-mode test strip for detecting OA and its preparation method, belonging to the field of biodetection technology. Background Technology

[0002] With the rapid development of point-of-care testing (POCT) technology, lateral chromatography strips (LFAs) have been widely used in disease diagnosis, food safety monitoring, and other fields due to their advantages such as ease of operation and rapid response. Traditional LFAs mainly rely on colorimetric detection for qualitative or semi-quantitative analysis, but their insufficient sensitivity and low quantitative accuracy limit their application in trace target analyte detection. Especially in complex samples (such as biological fluids and food matrices), background interference can easily lead to false positive or false negative results, making it difficult to meet the requirements of high-precision detection.

[0003] In recent years, researchers have proposed various improvement strategies to overcome the limitations of traditional colorimetric detection. For example, introducing single-mode enhancement techniques such as fluorescent labeling, electrochemical sensing, or surface-enhanced Raman scattering (SERS) has significantly improved detection sensitivity and quantification capabilities. However, such single-mode systems still suffer from signal susceptibility to interference from sample optical properties (such as color and turbidity) or reliance on complex instruments. Furthermore, some studies have attempted to develop dual-mode LFAs, achieving cross-validation through the synergistic output of colorimetric and electrochemical / fluorescent signals. However, existing schemes often require step-by-step sample addition or additional operations to activate the second signal module, resulting in cumbersome detection procedures and deviating from the core advantage of LFAs' "one-step" approach. In addition, the design of dual-signal probes often faces challenges such as interference between signal carriers and low loading efficiency, limiting their practical application potential.

[0004] Therefore, existing technologies struggle to balance high-sensitivity detection with resistance to matrix interference, and dual-mode systems generally suffer from operational complexity and signal crosstalk. There is an urgent need to develop an integrated dual-signal tag and synchronous detection mechanism that can achieve reliable and accurate analysis while maintaining ease of operation. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a colorimetric / electrochemical dual-mode test strip for detecting OA and its preparation method. The test strip is based on a hollow bimetallic organic framework (MOF)-derived nano-hybrid material to construct a dual-functional signal probe and employs an integrated electrode design. This enables simultaneous triggering of colorimetric and electrochemical dual-mode signal output after a single sample is loaded, combining rapid screening with high-precision quantification. This effectively solves the problems of cumbersome operation, signal crosstalk, and insufficient anti-interference capabilities in existing dual-mode detection methods.

[0006] To achieve the objective of this invention, the technical solution adopted is as follows: a colorimetric / electrochemical dual-mode test strip for detecting OA, wherein the colorimetric / electrochemical dual-mode test strip for detecting OA comprises a dual-functional signal probe, a chromatography matrix, and an integrated detection module; the preparation method of the dual-functional signal probe includes the following steps: S1 added zinc acetate dihydrate, copper acetylacetonate, and 2-imidazolium formaldehyde to the reactor in a molar ratio of 2:1:2. The reaction was carried out by solvothermal reaction at 80-90℃ for 20-24 hours. 150-250 mg of polyvinylpyrrolidone (PVP) was added to adjust the MOF particle size to 60-150 nm. Hollow Cu / Zn-MOF was obtained after centrifugation, washing with ethanol, and vacuum drying. S2 disperses hollow Cu / Zn-MOF in chloroauric acid solution to adsorb Au. 3+ After 12-24 hours of adsorption, sodium borohydride at a concentration of 1-2 mg / mL is added as a reducing agent, and the reaction is carried out for 20-40 minutes at a temperature of 4-25℃ to obtain hollow Cu / Zn-MOF@Au. S3 activated the surface carboxyl groups of hollow Cu / Zn-MOF@Au at pH 5.5-6.5 using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide (EDC / NHS). After activation, it underwent an amidation reaction with Fe-NH2 and reacted at 4-25℃ for 8-12 hours to obtain a bifunctional signal probe composed of gold nanoparticles loaded with hollow bimetallic Cu / Zn-MOF-derived nanohybrid material and aminoferrocene. The chromatography matrix is ​​chitosan-modified Whatman No. 1 paper with a chitosan concentration of 0.01~0.05% (w / v). The integrated detection module is a circular screen-printed electrode array with a diameter of 4~10 mm, used for synchronous acquisition of electrochemical signals.

[0007] According to a colorimetric / electrochemical dual-mode test strip for detecting OA, the preparation method of the colorimetric / electrochemical dual-mode test strip for detecting OA includes the following steps: S4 disperses a bifunctional signal probe with a concentration of 0.1~0.5 mg / mL and a volume of 5~15 μL on glass fiber, and obtains a probe binding pad after drying; S5 uses chitosan to modify cellulose paper, which is then dried to obtain the chromatography matrix; S6 involves immobilizing capture antibody (0.5–1 mg / mL, 0.5–1.5 μL) and goat anti-mouse IgG (0.5–1 mg / mL, 0.5–1.5 μL) onto a chromatography matrix to obtain the detection zone and the quality control zone, respectively. S7 sequentially stacks the sample pad, conjugation pad, chromatography matrix, and absorption pad onto a polyvinyl chloride (PVC) backing plate, and embeds an integrated detection module below the detection area to obtain the colorimetric / electrochemical dual-mode test strip.

[0008] This invention also provides a detection method for the above-mentioned colorimetric / electrochemical dual-mode test strip for detecting OA, the detection method comprising the following steps: S8 dilutes the known concentration of the OA target analyte into different concentration gradients with the test solution, then drops the diluted target analyte onto the sample pad of the immunochromatographic test strip, and then measures the colorimetric intensity and electrochemical signal of the detection area, and calculates the intensity ratio and current difference, and establishes standard curves of intensity ratio and current difference versus target analyte concentration, respectively. S9 adds the sample to be tested onto the sample pad of the immunochromatographic test strip. After 10-20 minutes, the color intensity of the detection area is measured by a smartphone, and the electrochemical signal is measured by a portable potentiostat and terminal device. S10 calculates the colorimetric intensity ratio and current difference, and compares them with the standard curve to obtain the concentration of OA in the sample to be tested.

[0009] According to a detection method for a colorimetric / electrochemical dual-mode test strip for detecting OA, in step S8, the OA concentration gradient range is 0.2~100 ng / mL, the sample volume is 50~100 μL, and the test solution is 0.1M PBS buffer.

[0010] According to a detection method for a colorimetric / electrochemical dual-mode test strip for detecting OA, in step S9, the electrochemical signal is a differential pulse voltammetry signal, and the current difference is ∆I.

[0011] (1) The colorimetric / electrochemical dual-mode test strip of the present invention achieves synchronous signal activation after sample loading. Through the multifunctional integration of hollow Cu / Zn-MOF-derived nano-hybrids, colorimetric and electrochemical signals are generated synchronously after sample loading, without the need for secondary operations or additional steps. This design simplifies the operation process, reduces human error, and is particularly suitable for rapid on-site detection.

[0012] (2) This invention significantly improves detection sensitivity by optimizing the structure and function of the nano-hybrids. The detection limit in electrochemical mode is as low as 0.06 ng / mL, and the detection limit in colorimetric mode is 0.89 ng / mL, covering a detection range of 0.2~100 ng / mL. This improved sensitivity enables the test strip to simultaneously meet the needs of rapid screening and accurate quantitative analysis, making it suitable for various scenarios from preliminary detection to trace analysis.

[0013] (3) The dual-mode test strip of this invention has excellent specific recognition ability. By optimizing antibody binding and the surface chemistry of nano-hybrids, the test strip can specifically recognize OA and has no cross-reactivity with common marine toxins such as microcystin-WR and brevicornuate toxin (BTX-1), maintaining high selectivity even in the presence of high concentrations of interfering substances. This anti-interference ability ensures the reliability of the test results and is particularly suitable for the detection of complex biological samples.

[0014] (4) The dual-mode test strip described in this invention exhibits excellent performance in actual shellfish sample testing. In mussels, oysters, and clams, the recovery rate of OA ranged from 83.5% to 115.5%, with a relative standard deviation (RSD) ≤ 8.6%. The test results were highly consistent with commercial ELISA kits, verifying the practicality of this invention in food safety monitoring and on-site testing. Combining its low cost and rapid response (testing completed in 15 minutes), this invention is particularly suitable for the immediate testing needs of primary healthcare institutions and resource-constrained environments. Attached Figure Description

[0015] Figure 1 This diagram illustrates the synthesis of hollow Cu / Zn-MOF@Au / Fe nanoprobes and the principle of dual-mode detection. Figure 2 SEM images of hollow Cu / Zn-MOFs synthesized with different PVP addition amounts; Figure 3 TEM images, elemental distribution, and spectral characterization results of hollow Cu / Zn-MOF@Au / Fe; Figure 4 Optimization parameters (chitosan concentration, probe concentration, antigen coating concentration) and electrochemical performance testing for the dual-mode LFA device; Figure 5 The detection performance of dual-mode LFA for OA (linear range of colorimetric and electrochemical signals, detection limit). Detailed Implementation

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention. Furthermore, unless otherwise specified, all raw materials, reagents, instruments, and equipment used in the present invention can be obtained commercially or prepared using existing methods.

[0017] Example 1 (PVP dosage: 150 mg) Weigh 43.8 mg of zinc acetate dihydrate, 26.1 mg of copper acetylacetonate, and 150 mg of PVP, dissolve them in 4 mL of DMF, and stir magnetically to form a homogeneous solution; rapidly inject 4 mL of DMF solution containing 19.2 mg of 2-imidazolium carboxaldehyde (2-ICA) into this solution, and stir vigorously for 5 min; add 6 mL of DMF dilution system, and continue stirring for 30 min; transfer the mixture to an 85℃ reactor for constant temperature crystallization for 24 h; after the reaction is complete, cool to room temperature, centrifuge at 10000 rpm to separate the precipitate, wash three times with ethanol, and vacuum dry overnight at 60℃ to obtain the target product, hollow Cu / Zn-MOF (… Figure 2 a,b).

[0018] Example 2 (PVP dosage: 200 mg) 43.8 mg zinc acetate dihydrate, 26.1 mg copper acetylacetonate, and 200 mg PVP were dissolved in 4 mL DMF and magnetically stirred until homogeneous. 4 mL of DMF containing 19.2 mg 2-ICA was rapidly added, and the mixture was vigorously stirred for 5 min. 6 mL of DMF was added for dilution, and stirring continued for 30 min. The mixture was then transferred to an 85°C reactor and reacted at this temperature for 24 h. After cooling, the precipitate was collected by centrifugation at 10000 rpm, washed three times with ethanol, and dried under vacuum at 60°C overnight to obtain the target product, hollow Cu / Zn-MOF. Figure 2 c,d).

[0019] Example 3 (PVP dosage: 250 mg) 43.8 mg of zinc acetate dihydrate, 26.1 mg of copper acetylacetonate, and 250 mg of PVP were dissolved in 4 mL of DMF by magnetic stirring. 4 mL of DMF containing 19.2 mg of 2-ICA was rapidly added, and the mixture was stirred vigorously for 5 min. The solution was diluted with 6 mL of DMF and stirred for 30 min. The mixture was then crystallized in an 85℃ reactor for 24 h. After cooling to room temperature, the mixture was centrifuged at 10,000 rpm, washed three times with ethanol, and dried under vacuum at 60℃ for 12 h to obtain the final product, hollow Cu / Zn-MOF. Figure 2 e,f). Example 4

[0020] This embodiment is based on the hollow Cu / Zn-MOF described in Example 3. First, 40 mg of hollow Cu / Zn-MOF was uniformly dispersed in 30 mL of deionized water and sonicated for 10 min to ensure complete dispersion. Then, 20 mg of potassium tetrachloroaurate (hereinafter abbreviated as KAuCl4) was added to the suspension, and the mixture was magnetically stirred at room temperature for 24 h to promote Au dispersion. 3+Adsorption on the MOF surface. After adsorption, the solid product was collected by centrifugation at 10,000 rpm and washed three times with deionized water to remove unbound KAuCl4 residue. The purified intermediate was redispersed in 15 mL of deionized water, and 1 mL of freshly prepared 1.5 mg / mL sodium borohydride (hereinafter referred to as NaBH4) aqueous solution was rapidly added under vigorous stirring to initiate the adsorption of Au. 3+ The reduction reaction was carried out. After the reaction lasted for 30 min, the dark precipitate was separated by centrifugation and washed three times alternately with deionized water and ethanol to remove byproducts. Finally, the product was dried in a vacuum oven at 60 °C for 24 h to obtain the final hollow Cu / Zn-MOF@Au composite material. For further modification, Fe-NH2 was covalently bonded to the carboxyl groups on the hollow Cu / Zn-MOF@Au. First, 30 mg of hollow Cu / Zn-MOF@Au was dispersed in 10 mL of MES buffer at pH 5.5 and stirred at room temperature for 2 h. Then, 76.7 mg of EDC and 11.5 mg of NHS were added to activate the carboxyl groups. After activation, 5 mg of Fe-NH2 was added, and the mixture was incubated at 4 °C for 10 h. After the reaction was completed, the product was separated by centrifugation and washed repeatedly with deionized water. Finally, it was redispersed in 10 mL of PBS for later use.

[0021] As shown in Figure 3, gold nanoparticles (hereinafter referred to as AuNPs) exhibit a uniform distribution on the MOF surface. High-resolution transmission electron microscopy images clearly show a lattice spacing of 0.235 nm, consistent with the characteristic parameters of the Au(111) crystal plane. Based on Cu²⁺-mediated oxidation, Fc-NH₂ was successfully covalently immobilized on the surface of the hollow Cu / Zn-MOF@Au nanocomposite using a carbodiimide / N-hydroxysuccinimide coupling system to generate carboxyl groups through the conversion of aldehyde groups within the metal-organic framework. Fourier transform infrared spectroscopy data show that the immobilized carboxyl groups (-COOH) are located at 1589 cm⁻¹. −1 The intensity of the characteristic stretching vibration peak at the point of origin decreased significantly, confirming the consumption of reactive groups during covalent coupling. This differentiated immobilization strategy achieved effective loading and spatial separation of the two signal reporter molecules on the hollow framework of Cu / Zn-MOF, significantly improving the stability of the composite structure and providing technical support for independent output of dual signals during detection. As shown in the figure, the hollow morphology of Cu / Zn-MOF remained intact during the multi-step modification process. Scanning electron microscopy combined with particle size distribution characterization showed that the particle size distribution curves of the original substrate material, the AuNPs modified body, and the Fc functionalized nanostructure were highly consistent, fully verifying the good maintenance of the metal-organic framework structure integrity during the construction of the dual-signal carrier. Example 5

[0022] This embodiment is based on the hollow Cu / Zn-MOF@Au / Fc described in Example 2. First, 1 mL of hollow Cu / Zn-MOF@Au / Fe suspensions (0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively) in PBS was mixed with 10 μL of anti-OA mAb solution (1 mg / mL). The mixture was incubated at room temperature with continuous stirring for 1 h to promote antibody attachment. To block residual non-specific binding sites, 100 μL of 10 wt% bovine serum albumin (hereinafter referred to as BSA) solution was added, and the mixture was stirred for another 1 h. Then, the conjugated hollow Cu / Zn-MOF@Au / Fe-mAb was collected by centrifugation (10000 rpm, 4℃, 5 min) and washed three times with PBS to remove unbound mAb and excess BSA. The final product was redispersed in a PBS stabilization buffer (0.01M, pH 7.4) containing 5% sucrose, 1% polyethylene glycol (PEG, Mw = 6000), 1% BSA and 0.4% Tween-20, and stored at 4°C for subsequent use. Example 6

[0023] This embodiment is based on the hollow Cu / Zn-MOF@Au / Fe-mAb described in Embodiment 3. For example... Figure 1 and Figure 4 The colorimetric / electrochemical dual-mode test strip shown is fabricated by sequentially assembling a sample pad, conjugate pad, nitrocellulose membrane, absorbent pad, and screen-printed electrode (hereinafter referred to as SPE) on a PVC base card. Before assembly, 1 μL of chitosan solution (0.025%, w / v) is dispensed into the predetermined test area (T) and control area (C), then dried at room temperature. Next, 1 μL of OA-BSA antigen (1 mg / mL) and goat anti-mouse IgG antibody (1 mg / mL) are immobilized onto T and C, respectively, thereby functionalizing the cellulose paper. The conjugate pad was pretreated by sequentially immersing it in PBS containing 0.5% BSA, 0.1% Tween-20, 0.1% PEG 6000, and 5% sucrose (30 min, dried at 37°C) and a hollow Cu / Zn-MOF@Au / Fc-mAb nanoprobe suspension (30 s, dried at 37°C for 1 h). The sample pad was functionalized by immersing it in PBS containing 0.5% BSA / 0.1% Tween-20 (30 min) and then drying at 37°C for 2 h. The final assembly involved overlapping the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad by 2 mm, precisely aligning the SPE below the T-line of the electrochemical detection to form a 5 mm wide dual-mode OA detection strip, which was then dried and stored at 4°C. Example 7

[0024] This embodiment is based on the colorimetric / electrochemical dual-mode test strip described in Example 4. For quantitative analysis of OA, 50 μL of a detection solution containing OA (serially diluted in 0.1M PBS buffer, pH 7.0) was first loaded onto the sample pad. After 20 minutes of lateral flow migration, colorimetric and electrochemical signals were simultaneously detected in the T region of the test strip. The colorimetric signal was captured by photographing the test strip under standard illumination conditions using a smartphone, and grayscale analysis was performed using ImageJ software to quantify the intensity of the colorimetric signal. Electrochemical detection was performed using a PalmSens BV electrochemical workstation (CHInstruments) on an integrated screen-printed electrode (SPE) using differential pulse voltammetry. The scanning parameters were set as follows: potential window from −0.2 V to 0.6 V (relative to the Ag / AgCl reference electrode), pulse amplitude of 0.01 V, potential increment of 200 mV, and pulse width of 0.02 s. Example 8

[0025] To optimize the performance of the dual-mode test strip constructed in Example 4, key parameters were systematically optimized prior to OA detection to ensure optimal analytical performance of the dual-mode colorimetric / electrochemical LFA. Signal homogeneity (expressed as the T0 / C0 ratio) and signal intensity (expressed as the T0 value) were selected as dual performance criteria to guide parameter adjustments. Here, T0 and C0 represent the color intensity (quantized in 8-bit grayscale values) of regions T and C, respectively, in the absence of OA. A T0 / C0 ratio close to 1.0 indicates uniform signal distribution in both regions. This homogeneity is crucial for minimizing false positives, improving interpretation accuracy, and establishing a clear baseline for analyte-dependent signal contrast in competitive LFA formats. Figure 4 As shown in b, chitosan-mediated OA-BSA immobilization exhibited the best capture efficiency at a chitosan concentration of 0.05% (w / v). Due to the inverse correlation between optical density and grayscale quantification, the T0 value gradually decreased with increasing chitosan concentration, stabilizing at 158.14 at 0.05%. Although a brown T-region was clearly visible at all chitosan concentrations, no significant color enhancement was observed at concentrations above 0.05%. 0.05% chitosan modification simultaneously achieved a uniform signal with a T0 / C0 ratio of 1.03. Next, the performance of the hollow Cu / Zn-MOF@Au / Fe conjugate detection probe was systematically evaluated at a fixed anti-OA-mAb dose (1 mg / mL). Figure 4As shown in c, the loading concentration of nanohybrids significantly affects both the uniformity and intensity of the signal. At 0.4 mg / mL, the optimal T0 / CO ratio of the system is 0.96, indicating that the signal distribution between the T and C regions is close to ideal. This concentration also produces the strongest colorimetric signal in the T region, with a corresponding minimum T0 value of 149.28. Example 9

[0026] After optimizing key parameters using colorimetric signals, the performance of the electrochemical module was verified to confirm its dual-mode functionality. For example... Figure 4 As shown, a charge transfer resistance of 2.09 kΩ and a clear CV redox peak were observed on the bare electrode. After integration with chitosan-modified cellulose paper, the CV redox peak showed a significant attenuation, while Rct increased significantly to 2.99 kΩ. This phenomenon may be due to the introduction of a poorly conductive paper substrate, hindering electron transfer on the electrode surface. After immobilizing the trapping probe (OA-BSA, 1 mg / mL) in the T region, the redox peak current further decreased, while Rct increased to 3.32 kΩ, possibly due to the increased electron transport resistance from the OA-BSA conjugate. Notably, when the immune complex was fully formed, the redox peak current showed a recovery trend, with Rct significantly decreasing to 1.17 kΩ, indicating enhanced electron transport mediated by the Fc-NH2 labeling site. Furthermore, DPV analysis of the T region showed a significant shift in the electrochemical reaction after immune complex formation. Specifically, before the immune reaction, the T region, without [Fe(CN)6], showed a significant change in the electrochemical reaction. 3− / 4− The near-flat baseline observed in the redox medium indicates minimal background signal. The hollow Cu / Zn-MOF@Au / Fc-mAb complex, upon specific binding to the immobilized capture probe, exhibited a characteristic Fc oxidation peak at 0.26 V (relative to Ag / AgCl), confirming successful specific immunoassay. To further determine the optimal detection time, the current response in the T region was systematically monitored from 5 to 25 min. Signal saturation was achieved at 20 min, with a current plateau of 12.18 μA. Beyond 20 min, the signal enhancement was minimal, but the signal dispersion significantly increased, indicating compromised detection stability. These findings demonstrate the successful construction of the dual-mode LFA electrochemical detection module. Example 10

[0027] To examine the performance of the colorimetric / electrochemical dual-mode test strip in Example 5, the test strip constructed in Example 5 was used with OA as a model for performance verification. (Reference) Figure 5 OA competes with the OA-BSA conjugate immobilized in the T region for binding to the hollow Cu / Zn-MOF@Au / Fe-mAb probe. For example... Figure 5As shown in b, in continuous standard samples, the brown spots in the T region gradually weakened with increasing OA concentration. Specifically, in the blank sample, strong color development appeared in the T region due to the highest probe capture. In contrast, in OA-containing samples, the color development was weakened because free OA occupied the probe binding sites, causing more probe to bypass the T region. Complete color disappearance was observed at ≥50 ng / mL, while the visual detection limit reached 2 ng / mL, and the signal was clearly distinguishable from the blank. Figure 5 As shown, quantitative analysis further revealed a semi-logarithmic relationship between the optical density ratio in region T (T / T0, where T is the density of sample region T and T0 is the blank) and the OA concentration (0.2~100 ng / mL), exhibiting an S-shaped curve. Within the range of 2~50 ng / mL, the two showed a linear correlation (…). Figure 5 d). The limit of detection (LOD) calculated based on 3δ / S is 0.89 ng / mL. Although the colorimetric module has a detection limit of 2 ng / mL, allowing for rapid visual screening and semi-quantitative analysis down to 0.89 ng / mL, its linear range (2–50 ng / mL) is limited by optical signal saturation at higher concentrations. Figure 5 As shown, to overcome this limitation and achieve ultrasensitive quantification over a wider concentration range, an electrochemical mode was employed to test current changes in the range of 0–100 ng / mL, where the redox current of the hollow Cu / Zn-MOF@Au / Fe-mAb probe provides a linear response unaffected by optical interference. Figure 5 As shown, within the range of 0.2–80 ng / mL, the calibration plots revealed a linear correlation between the current difference ΔI (blank vs. sample) and the logarithm of the OA concentration (R0). 2 = 0.9957). The sensitivity of the electrochemical mode was improved by 15 times, with a detection limit of 0.06 ng / mL.

Claims

1. A colorimetric / electrochemical dual-mode test strip for detecting OA, characterized in that, The colorimetric / electrochemical dual-mode test strip for detecting OA consists of a dual-functional signal probe, a chromatographic matrix, and an integrated detection module; the preparation method of the dual-functional signal probe includes the following steps: S1 Zinc acetate dihydrate, copper acetylacetonate, and 2-imidazolium formaldehyde were added to the reactor in a molar ratio of 2:1:

2. The reaction was carried out at 80-90℃ for 20-24 hours. Then, 150-250 mg of polyvinylpyrrolidone (PVP) was added to adjust the MOF particle size to 60-150 nm. After centrifugation, washing with ethanol, and vacuum drying, hollow Cu / Zn-MOF was obtained. S2. Hollow Cu / Zn-MOF is dispersed in chloroauric acid solution to adsorb Au. 3+ After 12-24 hours of adsorption, sodium borohydride at a concentration of 1-2 mg / mL is added as a reducing agent, and the reaction is carried out for 20-40 minutes at a temperature of 4-25℃ to obtain hollow Cu / Zn-MOF@Au. S3 Hollow Cu / Zn-MOF@Au was activated at pH 5.5-6.5 with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide to activate the surface carboxyl groups. After activation, it was subjected to an amidation reaction with Fe-NH2 and reacted at 4-25℃ for 8-12 hours to obtain a bifunctional signal probe composed of gold nanoparticles and aminoferrocene loaded on hollow bimetallic Cu / Zn-MOF-derived nanohybrid material. The chromatography matrix is ​​chitosan-modified Whatman No. 1 paper with a chitosan concentration of 0.01~0.05% (w / v). The integrated detection module is a circular screen-printed electrode array with a diameter of 4~10 mm, used for synchronous acquisition of electrochemical signals.

2. The colorimetric / electrochemical dual-mode test strip for detecting OA as described in claim 1, characterized in that, The preparation method of the colorimetric / electrochemical dual-mode test strip for detecting OA includes the following steps: S4 disperses a bifunctional signal probe with a concentration of 0.1~0.5 mg / mL and a volume of 5~15 μL on glass fiber, and obtains a probe binding pad after drying; S5 uses chitosan to modify Whatman No. 1 paper, which is then dried to obtain the chromatography matrix; S6 involves immobilizing capture antibody (0.5–1 mg / mL, 0.5–1.5 μL) and goat anti-mouse IgG (0.5–1 mg / mL, 0.5–1.5 μL) onto a chromatography matrix to obtain the detection zone and the quality control zone, respectively. S7 sequentially stacks the sample pad, binding pad, chromatography matrix, and absorption pad onto a polyvinyl chloride backing plate, and embeds an integrated detection module below the detection area to obtain the colorimetric / electrochemical dual-mode test strip.

3. The colorimetric / electrochemical dual-mode test strip for detecting OA as described in claim 1, characterized in that, The detection method for the colorimetric / electrochemical dual-mode test strip for detecting OA includes the following steps: S8 dilutes the known concentration of the OA target analyte into different concentration gradients with the test solution, then drops the diluted target analyte onto the sample pad of the immunochromatographic test strip, and then measures the colorimetric intensity and electrochemical signal of the detection area, and calculates the intensity ratio and current difference, and establishes standard curves of intensity ratio and current difference versus target analyte concentration, respectively. S9 adds the sample to be tested onto the sample pad of the immunochromatographic test strip. After 10-20 minutes, the color intensity of the detection area is measured by a smartphone, and the electrochemical signal is measured by a portable potentiostat and terminal device. S10 calculates the colorimetric intensity ratio and current difference, and compares them with the standard curve to obtain the concentration of OA in the sample to be tested.

4. The detection method for OA using a colorimetric / electrochemical dual-mode test strip as described in claim 3, characterized in that, In step S8, the OA concentration gradient range is 0.2~100 ng / mL, and the sample volume is 50~100 μL; the test solution is 0.1M PBS buffer.

5. The detection method for OA using a colorimetric / electrochemical dual-mode test strip as described in claim 3, characterized in that, In step S9, the electrochemical signal is a differential pulse voltammetry signal, and the current difference is ∆I.

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