A photoelectrochemical-visualization dual-mode sensor for detecting Cry1Ab protein based on proximity hybridization technology

Abstract

The present invention belongs to the field of biosensor technology and discloses a method for detecting Cry1Ab protein using a photoelectrochemical-visualization dual-mode sensor based on close-range hybridization technology. Through the regulation of close-range hybridization technology, gold nanoparticles AuNPs with localized plasma resonance effect and catalytic performance are introduced as signal probes, thereby realizing PEC-visualization dual-mode detection based on a single sensitive element. With the assistance of Cry1Ab and antibody-labeled single-stranded DNA (Ab2-S2), the two probes (Ab1-S1-AuNPs and Ab2-S2) undergo close hybridization, causing Ab1-S1-AuNPs to be released from the electrode interface, weakening the PEC enhancement effect and significantly reducing the photocurrent signal. At the same time, the released Ab1-S1-AuNPs catalyze the color development of the chromogenic substrate. A portable colorimeter is introduced to transmit the solution color to the mobile phone software, and the captured color is converted into RGB digital signals to realize accurate visual analysis of Cry1Ab. This PEC-visualization dual-mode sensing strategy can be used for preliminary screening of on-site monitoring and can also be extended to high-sensitivity analysis of PEC.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and particularly relates to a method for detecting Cry1Ab protein by constructing a photoelectrochemical-visualization dual-mode sensor based on close-range hybridization technology to drive a DNA-antibody complex. Background Art

[0002] The Cry1Ab protein, produced by the Gram-positive bacillus Bacillus thuringiensis, is one of the most widely distributed transgenic proteins in genetically modified crops, such as corn, rice, and cotton. Currently, many countries have implemented mandatory labeling measures for genetically modified products. Therefore, achieving simple, rapid, and accurate detection of the Cry1Ab protein is crucial for the safety and regulation of genetically modified products. In recent years, the combination of visualization and photoelectrochemical (PEC) technology has attracted increasing attention due to its ability to achieve rapid on-site determination and sensitive quantitative detection. The difference in energy form significantly reduces background signals, giving this emerging visualization and PEC dual-mode biosensing technology advantages such as low instrument cost, ease of miniaturization, and high sensitivity. However, achieving visualization and PEC dual-mode detection based on a single sensor while freely switching between different modes according to test requirements remains a challenging task.

[0003] Currently, antibodies that recognize Cry1Ab proteins are easily constrained by the electrode sensing interface, making it difficult to freely control the antibody at this interface. Inspired by proximity hybridization technology for nucleic acid-driven probes, a DNA-assisted antibody detection method was developed. Chen Jinhua's research group utilized proximity hybridization to trigger the target, driving the antibody from the sensing platform via DNA, thereby freeing the antibody from the interface and enabling sensitive detection of the target. Therefore, using proximity hybridization to drive a multifunctional signaling probe carrying an antibody has the potential to achieve PEC-visualization synergistic detection based on a single sensor. The selection of a multifunctional signaling probe is essential for establishing dual-mode detection. By controlling proximity hybridization, gold nanoparticles (AuNPs) with localized plasmon resonance (LSPR) and catalytic properties were introduced as signaling probes, enabling PEC-visualization dual-mode detection based on a single sensor. Traditional detection methods rely solely on visual judgment and are susceptible to user interpretation. Therefore, a colorimeter, which is immune to operator subjective interpretation and external environmental noise, was introduced to further improve the accuracy of visual detection.

[0004] Based on the above research, we developed a PEC-visualization dual-mode biosensor based on proximity hybridization technology for convenient and sensitive detection of Cry1Ab protein in genetically modified crops. Summary of the Invention

[0005] The present invention aims to use close-range hybridization technology to drive the antibody-DNA complex carrying the signal probe to develop a PEC-visualization dual-mode sensor based on a single sensitive element for Cry1Ab protein detection. First, through hybridization between single-stranded DNA S0 and S1, the primary antibody-labeled S1-AuNPs (Ab1-S1-AuNPs) are introduced into the sensing interface as a multifunctional signal probe. At this time, AuNPs with LSPR effect approach cadmium selenide quantum dots (CdSe QDs) to improve the electron-hole separation efficiency, thereby amplifying the PEC signal. With the assistance of Cry1Ab and the secondary antibody-labeled single-stranded DNA S2 (Ab2-S2), the two probes (Ab1-S1-AuNPs and Ab2-S2) undergo proximity hybridization, causing Ab1-S1-AuNPs to be released from the electrode interface, weakening the PEC enhancement effect, and significantly reducing the photocurrent signal. Simultaneously, the released Ab1-S1-AuNPs catalyze the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) to form a colored product. A portable colorimeter with Bluetooth transmission was introduced to transmit the solution color to a smartphone app. Finally, the captured color was converted into an RGB digital signal via the mobile app, enabling precise visual analysis of Cry1Ab. Using Cry1Ab as a proof-of-concept, this PEC-visualization dual-mode sensing strategy can be used for preliminary screening in field monitoring and can also be expanded to highly sensitive analysis in the laboratory.

[0006] The method for detecting Cry1Ab protein based on a photoelectrochemical-visualization dual-mode sensor using proximity hybridization technology comprises the following steps:

[0007] (1) Preparation of cadmium selenide quantum dots (CdSe QDs) nanomaterials using microwave method:

[0008] First, under nitrogen protection, 7.9 mg of selenium powder and 7.6 mg of sodium borohydride were dissolved in 5 mL of ultrapure water to obtain a fresh sodium selenide NaHSe precursor solution; then, under nitrogen protection, 45.7 mg of chromium chloride CdCl2·2.5H2O was dissolved in 50 mL of H2O, 44 μL of 3-mercaptopropionic acid MPA was added as a stabilizer, and the pH of the solution was adjusted to 11 with 1 M sodium hydroxide. After nitrogen was passed through for 30 minutes, freshly prepared NaHSe was quickly added to the above solution. Subsequently, the mixed solution was reacted at 100°C in a microwave synthesizer for 4 hours to obtain CdSe QDs. Finally, the product was purified three times with anhydrous ethanol, then centrifuged, washed, dried at 50°C, and redispersed in ultrapure water and stored at 4°C in the dark.

[0009] (2) Preparation of gold nanoparticles Au NPs:

[0010] 200 μL of 0.1 M HAuCl4·3H2O solution was added to 25 mL of H2O and heated to boiling at 150 °C. Then, 250 μL of 100 mg mL -1 Trisodium citrate was quickly added to the mixture and reacted for 15 min to obtain a bright red AuNPs solution, which was stored at 4 °C in the dark;

[0011] (3) Preparation of Ab1-S1-AuNPs and Ab2-S2 composite materials respectively:

[0012] 40 μg mL -1 The primary antibody Ab1 of Cry1Ab was reacted with 2.5% glutaraldehyde GA at 4°C for 15 min to obtain GA-Ab1; then, 250 μL of 20 μg mL -1 GA-Ab1 was mixed with 250 μL of 4 μM S1 under stirring for 30 minutes to obtain Ab1-S1 through chemical cross-linking reaction between GA and amino-modified S1. Subsequently, 1 mL of 6 nM AuNPs was added to the Ab1-S1 solution and incubated in the dark for 16 hours. Finally, the solution was centrifuged at 10,000 rpm for 10 minutes to remove excess Ab1-S1, and the precipitate was redispersed in 500 μL of ultrapure water.

[0013] The secondary antibody-labeled S2, i.e., Ab2-S2, was synthesized according to the above-mentioned Ab1-S1 synthesis steps;

[0014] The obtained Ab1-S1-AuNPs and Ab2-S2 were stored at 4 °C for further use.

[0015] (4) Pretreatment of ITO glass electrode: The ITO glass electrode with a diameter of 6 mm was boiled in 1 M NaOH solution for 20 min, then ultrasonicated in anhydrous ethanol and ultrapure water for 15 min, and finally dried in air.

[0016] (5) modifying the CdSe QDs nanomaterial prepared in step (1) onto the surface of the indium tin oxide glass ITO electrode pretreated in step (4) and drying at room temperature. At this time, the product is marked as CdSe QDs / ITO;

[0017] In step (5), the concentration of CdSe QDs is 0.1-1.25 mg mL -1 , the dosage is 20μL.

[0018] (6) Modifying the surface of the electrode obtained in step (5) with single-stranded DNA S0, incubating the electrode at a certain temperature for a period of time, and then washing the product with PBS. At this time, the product is labeled as S0 / CdSe QDs / ITO;

[0019] In step (6), the concentration of S0 DNA is 0.1-2.5 μM, the dosage is 15 μL; the reaction temperature is 4° C., and the reaction time is 12 h.

[0020] (7) Modifying the sensor surface obtained in step (6) with 6-mercapto-1-hexanol MCH to block unbound nonspecific sites, and then washing the product with PBS. At this time, the product is labeled as MCH / S0 / CdSe QDs / ITO;

[0021] In step (7), the MCH concentration was 1 mM, the dosage was 15 μL, the reaction temperature was room temperature, and the reaction time was 1 h.

[0022] (8) Modifying the Ab1-S1-AuNPs material prepared in step (3) onto the surface of the sensor prepared in step (7), incubating the product at a certain temperature for a period of time, and then washing the product with PBS. At this time, the product is labeled as Ab1-S1-AuNPs / MCH / S0 / CdSe QDs / ITO;

[0023] In step (8), the amount of Ab1-S1-AuNPs used was 15 μL, the reaction temperature was 37° C., and the reaction time was 2 h.

[0024] (9) Ab2-S2 prepared in step (3) and different concentrations of Cry1Ab protein were added dropwise to the electrode surface prepared in step (8) and incubated for a period of time. At this time, the product was labeled Cry1Ab+Ab2-S2 / Ab1-S1-AuNPs / MCH / S0 / CdSe QDs / ITO;

[0025] In step (9), the concentration of Ab2-S2 is 1 μM and the dosage is 7.5 μL; the concentration of Cry1Ab is 0.001-100 ng mL -1 The dosage was 7.5 μL; the reaction temperature was 37°C and the reaction time was 1 h.

[0026] (10) The sensor prepared in step (9) was continuously cleaned with a NaAc-HAc buffer solution on the surfaces of the two ITO electrodes, and the cleaned sensor was subjected to a photoelectrochemical test; H2O2 and TMB were added to the mixed solution collected after cleaning, and the solution was placed in a customized quartz tube, and the RGB color was measured using a colorimeter.

[0027] In step (10), the pH is 4, the volume of NaAc-HAc is 30 μL, and the concentration is 0.2 M; the volume of H2O2 is 15 μL, and the concentration is 4 M; the volume of TMB is 15 μL, and the concentration is 8 mM; and the reaction time is 10 min.

[0028] In step (10), during the photoelectrochemical test, the cleaned sensor is used as the working electrode, the saturated Ag / AgCl electrode is used as the reference electrode, and the platinum wire electrode is used as the counter electrode. The photoelectrochemical signal is recorded and detected by a PLS-FX300HU xenon lamp and an Autolab PGSTAT 302N electrochemical workstation. The test is carried out in a 0.1 M PBS (pH = 7.4) buffer solution containing 0.1 M ascorbic acid (AA), and the applied bias voltage is 0 V.

[0029] The sequence of the present invention is as follows:

[0030] S0:5′-CGC GTT AAC ATA CAA TAG ATC GCG-(CH2)6-SH-3′

[0031] S1:5′-HS-GCG GAT CTA TTG TAT CAC ATA TTT TTT TTT TTT TTT TTT CAC CGTATG CTA CTG TAG AT-NH2-3′

[0032] S2:5′-NH2-TAG GAA AAG GAG GAG GGT GGT TTT TTT TTT TTT TTT TTT TTA GATACA ATA GAT C-3′

[0033] Beneficial effects of the present invention:

[0034] (1) Using close-range hybridization technology to drive the antibody-DNA complex, the antibody is released from the interface and the free switching of PEC-visualization dual-mode signals is achieved.

[0035] (2) AuNPs, as signal probes, have LSPR effect and good catalytic performance, affecting PEC and visualizing signal changes.

[0036] (3) The present invention introduces a portable colorimeter with Bluetooth transmission mode to transmit the solution color to a smart phone, which is converted into an RGB digital signal to achieve accurate visual analysis of Cry1Ab.

[0037] (4) The present invention introduces a specific recognition antibody for the Cry1Ab protein, improves the selectivity of the dual-mode PEC-visualization sensor, reduces the interference of other proteins in genetically modified crops, and realizes the specific analysis of the Cry1Ab protein.

[0038] (5) The PEC-visualization dual-mode sensor constructed in the present invention realizes convenient and sensitive detection of Cry1Ab with high sensitivity, good selectivity, and wide linear range, with a detection range of 0.001-100 ng mL -1. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the construction of the photoelectrochemical-visualization dual-mode sensor and the detection of Cry1Ab.

[0040] Figure 2 (A) Electrochemical impedance spectroscopy (EIS) and (B) photocurrent response diagram, including (a) ITO, (b) CdSe QDs / ITO, (c) S0 / CdSe QDs / ITO, (d) MCH / S0 / CdSe QDs / ITO, (e) Ab1-S1-AuNPs / MCH / S0 / CdSe QDs / ITO, and (f) Cry1Ab+Ab2-S2 / Ab1-S1-AuNPs / MCH / S0 / CdSe QDs / ITO.

[0041] Figure 3 (A)(a)0ng mL -1 Cry1Ab,(b)1ng mL -1 Cry1Ab,(c)100ng mL -1 UV-visible absorption spectrum of Cry1Ab in TMB-H2O2 mixed solution, (B) visualization test steps and corresponding photos.

[0042] Figure 4 (A) Visual color response of the sensor to different concentrations of Cry1Ab (a to h: 0, 0.1, 0.5, 1, 5, 10, 50, 100 ng mL -1 (B) Calibration curve for detecting Cry1Ab (△C vs. logarithm of Cry1Ab concentration). (C) Reproducibility of six parallel measurements. (D) Sensor response to 100 ng mL -1 Visual selectivity study of Cry1Ab, the interfering substances are PAT / bar protein, PAT / pat protein, CP4-EPSPS protein, and their mixture.

[0043] Figure 5 (A) PEC responses of the sensor to different concentrations of Cry1Ab (a to j: 0, 0.001, 0.005, 0.01, 0.05, 0.1, 1, 10, 50, 100 ng mL -1 (B) Linear regression curve of Cry1Ab detection (I vs. logarithm of Cry1Ab concentration). (C) Reproducibility of six parallel measurements. (D) Sensor response to 100 ng mL -1 Photoelectrochemical selectivity study of Cry1Ab, the interfering substances were PAT / bar protein, PAT / pat protein, CP4-EPSPS protein, and their mixture. DETAILED DESCRIPTION

[0044] The present invention will be further explained below with reference to specific embodiments and accompanying drawings.

[0045] Example 1

[0046] according to Figure 1 Described preparation process:

[0047] (1) Preparation of CdSe QDs nanomaterials:

[0048] CdSe QDs were synthesized using a microwave method. First, under nitrogen protection, 7.9 mg of Se powder and 7.6 mg of NaBH4 were dissolved in 5 mL of H2O to obtain a fresh NaHSe precursor solution. Then, under nitrogen protection, 45.7 mg of CdCl2·2.5H2O was dissolved in 50 mL of H2O, and 44 μL of MPA was added as a stabilizer. The pH of the solution was adjusted to 11 with 1 M NaOH. After nitrogen was passed through for 30 minutes, freshly prepared NaHSe was quickly added to the above solution. Subsequently, the mixed solution was reacted at 100°C in a microwave synthesizer for 4 hours to obtain CdSe QDs. Finally, the product was purified three times with anhydrous ethanol, then centrifuged, washed, dried at 50°C, and redispersed in ultrapure water and stored at 4°C in the dark.

[0049] (2) Preparation of AuNPs nanomaterials:

[0050] 200 μL of 0.1 M HAuCl4·3H2O solution was added to 25 mL of H2O and heated to boiling at 150°C. Then, 250 μL of 100 mg mL -1 Trisodium citrate was quickly added to the mixture and reacted for 15 min to obtain a bright red AuNPs solution, which was stored at 4°C in the dark.

[0051] (3) Preparation of Ab1-S1-AuNPs and Ab2-S2 composites:

[0052] 40 μg mL -1 Ab1 was reacted with 2.5% GA at 4°C for 15 min to obtain GA-Ab1. Then, 250 μL of 20 μg mL -1GA-Ab1 was mixed with 250 μL of 4 μM S1 under stirring for 30 minutes. Ab1-S1 was obtained through chemical cross-linking of GA with the amino-modified S1. Subsequently, 1 mL of 6 nM AuNPs was added to the Ab1-S1 solution and incubated in the dark for 16 hours. Finally, the solution was centrifuged at 10,000 rpm for 10 minutes to remove excess Ab1-S1, and the precipitate was redispersed in 500 μL of HO.

[0053] Ab2-S2 was synthesized according to the above synthesis steps of Ab1-S1. The obtained Ab1-S1-AuNPs and Ab2-S2 were stored at 4°C for further use.

[0054] (4) Pretreatment of ITO electrodes: The ITO electrodes (6 mm in diameter) were boiled in 1 M NaOH solution for 20 min, then ultrasonicated in anhydrous ethanol and ultrapure water for 15 min, respectively, and finally dried in air.

[0055] (5) Add 20 μL of 0.5 mg mL prepared in step (1) -1 CdSe QDs nanomaterials are modified onto the surface of the ITO electrode pretreated in step (4) and dried at room temperature. At this point, the product is labeled as CdSe QDs / ITO;

[0056] (6) 15 μL of 1 μM single-stranded S0 DNA was modified on the electrode surface and incubated at 4°C for 12 h. The product was then washed with PBS. At this point, the product was labeled S0 / CdSe QDs / ITO.

[0057] (7) Modify the sensor surface obtained in step (6) with 15 μL of 1 mM MCH to block unbound nonspecific sites, and then wash the product with PBS. At this time, the product is labeled as MCH / S0 / CdSe QDs / ITO;

[0058] (8) 15 μL of Ab1-S1-AuNPs prepared in step (3) was modified onto the sensor surface prepared in step (7), and the mixture was incubated at 37°C for 2 h. The product was then washed with PBS. At this point, the product was labeled Ab1-S1-AuNPs / MCH / S0 / CdSe QDs / ITO.

[0059] (9) 15 μL of 1 μM Ab2-S2 prepared in step (3) and different concentrations of Cry1Ab protein were added dropwise to the electrode surface prepared in step (8) and incubated at 37°C for 1 h. At this time, the product was labeled Cry1Ab+Ab2-S2 / Ab1-S1-AuNPs / MCH / S0 / CdSe QDs / ITO;

[0060] (10) The sensor prepared in step (9) was rinsed continuously on the two ITO electrodes with 30 μL of 0.2 M NaAc-HAc (pH 4) buffer solution, and the mixed solution was collected. Then, 15 μL of 4 M H₂O₂ and 15 μL of 8 mM TMB were added to the solution. After a 10-min reaction, the solution was placed in a custom quartz tube, and color (RGB) was measured using a colorimeter.

[0061] (11) The sensor cleaned with the buffer solution in step (10) was subjected to a photoelectrochemical test. The sensor prepared in the present invention was used as the working electrode, a saturated Ag / AgCl electrode was used as the reference electrode, and a platinum wire electrode was used as the counter electrode. The photoelectrochemical signal was recorded and detected using a PLS-FX300HU xenon lamp and an Autolab PGSTAT 302N electrochemical workstation. The test was conducted in a 0.1 M PBS (pH = 7.4) buffer solution containing 0.1 M AA, with an applied bias voltage of 0 V.

[0062] The surface of the immunosensor prepared above was modified with Cry1Ab protein at different concentrations. The binding time was 60 min at room temperature, and the Cry1Ab concentration was 0.001 ng mL -1 ,0.005ng mL -1 ,0.01ng mL -1 ,0.05ng mL -1 ,0.1ngmL -1 ,0.5ng mL -1 ,1ng mL -1 ,5ng mL -1 ,10ng mL -1 ,50ng mL -1 ,100ng mL -1 .

[0063] Example 2

[0064] according to Figure 1 Described preparation process:

[0065] (1) Preparation of CdSe QDs nanomaterials:

[0066] CdSe QDs were synthesized using a microwave method. First, under nitrogen protection, 7.9 mg of Se powder and 7.6 mg of NaBH4 were dissolved in 5 mL of H2O to obtain a fresh NaHSe precursor solution. Then, under nitrogen protection, 45.7 mg of CdCl2·2.5H2O was dissolved in 50 mL of H2O, and 44 μL of MPA was added as a stabilizer. The pH of the solution was adjusted to 11 with 1 M NaOH. After nitrogen was passed through for 30 minutes, freshly prepared NaHSe was quickly added to the above solution. Subsequently, the mixed solution was reacted at 100°C in a microwave synthesizer for 4 hours to obtain CdSe QDs. Finally, the product was purified three times with anhydrous ethanol, then centrifuged, washed, dried at 50°C, and redispersed in ultrapure water and stored at 4°C in the dark.

[0067] (2) Preparation of AuNPs nanomaterials:

[0068] 200 μL of 0.1 M HAuCl4·3H2O solution was added to 25 mL of H2O and heated to boiling at 150°C. Then, 250 μL of 100 mg mL -1 Trisodium citrate was quickly added to the mixture and reacted for 15 min to obtain a bright red AuNPs solution, which was stored at 4°C in the dark.

[0069] (3) Preparation of Ab1-S1-AuNPs and Ab2-S2 composites:

[0070] 40 μg mL -1 Ab1 was reacted with 2.5% GA at 4°C for 15 min to obtain GA-Ab1. Then, 250 μL of 20 μg mL -1 GA-Ab1 was mixed with 250 μL of 4 μM S1 under stirring for 30 minutes. Ab1-S1 was obtained through chemical cross-linking of GA with the amino-modified S1. Subsequently, 1 mL of 6 nM AuNPs was added to the Ab1-S1 solution and incubated in the dark for 16 hours. Finally, the solution was centrifuged at 10,000 rpm for 10 minutes to remove excess Ab1-S1, and the precipitate was redispersed in 500 μL of HO.

[0071] Ab2-S2 was synthesized according to the above synthesis steps of Ab1-S1. The obtained Ab1-S1-AuNPs and Ab2-S2 were stored at 4°C for further use.

[0072] (4) Pretreatment of ITO electrodes: The ITO electrodes (6 mm in diameter) were boiled in 1 M NaOH solution for 20 min, then ultrasonicated in anhydrous ethanol and ultrapure water for 15 min, respectively, and finally dried in air.

[0073] (5) Add 20 μL of 0.1 mg mL prepared in step (1) -1 CdSe QDs nanomaterials are modified onto the surface of the ITO electrode pretreated in step (4) and dried at room temperature. At this point, the product is labeled as CdSe QDs / ITO;

[0074] (6) 15 μL of 0.1 μM single-stranded S0 DNA was modified on the electrode surface and incubated at 4 °C for 12 h. The product was then washed with PBS. At this point, the product was labeled S0 / CdSe QDs / ITO.

[0075] (7) Modify the sensor surface obtained in step (6) with 15 μL of 1 mM MCH to block unbound nonspecific sites, and then wash the product with PBS. At this time, the product is labeled as MCH / S0 / CdSe QDs / ITO;

[0076] (8) 15 μL of Ab1-S1-AuNPs prepared in step (3) was modified onto the sensor surface prepared in step (7), and the mixture was incubated at 37°C for 2 h. The product was then washed with PBS. At this point, the product was labeled Ab1-S1-AuNPs / MCH / S0 / CdSe QDs / ITO.

[0077] (9) 15 μL of 1 μM Ab2-S2 prepared in step (3) and different concentrations of Cry1Ab protein were added dropwise to the electrode surface prepared in step (8) and incubated at 37°C for 1 h. At this time, the product was labeled Cry1Ab+Ab2-S2 / Ab1-S1-AuNPs / MCH / S0 / CdSe QDs / ITO;

[0078] (10) The sensor prepared in step (9) was rinsed continuously on the two ITO electrodes with 30 μL of 0.2 M NaAc-HAc (pH 4) buffer solution, and the mixed solution was collected. Then, 15 μL of 4 M H₂O₂ and 15 μL of 8 mM TMB were added to the solution. After a 10-min reaction, the solution was placed in a custom quartz tube, and color (RGB) was measured using a colorimeter.

[0079] (11) The sensor cleaned with the buffer solution in step (10) was subjected to a photoelectrochemical test. The sensor prepared in the present invention was used as the working electrode, a saturated Ag / AgCl electrode was used as the reference electrode, and a platinum wire electrode was used as the counter electrode. The photoelectrochemical signal was recorded and detected using a PLS-FX300HU xenon lamp and an Autolab PGSTAT 302N electrochemical workstation. The test was conducted in a 0.1 M PBS (pH = 7.4) buffer solution containing 0.1 M AA, with an applied bias voltage of 0 V.

[0080] The surface of the immunosensor prepared above was modified with Cry1Ab protein at different concentrations. The binding time was 60 min at room temperature, and the Cry1Ab concentration was 0.001 ng mL -1 ,0.005ng mL -1 ,0.01ng mL -1 ,0.05ng mL -1 ,0.1ngmL -1 ,0.5ng mL -1 ,1ng mL -1 ,5ng mL -1 ,10ng mL -1 ,50ng mL -1 ,100ng mL -1 .

[0081] Example 3

[0082] according to Figure 1 Described preparation process:

[0083] (1) Preparation of CdSe QDs nanomaterials:

[0084] CdSe QDs were synthesized using a microwave method. First, under nitrogen protection, 7.9 mg of Se powder and 7.6 mg of NaBH4 were dissolved in 5 mL of H2O to obtain a fresh NaHSe precursor solution. Then, under nitrogen protection, 45.7 mg of CdCl2·2.5H2O was dissolved in 50 mL of H2O, and 44 μL of MPA was added as a stabilizer. The pH of the solution was adjusted to 11 with 1 M NaOH. After nitrogen was passed through for 30 minutes, freshly prepared NaHSe was quickly added to the above solution. Subsequently, the mixed solution was reacted at 100°C in a microwave synthesizer for 4 hours to obtain CdSe QDs. Finally, the product was purified three times with anhydrous ethanol, then centrifuged, washed, dried at 50°C, and redispersed in ultrapure water and stored at 4°C in the dark.

[0085] (2) Preparation of AuNPs nanomaterials:

[0086] 200 μL of 0.1 M HAuCl4·3H2O solution was added to 25 mL of H2O and heated to boiling at 150°C. Then, 250 μL of 100 mg mL -1 Trisodium citrate was quickly added to the mixture and reacted for 15 min to obtain a bright red AuNPs solution, which was stored at 4°C in the dark.

[0087] (3) Preparation of Ab1-S1-AuNPs and Ab2-S2 composites:

[0088] 40 μg mL-1 Ab1 was reacted with 2.5% GA at 4°C for 15 min to obtain GA-Ab1. Then, 250 μL of 20 μg mL -1 GA-Ab1 was mixed with 250 μL of 4 μM S1 under stirring for 30 minutes. Ab1-S1 was obtained through chemical cross-linking of GA with the amino-modified S1. Subsequently, 1 mL of 6 nM AuNPs was added to the Ab1-S1 solution and incubated in the dark for 16 hours. Finally, the solution was centrifuged at 10,000 rpm for 10 minutes to remove excess Ab1-S1, and the precipitate was redispersed in 500 μL of HO.

[0089] Ab2-S2 was synthesized according to the above synthesis steps of Ab1-S1. The obtained Ab1-S1-AuNPs and Ab2-S2 were stored at 4°C for further use.

[0090] (4) Pretreatment of ITO electrodes: The ITO electrodes (6 mm in diameter) were boiled in 1 M NaOH solution for 20 min, then ultrasonicated in anhydrous ethanol and ultrapure water for 15 min, respectively, and finally dried in air.

[0091] (5) Add 20 μL of 1.25 mg mL prepared in step (1) -1 CdSe QDs nanomaterials are modified onto the surface of the ITO electrode pretreated in step (4) and dried at room temperature. At this point, the product is labeled as CdSe QDs / ITO;

[0092] (6) 15 μL of 2.5 μM single-stranded S0 DNA was modified on the electrode surface and incubated at 4 °C for 12 h. The product was then washed with PBS. At this point, the product was labeled S0 / CdSe QDs / ITO.

[0093] (7) Modify the sensor surface obtained in step (6) with 15 μL of 1 mM MCH to block unbound nonspecific sites, and then wash the product with PBS. At this time, the product is labeled as MCH / S0 / CdSe QDs / ITO;

[0094] (8) 15 μL of Ab1-S1-AuNPs prepared in step (3) was modified onto the sensor surface prepared in step (7), and the mixture was incubated at 37°C for 2 h. The product was then washed with PBS. At this point, the product was labeled Ab1-S1-AuNPs / MCH / S0 / CdSe QDs / ITO.

[0095] (9) 15 μL of 1 μM Ab2-S2 prepared in step (3) and different concentrations of Cry1Ab protein were added dropwise to the electrode surface prepared in step (8) and incubated at 37°C for 1 h. At this time, the product was labeled Cry1Ab+Ab2-S2 / Ab1-S1-AuNPs / MCH / S0 / CdSe QDs / ITO;

[0096] (10) The sensor prepared in step (9) was rinsed continuously on the two ITO electrodes with 30 μL of 0.2 M NaAc-HAc (pH 4) buffer solution, and the mixed solution was collected. Then, 15 μL of 4 M H₂O₂ and 15 μL of 8 mM TMB were added to the solution. After a 10-min reaction, the solution was placed in a custom quartz tube, and color (RGB) was measured using a colorimeter.

[0097] (11) The sensor cleaned with the buffer solution in step (10) was subjected to a photoelectrochemical test. The sensor prepared in the present invention was used as the working electrode, a saturated Ag / AgCl electrode was used as the reference electrode, and a platinum wire electrode was used as the counter electrode. The photoelectrochemical signal was recorded and detected using a PLS-FX300HU xenon lamp and an Autolab PGSTAT 302N electrochemical workstation. The test was conducted in a 0.1 M PBS (pH = 7.4) buffer solution containing 0.1 M AA, with an applied bias voltage of 0 V.

[0098] The surface of the immunosensor prepared above was modified with Cry1Ab protein at different concentrations. The binding time was 60 min at room temperature, and the Cry1Ab concentration was 0.001 ng mL -1 ,0.005ng mL -1 ,0.01ng mL -1 ,0.05ng mL -1 ,0.1ngmL -1 ,0.5ng mL -1 ,1ng mL -1 ,5ng mL -1 ,10ng mL -1 ,50ng mL -1 ,100ng mL -1 .

[0099] from Figure 2 (A) It can be seen that the ITO electrode shows a relatively small impedance value (curve a) due to its good electron transfer ability. When CdSe QDs are assembled on the electrode, R et The value increases (curve b), which is attributed to the weak conductivity of the semiconductor. After the gradual assembly of S0 (curve c) and MCH (curve d), due to the electrostatic repulsion of nucleic acids and the non-conductivity of organic molecules, Ret When the probe Ab1-S1-AuNPs hybridizes with S0, R et The value further increases (curve e), which is attributed to the steric hindrance of Ab1 and S1 and the electrostatic repulsion of negatively charged AuNPs. However, after incubation with Cry1Ab and Ab2-S2 solutions, R et The value decreased (curve f), which was due to the pull-off of Ab1-S1-AuNPs from the electrode surface. The EIS results showed that the PEC-visualization dual-mode biosensor was successfully constructed.

[0100] from Figure 2 (B) It can be seen that bare ITO has almost no photocurrent response (curve a). After modification with CdSe QDs, the photocurrent increases significantly due to the excellent photoelectric activity of CdSe QDs (curve b). When S0 (curve c) and MCH (curve d) are assembled on the electrode, the photocurrent output gradually decreases, which is due to the steric effect of DNA and organic molecules hindering the transfer of electron donor AA to the electrode. However, after incubation with Ab1-S1-AuNPs, the photocurrent response increases significantly (curve e), and its current value is 1.4 times that of CdSe QD. The reasons may be as follows: (1) Due to the hybridization of S1 and S0, the direct contact between AuNPs and CdSe QDs promotes the charge transfer between the two particles, thereby enhancing the anode photocurrent; (2) The LSPR effect of AuNPs enhances the local electric field and promotes the generation of electron-holes, resulting in an increase in the photocurrent response. When Cry1Ab and Ab2-S2 are present, Ab1-S1-AuNPs move away from the electrode surface and reduce the LSPR effect, resulting in a significant decrease in the photocurrent output (curve f).

[0101] from Figure 3 (A, B) It can be seen that when there is no target, no Ab1-S1-AuNPs are collected. After 10 minutes of reaction, the color of the TMB / H2O2 reaction system does not change when the colorimeter is detected. Figure 3 Inset a of (B), TMB / H2O2 solution showed low absorbance at 652 nm ( Figure 3 (A) curve a), which is attributed to the production of a small amount of oxidized TMB (oxTMB). - 1 After incubation with Cry1Ab, the released Ab1-S1-AuNPs catalyzed the oxidation of TMB in the presence of H2O2, and the solution changed from colorless to blue. Figure 3 (B) Illustration b. When the concentration of Cry1Ab was increased from 1 ng mL -1 Increase to 100 ng mL -1 When the absorbance at 652 nm is significantly enhanced ( Figure 3(A) curve c), the blue changes from light to dark, see Figure 3 (B) Illustration c. This visual detection has the obvious advantages of simple operation, low cost, and time saving, and can be widely used in preliminary screening on site.

[0102] like Figure 4 As shown in (A), as the concentration of Cry1Ab increases, Ab1-S1-AuNPs separate from the electrode, and the concentration of AuNPs with enzyme-like catalytic activity increases. Consequently, more H2O2 is decomposed into ·OH radicals, enhancing the catalytic oxidation of TMB and gradually deepening the blue-green color of the colorimetric card. Figure 4 (B) shows that there is a linear relationship between the visualized signal response (ΔC value) and the logarithm of the Cry1Ab concentration. The linear regression equation is ΔC = 65.3693lgc + 91.3529 (R 2 =0.9992), with a detection range of 0.1-100 ng mL -1 The limit of detection (LOD) was 0.1 ng mL -1 .from Figure 4 As can be seen in (C), the reproducibility of the sensor was tested by six parallel experiments, and the relative standard deviation (RSD) was 5.6%, indicating that its reproducibility is acceptable. Figure 4 As shown in (D), when the target compound (100 ng mL -1 In the presence of Cry1Ab, the sensor produced a significant change in signal response. However, when 10-fold concentrations of common interfering reagents (PAT / pat protein, PAT / bar protein, and CP4-EPSPS protein) were added, the signal response changed little compared to the blank. Therefore, the sensor exhibited excellent selectivity for Cry1Ab detection.

[0103] like Figure 5 As shown in (A), as the concentration of Cry1Ab increases, the photocurrent after adding the target gradually decreases. Figure 5 (B) is the linear regression curve of the photoelectric signal and the logarithm of Cry1Ab concentration. The results show that the linear range of the sensor for Cry1Ab detection is 0.001 ng mL -1 -100ng mL -1 The linear regression equation is I = -3.5719lg c + 15.8478, and the correlation coefficient (R 2 ) is 0.9984. Figure 5 As can be seen in (C), the reproducibility of the sensor is tested by six independent electrodes, and the RSD is 2.3%, indicating good reproducibility. Figure 5As shown in (D), the photoelectric signal generated by the sensor changes significantly only when the target is present. When common interfering reagents (PAT / pat protein, PAT / bar protein, CP4-EPSPS protein) are added, the photocurrent changes very little compared to the blank. Therefore, the sensor has good selectivity for Cry1Ab detection.

Claims

1. A method for detecting Cry1Ab protein using a photoelectrochemical-visualization dual-mode sensor based on close-range hybridization technology, characterized in that: Here are the steps: (1) preparing cadmium selenide quantum dot CdSe QDs nanomaterials for standby use; (2) preparing gold nanoparticles AuNPs for later use; (3) preparing Ab1-S1-AuNPs and Ab2-S2 composite materials respectively for later use; (4) The indium tin oxide glass (ITO) electrode is pre-treated and ready for use; (5) modifying the CdSe QDs nanomaterial prepared in step (1) onto the surface of the indium tin oxide glass ITO electrode pretreated in step (4) and drying at room temperature. At this time, the product is marked as CdSe QDs / ITO; (6) Modifying the surface of the electrode obtained in step (5) with single-stranded DNA S0, incubating the electrode at a certain temperature for a period of time, and then washing the product with PBS. At this time, the product is labeled as S0 / CdSe QDs / ITO; (7) Modifying the sensor surface obtained in step (6) with 6-mercapto-1-hexanol MCH to block unbound nonspecific sites, and then washing the product with PBS. At this time, the product is labeled as MCH / S0 / CdSe QDs / ITO; (8) Modifying the Ab1-S1-AuNPs material prepared in step (3) onto the surface of the sensor prepared in step (7), incubating the product at a certain temperature for a period of time, and then washing the product with PBS. At this time, the product is labeled as Ab1-S1-AuNPs / MCH / S0 / CdSe QDs / ITO; (9) Ab2-S2 prepared in step (3) and different concentrations of Cry1Ab protein were added dropwise to the electrode surface prepared in step (8) and incubated for a period of time. At this time, the product was labeled Cry1Ab+Ab2-S2 / Ab1-S1-AuNPs / MCH / S0 / CdSe QDs / ITO; (10) The sensor prepared in step (9) was continuously cleaned with a NaAc-HAc buffer solution on the surfaces of two ITO electrodes, and the cleaned sensor was subjected to a photoelectrochemical test; H2O2 and TMB were added to the mixed solution collected after cleaning, and the solution was placed in a customized quartz tube, and the color was measured RGB using a colorimeter; in, Antibodies Ab1 and Ab2 are used to specifically recognize Cry1Ab protein; S0:5′-CGC GTT AAC ATA CAA TAG ATC GCG-(CH2)6-SH-3′ S1:5′-HS-GCG GAT CTA TTG TAT CAC ATA TTT TTT TTT TTT TTT TTT CAC CGT ATGCTA CTG TAG AT-NH2-3′ S2: 5′-NH2-TAG GAA AAG GAG GAG GGT GGT TTT TTT TTT TTT TTT TTT TTA GATACAATA GAT C-3′.

2. The method according to claim 1, characterized in that In step (1), cadmium selenide quantum dot CdSe QDs nanomaterials are synthesized using a microwave method, the steps being: first, under nitrogen protection, 7.9 mg of selenium powder and 7.6 mg of sodium borohydride are dissolved in 5 mL of ultrapure water to obtain a fresh sodium selenide NaHSe precursor solution; then, under nitrogen protection, 45.7 mg of chromium chloride CdCl2·2.5H2O is dissolved in 50 mL of H2O, 44 μL of 3-mercaptopropionic acid MPA is added as a stabilizer, and the solution is adjusted to pH 11 with 1 M sodium hydroxide. After nitrogen is passed for 30 minutes, the freshly prepared NaHSe is quickly added to the above solution, and then the mixed solution is reacted at 100° C. for 4 hours in a microwave synthesizer to obtain CdSe QDs. Finally, the product is purified three times with anhydrous ethanol, then centrifuged and washed, dried at 50° C., and then dispersed in ultrapure water again, and stored at 4° C. in the dark.

3. The method according to claim 1, characterized in that In step (2), the preparation steps of gold nanoparticles Au NPs are as follows: 200 μL of 0.1M HAuCl4·3H2O solution was added to 25 mL of H2O and heated to boiling at 150°C, and then 250 μL of 100 mg·mL -1 Trisodium citrate was quickly added to the mixture and reacted for 15 min to obtain a bright red AuNPs solution, which was stored at 4 °C in the dark; In step (3), the preparation steps for preparing Ab1-S1-AuNPs and Ab2-S2 composite materials are as follows: 40 μg·mL -1 The primary antibody Ab1 of Cry1Ab was reacted with 2.5% glutaraldehyde GA at 4°C for 15 min to obtain GA-Ab1; then, 250 μL of 20 μg mL -1 GA-Ab1 was mixed with 250 μL of 4 μM S1 under stirring for 30 minutes to obtain Ab1-S1 through chemical cross-linking reaction between GA and amino-modified S1. Subsequently, 1 mL of 6 nM AuNPs was added to the Ab1-S1 solution and incubated in the dark for 16 hours. Finally, the solution was centrifuged at 10,000 rpm for 10 minutes to remove excess Ab1-S1, and the precipitate was redispersed in 500 μL of ultrapure water. The secondary antibody-labeled S2, i.e., Ab2-S2, was synthesized according to the above-mentioned Ab1-S1 synthesis steps; The obtained Ab1-S1-AuNPs and Ab2-S2 were stored at 4 °C for further use; In step (4), pretreatment of the indium tin oxide glass electrode ITO: boil the indium tin oxide glass electrode with a diameter of 6 mm in 1M NaOH solution for 20 minutes, then ultrasonicate in anhydrous ethanol and ultrapure water for 15 minutes, and finally dry in air.

4. The method according to claim 1, wherein In step (5), the concentration of CdSe QDs is 0.1-1.25 mg·mL -1 , the dosage is 20μL.

5. The method according to claim 1, wherein In step (6), the concentration of S0 DNA is 0.1-2.5 μM, the dosage is 15 μL; the reaction temperature is 4° C., and the reaction time is 12 h.

6. The method according to claim 1, wherein In step (7), the MCH concentration was 1 mM, the dosage was 15 μL, the reaction temperature was room temperature, and the reaction time was 1 h.

7. The method according to claim 1, characterized in that In step (8), the amount of Ab1-S1-AuNPs used was 15 μL, the reaction temperature was 37° C., and the reaction time was 2 h.

8. The method according to claim 1, characterized in that In step (9), the concentration of Ab2-S2 is 1 μM and the dosage is 7.5 μL; the concentration of Cry1Ab is 0.001-100 ng·mL -1 The dosage was 7.5 μL; the reaction temperature was 37°C and the reaction time was 1 h.

9. The method according to claim 1, characterized in that In step (10), the pH is 4, the volume of NaAc-HAc is 30 μL, and the concentration is 0.2 M; the volume of H2O2 is 15 μL, and the concentration is 4 M; the volume of TMB is 15 μL, and the concentration is 8 mM; and the reaction time is 10 min.

10. The method according to claim 1, characterized in that In step (10), during the photoelectrochemical test, the cleaned sensor is used as the working electrode, the saturated Ag / AgCl electrode is used as the reference electrode, and the platinum wire electrode is used as the counter electrode. The photoelectrochemical signal is recorded and detected by a PLS-FX300HU xenon lamp and an Autolab PGSTAT 302N electrochemical workstation. The test is performed in a 0.1 M PBS buffer solution containing 0.1 M ascorbic acid AA, with an applied bias voltage of 0 V and a pH of 7.4.

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