Method and application of an optoelectrochemical aptasensor for detecting rabies virus

Abstract

The present invention discloses a method and application for detecting rabies virus by an optoelectrochemical aptasensor. The aptasensor is prepared through the following steps: CdTe QDs are assembled on the surface of a carbon-doped TiO2 nanoparticle electrode by electrostatic adsorption; NH2-RNA-SH / Au NPs are immobilized on the TiO2:C-NP / CdTe electrode through a classical EDC coupling reaction between the carbonyl group on CdTe QDs and the amino group of the probe, thereby constructing the aptasensor TiO2:C-NP / CdTe / AuNPs / PRNA; then, monoethanolamine is used to block the unbound sites on the electrode surface to avoid false positives caused by non-specific binding. The present invention utilizes the efficient exciton energy transfer between cadmium telluride quantum dots and gold nanoparticles, and can quickly and reliably diagnose rabies virus, with the lowest detection concentration limit being approximately 2.6 ffu / mL.

Description

Technical Field

[0001] The present invention relates to a method for detecting rabies virus, and particularly to a method and application for detecting rabies virus by an optoelectrochemical aptasensor. Background Art

[0002] Rabies is an acute zoonotic disease with a mortality rate of almost 100%. Rapid diagnosis of rabies is an important step in preventing and controlling rabies. Currently, its detection methods include conventional detection methods and nucleic acid diagnosis methods. Among them, the conventional methods include direct fluorescent antibody test (DFAT) and mouse inoculation test (MIT). DFAT has high sensitivity and specificity, but requires trained personnel and expensive fluorescence microscopes. When the brain tissue begins to decompose, DFAT may also produce false negative results. MIT requires animal facilities and good containment facilities. The main disadvantage is the delay in obtaining results, which may even take 4 weeks. Therefore, there is an urgent need to develop methods for nucleic acid diagnosis of rabies virus.

[0003] Viral RNA quantitative PCR detection is another method for confirming whether it is a negative result of RABV. Viral RNA can be effectively quantified by quantitative PCR, such as reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and semi-nested RT-PCR (hnRT-PCR). The RABV genome contains the nucleoprotein gene (N), and the N gene has been identified as a diagnostic marker because it is the most conserved region in the RABV genome. Although the amplification methods are versatile, due to a single sequence mismatch between the primer or probe sequence designed according to the N gene and the target virus sequence, these amplification methods usually result in false negative results, thus changing the sensitivity of the test. In addition, these methods require expensive experimental equipment, professional personnel, and nucleic acid extraction, which limits the places where the detection can be applied.

[0004] Photoelectrochemical technology is a newly emerging and powerful analytical method in biological research. According to the source of the detection signal and the type of the signal, due to its superior performance such as convenient construction, wide detection range, and specific and sensitive analysis strategy, the amperometric photoelectrochemical sensor has developed rapidly. The detection of rabies virus nucleic acid in this experiment is based on this strategy. The two essential devices for constructing an amperometric photoelectrochemical sensor include: a biorecognition element (photoelectroactive material) and a biorecognition probe. When the amperometric photoelectrochemical sensor is excited by light, the photoelectroactive material generates photogenerated electrons that transfer directionally to the electrode of the sensor, forming a current signal, and the analyte is specifically detected through the change of the signal. Compared with electrochemical and optical methods, photoelectrochemical technology shows the advantages of simple equipment, low cost, and easy miniaturization. This strategy uses different forms of energy for excitation and detection, with lower background signals and higher sensitivity. Therefore, photoelectrochemical technology has attracted the interest of many researchers and has been successfully used for the analysis, detection, and identification in various biomedical fields. The biorecognition probe in the constructed device is basically the same in all types of biosensors, usually protein or nucleic acid molecules. Therefore, in order to improve the performance of the sensor, researchers have conducted a lot of research on the biorecognition element. The core of improving the sensor performance is to increase the generation of current signals. According to the current types of current generation strategies, there are mainly the following four types of sensors: sensors constructed based on the change of current signals generated by steric hindrance effects, sensors constructed based on the change of current signals generated by enzyme catalysis, sensors constructed by using a photoelectroactive material as a signal amplification element, and sensors constructed by using energy transfer. The sensor constructed in this experiment for detecting rabies virus nucleic acid is based on this principle.

[0005] For the sensor constructed by using energy transfer, when the photoelectroactive material on the sensor is excited by light, the energy generated by the excitation of the energy donor is transferred to the adjacent energy acceptor. This is the basic process of energy transfer. The two key words that need to be noted in this process are: one is the existence of an energy donor and an energy acceptor, and the energy relationship between the two is that one can emit and the other can absorb; the other is the positional relationship between the two is adjacent. Generally speaking, the generation of energy transfer in this type of sensor needs to meet these two conditions simultaneously: one is that the distance from the energy donor to the energy acceptor is between 10±2 nm; the other is that the emission spectrum of the energy donor overlaps with the absorption spectrum of the energy acceptor. Summary of the Invention

[0006] To solve the above problems, the present invention provides a method and application for detecting rabies virus using a photoelectrochemical aptasensor. This method utilizes the efficient exciton energy transfer between cadmium telluride quantum dots and gold nanoparticles, which is simple, low-cost, easy to miniaturize, and can diagnose rabies virus quickly and reliably.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] An optoelectrochemical aptasensor for detecting rabies virus is prepared by the following steps: CdTe QDs are assembled on the surface of a carbon-doped TiO2 nanoparticle (TiO2:C-NPs) electrode through electrostatic adsorption; NH2-RNA-SH / Au NPs are fixed on the TiO2:C-NP / CdTe electrode through a classical EDC coupling reaction between the carbonyl group on CdTe QDs and the amino group of the probe, thereby constructing the aptasensor TiO2:C-NP / CdTe / AuNPs / PRNA; immediately afterwards, monoethanolamine (MFA) is used to block the unbound sites on the electrode surface to avoid false positives caused by non-specific binding.

[0009] Furthermore, it specifically includes the following steps:

[0010] S1. Synthesis of CdTe QDs

[0011] S2. Pretreatment of ITO conductive glass

[0012] The ITO conductive glass is placed in an acetone solution and ultrasonically treated for 15 min, then placed in a NaOH solution (10 mL ethanol, 10 mL deionized water, 0.49 g NaOH) and ultrasonically treated for 15 min to completely remove organic substances, then placed in deionized water and ultrasonically treated for 15 min, then washed 5 times with deionized water, ultrasonically treated with deionized water for 15 min again, and washed 5 times with deionized water; dried in an argon atmosphere at 90 °C for 6 hours, cooled, and reserved for use;

[0013] S3. Preparation of TiO2:C-NPs / CdTe electrode

[0014] 4 mg of TiO2:C-NPs powder is ultrasonically dispersed in 2 mL of deionized water to prepare a TiO2:C-NPs suspension with a concentration of 2.0 mg / mL;

[0015] Using a pipette, 20 μL of the TiO2:C-NPs suspension is quantitatively aspirated and dropped from one end of the ITO glass, and slightly tilted and mixed evenly to make the TiO2:C-NPs suspension evenly coated on the 0.5 cm × 0.5 cm ITO electrode. After drying at room temperature, it is calcined in a muffle furnace at 450 °C for 30 min and left to stand on a clean bench to slowly cool to room temperature to obtain the required TiO2:C-NPs / ITO electrode;

[0016] Then, immerse the TiO2:C-NPs electrode in a 1 wt% PDDA solution for 2 min, and then slowly immerse and wash it with sterilized distilled water; Next, place CdTe QDs in a petri dish and immerse the electrode in it for 10 min, slowly immerse and wash it with sterilized distilled water, suck off the excess water with sterilized blotting paper, and dry the electrode in a drying oven at 90 °C. At this time, the electrode with CdTe attached is obtained. Repeat this process four times to obtain the TiO2:C-NPs / CdTe electrode;

[0017] S4. Preparation of Au / SH-pRNA-NH2

[0018] Activate 280 μL of 10 μM pRNA probe (SH-pRNA-NH2) with 10 μL of 10 mM TCEP for 1 h to break the disulfide bonds between the thiol-functionalized pRNAs; Then, add 1000 μL of purified Au NPs solution to the pRNA probe solution and shake and incubate in the dark for 20 h; After that, inject 50 μL of 0.1 mM MCH into the above solution and continue to shake and incubate for 2 h. After centrifuging several times, the required Au NPs-pRNA conjugate is obtained;

[0019] S5. Construction of the aptasensor

[0020] Activate by dropping 25 μL of a mixture containing 20 mM EDC and 10 mM NHS onto the CdTe QDs-modified electrode at room temperature. After 30 min, slowly rinse with deionized water, and then rinse with Tris-HC1 buffer (10 mM, pH 7.4) to remove the excess water, and dry in an oven at 60 °C for 30 min;

[0021] Drop 20 μL of 1 μM NH2-RNA-SH-Au NP onto the electrode surface and incubate overnight at 4 °C, and then rinse the electrode with Tris-HCl buffer to remove the unbound NH2-RNA-SH-AuNP;

[0022] Block the electrode with 20 μL of 1 mM MEA at room temperature for 1 h and rinse thoroughly with Tris-HCl buffer.

[0023] The optoelectrochemical aptasensor for detecting rabies virus described in the present invention has a good difference in photocurrent signals, which can be used to detect rabies virus. During detection, incubate the obtained optoelectrochemical aptasensor with 20 μL of viruses at different concentrations at 37 °C for 1 h. Then, slowly rinse the electrode with Tris-HCl buffer in a petri dish. At this time, it can be seen with the naked eye that there are dense attachments on the electrode, and then the photocurrent can be detected immediately;

[0024] When detecting photocurrent, ascorbic acid (AA) was used as the electron donor in the photocurrent signal detection system. The ascorbic acid solution was deoxygenated with argon before use. Light source: light with a wavelength of 460 nm, the light source was turned on and off at intervals of 10 s each time, and the external voltage of the photoelectrochemical system was 0.0 V.

[0025] The present invention also provides a method for detecting rabies virus. Using semiconductor CdTe quantum dots as the energy donor and AuNPs as the energy transfer acceptor, an enhanced photoelectrochemical aptasensor platform for detecting rabies virus was constructed. The emission spectrum was regulated by controlling the ratio of Cd and Te and the reaction process conditions. As the energy transfer acceptor, AuNPs regulated its absorption spectrum by controlling the dosage of HAuCl4 added dropwise and its ratio with trisodium citrate and NaBH4. Preferably, the ratio of Cd∶Te was adjusted to 1∶0.1 (molar ratio), and the particle size of CdTe QDs obtained was 2.93±0.53 nm, the ultraviolet-visible light absorption peak was 510 nm, and the emission peak of the emission spectrum was 560 nm. The absorption peak of the absorption spectrum of the energy acceptor Au NCs was 538 nm.

[0026] Furthermore, the enhanced photoelectrochemical aptasensor platform was based on an aptasensor to detect rabies virus. The aptasensor was prepared through the following steps: CdTe QDs were assembled on the surface of a carbon-doped TiO2 nanoparticle (TiO2:C-NPs) electrode by electrostatic adsorption; NH2-RNA-SH / Au NPs were immobilized on the TiO2:C-NP / CdTe electrode through a classical EDC coupling reaction between the carbonyl group on CdTe QDs and the amino group of the probe, thereby constructing the aptasensor TiO2:C-NP / CdTe / AuNPs / PRNA; immediately afterwards, monoethanolamine (MFA) was used to block the unbound sites on the electrode surface to avoid false positives caused by non-specific binding.

[0027] Furthermore, the method for detecting rabies virus specifically includes the following steps:

[0028] S1. Synthesis of CdTe QDs

[0029] First, 120 mL of ultrapure water was added to a 250 mL three-necked flask, then 0.137 g of CdCl2 and 89 μL of MPA were added. Argon was introduced and stirred, and 1.0 M NaOH was added dropwise until the pH reached 11.8. Argon was continuously introduced for thorough deoxygenation, and 120 mg of NaBH4 and 0.0133 g of Na2TeO3 were added. At this time, the molar ratio of Cd 2+ ∶Te 2-∶MPA = 1∶0.1∶1.7; The obtained solution was refluxed in an oil bath at 100 °C for 3 h under argon protection, centrifuged at 12000 rpm for 10 min, purified three times, and the final solution was stored in a refrigerator at 4 °C after deoxygenation;

[0030] S2. Pretreatment of ITO conductive glass

[0031] The ITO conductive glass was ultrasonically treated in an acetone solution for 15 min, then placed in a NaOH solution (10 mL ethanol, 10 mL deionized water, 0.49 g NaOH) and ultrasonically treated for 15 min to completely remove organic substances. Then it was placed in deionized water and ultrasonically treated for 15 min, washed 5 times with deionized water, ultrasonically treated with deionized water for 15 min again, and washed 5 times with deionized water; dried in an argon atmosphere at 90 °C for 6 h, cooled, and reserved for use;

[0032] S3. Preparation of TiO2:C-NPs / CdTe electrode

[0033] 4 mg of TiO2:C-NPs powder was ultrasonically dispersed in 2 mL of deionized water to prepare a TiO2:C-NPs suspension with a concentration of 2.0 mg / mL;

[0034] 20 μL of the TiO2:C-NPs suspension was quantitatively pipetted and dropped from one end of the ITO glass, and slightly tilted and mixed evenly to allow the TiO2:C-NPs suspension to be evenly coated on the ITO electrode with a size of 0.5 cm × 0.5 cm. After drying at room temperature, it was calcined in a muffle furnace at 450 °C for 30 min, and then left to stand on a clean bench and slowly cooled to room temperature to obtain the required TiO2:C-NPs / ITO electrode;

[0035] Then, the TiO2:C-NPs electrode was immersed in a 1 wt% PDDA solution for 2 min, and then slowly immersed and washed with sterilized distilled water; then a Petri dish was filled with CdTe QDs, and the electrode was placed in it and immersed for 10 min, slowly immersed and washed with sterilized distilled water, and the excess water was blotted off with sterilized blotting paper, and the electrode was dried in a drying oven at 90 °C. At this time, the electrode with CdTe attached was obtained, and this was repeated four times to obtain the TiO2:C-NPs / CdTe electrode;

[0036] S4. Preparation of Au / SH-pRNA-NH2

[0037] Activate 280 μL of 10 μM pRNA probe (SH-pRNA-NH2) with 10 μL of 10 mM TCEP for 1 h to break the disulfide bonds between the thiol-functionalized pRNAs; then, add 1000 μL of the purified Au NPs solution to the pRNA probe solution and incubate with shaking in the dark for 20 h; after that, inject 50 μL of 0.1 mM MCH into the above solution and continue shaking and incubating for 2 h. After centrifuging several times, the desired Au NPs-pRNA conjugate is obtained;

[0038] S5. Construction of the aptamer sensor

[0039] Activate by dropping 25 μL of the mixture containing 20 mM EDC and 10 mM NHS onto the CdTe QDs-modified electrode at room temperature. After 30 min, slowly rinse with deionized water, and then rinse with Tris-HC1 buffer (10 mM, pH 7.4) to remove the excess water, and dry in an oven at 60 °C for 30 min.

[0040] Drop 20 μL of 1 μM NH2-RNA-SH-Au NP onto the electrode surface and incubate overnight at 4 °C, and then rinse the electrode with Tris-HCl buffer to remove the unbound NH2-RNA-SH-AuNP;

[0041] Block the electrode with 20 μL of 1 mM MEA at room temperature for 1 h and rinse thoroughly with Tris-HCl buffer;

[0042] S6. Incubate the obtained photoelectrochemical aptamer sensor with 20 μL of viruses at different concentrations at 37 °C for 1 h. Then, slowly rinse the electrode with Tris-HCl buffer in a petri dish. At this time, it can be seen with the naked eye that there are dense attachments on the electrode, and then the photocurrent can be detected immediately;

[0043] When detecting the photocurrent, ascorbic acid (AA) is used as the electron donor of the photocurrent signal detection system. The ascorbic acid solution is deoxygenated with argon before use. Light source: light with a wavelength of 460 nm, the light source is turned on and off at intervals of 10 s / time, and the external voltage of the photoelectrochemical system is 0.0 V.

[0044] In the above scheme, a rabies aptamer sensing platform was constructed. This sensor has a low detection limit, a wide linear detection range, good selectivity, good reproducibility, and good stability, and can achieve specific detection of rabies virus, with diagnostic value.

[0045] This platform can detect the viral RNA of rabies virus particles in cell culture supernatants, brain and lung tissue samples, and cultured cells. This tool may help to alleviate the complex and cumbersome traditional rabies virus diagnostic methods.

[0046] The rabies aptamer sensing platform has high sensitivity and specificity; the lowest detection concentration limit of rabies virus is about 2.6 ffu / mL. In addition, PBA can also detect rabies in the brain and lung tissues of rabid dogs and mice, with higher sensitivity than RT-PCR. Brief Description of the Drawings

[0047] Other features, objectives, and advantages of the present invention will become more apparent by reading the detailed description of the non-limiting embodiments with reference to the following drawings:

[0048] Figure 1 Photoelectrochemical mechanism of the aptasensor;

[0049] In the figure: A Construction process of the aptasensor (a is the current value detected by the electrode for the target; b is the current value detected by the electrode without the target); B Photoelectro-optical principle of the aptasensor.

[0050] Figure 2 HRTEM images and UV-vis absorption spectra of CdTeQDs and Au NPs;

[0051] In the figure: (A) HRTEM image, (B) size distribution, and (E) UV-vis absorption spectrum of CdTe quantum dots; (C) HRTEM image and (D) size distribution of Au NPs; (F) PL emission spectrum (blue curve) of CdTe QDs and absorption spectrum (red curve) of Au NPs.

[0052] Figure 3 SEM images and XPS characterization of TiO2:C-NPs;

[0053] (A) SEM image of TiO2:C-NPs, (B) XPS characterization of TiO2:C-NPs.

[0054] Figure 4 Corresponding photocurrent response of the electrode;

[0055] In the figure: Corresponding photocurrent response of the electrode: (a) TiO2:C-NPs, (b) TiO2:C-NPs / CdTe / AuNPs IPRNA / MEA, (c) TiO2:C-NPs / CdTe / AuNPs / PRNA, (d) +RABV, (e) TiO2:C-NPs / CdTe-PRNA, and (f) TiO2:C-NP / CdTe.

[0056] Figure 5 Response of the biosensor to different concentrations of RABV;

[0057] In the figure: (A) Photocurrent responses of different concentrations of rabies virus in cell lysates; (B) Linear regression equation of photocurrent response versus RABV concentration (the concentration of RABV ranges from 2.16×10 5 ffu / mL to 2.16 ffu / mL, and the error bars represent the standard deviation of ten replicates).

[0058] Figure 6 Photocurrent responses of the universal RABV aptamer sensor to different viruses;

[0059] In the figure: (a) rRC-HL; (b) CVS-11; (c) GX074; (d) CSFV; (e) PRRSV-5; (f) PRRSV-96; (g) PCV; and (h) VSV.

[0060] Figure 7 Detection of tissue samples using the photoelectrochemical biosensor method (PBA), RT-PCR, and MIT;

[0061] In the figure: MIT-1, PCR1, and PBA1 are the results from dog brain and lung samples. Positive indicates cell samples infected with the standard rabies virus strain (CVS), brain samples from four dogs (DB1, DB2, DB3, and DB4), normal dog lung samples (NDL), and dog lung sample (DL1). MIT-2, PCR2, and PBA2 show the test results of mouse brain and lung samples. Positive indicates cell samples infected with the standard rabies virus strain (CVS), two mouse brain samples (DM1, DM2), one normal mouse lung (NML), and two mouse lung samples (ML1, ML2). Detailed implementation manners

[0062] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made. These all fall within the protection scope of the present invention.

[0063] Test data:

[0064] 1 Materials and methods

[0065] 1.1 Reagents and instruments

[0066] 1.1.1 Reagents

[0067] Cadmium chloride (CdCl2·2.5H2O), sodium tellurite (Na2TeO3), chloroauric acid (HAuCl4·4H2O), sodium hydroxide (NaOH), and sodium chloride (NaCl) were purchased from Aladdin Reagent Inc. (China). Sodium citrate dehydrate and sodium borohydride (NaBH4) were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). Titanium sheets (purity 99.7%, thickness 0.127 mm), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), monoethanolamine (MFA), poly(diallyldimethylammonium chloride) (PDDA), 3-mercaptopropionic acid (MPA), and 6-mercaptohexanol (MCH) were ordered from Sigma-Aldrich (USA). Indium tin oxide (ITO) electrodes were purchased from Yijin New Materials, affiliated with Beijing Huimo Technology Co., Ltd. All solutions were prepared from deionized ultrapure water (DI water, 18 MΩ / cm) of the Milli-Q water purification system. The solvent for all RNA solutions was PBS, with a solution pH of 7.4 and a solution concentration of 20 mM.

[0068] The synthetic oligonucleotides were all synthesized by Shanghai Sangon Biotech Co., Ltd. and had the following sequences:

[0069] RABV universal probe (pRNA): 5'-SH-(CH2)3-TTT ACC ATA CGG CCG GGC AAT CTG AAGTTCGGT ATGGT-(CH2)6-NH2-3';

[0070] r-RC-HL RABV probe (pr-RNA): 5'-SH-(CH2)3-TTT ACC ATA CGG CCG GAG AAG CAGYGA CAA CAG TACC-(CH2)6-NH2-3';

[0071] GX074 wild RABV probe (p-w-RNA): 5-SH-(CH2)3-TTT ACC ATA CGG CCG GGC AATAGG AAT GAG GAA CAGC-(CH2)6-NH2-3';

[0072] CVS-11 RABV probe (pc-RNA): 5'-SH-(CH2)3-TTT ACC ATA CGG CCG GGC ATGTTTGTC TTGTAGTTGC-(CH2)6-NH2-3'.

[0073] 1.1.2 Instruments

[0074] The photocurrent value of the sensor was measured by a photoelectrochemical workstation, with a xenon lamp of about 400 μW / cm intensity at 500W as the light source; the absorption and emission spectra of the energy donor and energy acceptor were measured by a TECAN microplate reader. The field emission scanning electron microscope (FE-SEM) was carried out on a SUPRA 55 Sapphire scanning electron microscope from Carl Zeiss AG in Germany. The quantum dot characterization was performed using a transmission electron microscope (TEM) on a Talos-F200X transmission electron microscope. -2 The photocurrent value of the sensor was measured by a photoelectrochemical workstation, with a xenon lamp of about 400 μW / cm intensity at 500W as the light source; the absorption and emission spectra of the energy donor and energy acceptor were measured by a TECAN microplate reader. The field emission scanning electron microscope (FE-SEM) was carried out on a SUPRA 55 Sapphire scanning electron microscope from Carl Zeiss AG in Germany. The quantum dot characterization was performed using a transmission electron microscope (TEM) on a Talos-F200X transmission electron microscope.

[0075] 1.2 Synthesis of CdTe QDs

[0076] First, 120 mL of ultrapure water was added to a 250 mL three-necked flask, followed by 0.137 g of CdCl2 and 89 μL of MPA. Argon was introduced and stirred, and 1.0 M NaOH was added dropwise until the pH reached 11.8. Argon was continuously introduced to completely remove oxygen. Then, 120 mg of NaBH4 and 0.0133 g of Na2TeO3 were added. At this time, the molar ratio of Cd 2+ ∶Te 2- ∶MPA = 1∶0.1∶1.7. The resulting solution was refluxed in an oil bath at 100 °C for 3 h under argon protection, centrifuged at 12000 rpm for 10 min, and purified three times. The final solution was stored in a refrigerator at 4 °C after deoxygenation.

[0077] 1.3 Pretreatment of ITO conductive glass

[0078] The ITO conductive glass was placed in an acetone solution and ultrasonically treated for 15 min to remove the attached organic matter; then it was placed in a NaOH solution (10 mL of ethanol, 10 mL of deionized water, 0.49 g of NaOH) and ultrasonically treated for 15 min to completely remove the organic matter; then it was placed in deionized water and ultrasonically treated for 15 min, followed by washing 5 times with deionized water and then ultrasonically treated for 15 min with deionized water and washing 5 times with deionized water; it was dried in an argon atmosphere at 90 °C for 6 hours and could be used after cooling.

[0079] 1.4 Preparation of TiO2:C-NPs / CdTe electrode

[0080] Preparation of 2.0 mg / mL TiO2:C-NPs suspension: 4 mg of TiO2:C-NPs powder was added to 2 mL of deionized water, and the powder was uniformly dispersed by an ultrasonic instrument to prepare a TiO2:C-NPs suspension with a concentration of 2.0 mg / mL.

[0081] Quantitatively pipette 20 μL of TiO2:C-NPs suspension, drop it from one end of the ITO glass, tilt it slightly and mix well to evenly coat the TiO2:C-NPs suspension on the ITO electrode with a size of 0.5 cm × 0.5 cm. After drying at room temperature, calcine it in a muffle furnace at 450 °C for 30 min, and let it stand on the ultra-clean workbench to slowly cool to room temperature to obtain the required TiO2:C-NPs / ITO electrode. The modification of CdTe QDs adopts the layer-by-layer self-assembly method: First, immerse the TiO2:C-NPs electrode in 1 wt% PDDA solution for 2 min, and then slowly immerse and wash it with sterilized distilled water; then place CdTe QDs in a petri dish, immerse the electrode in it for 10 min, slowly immerse and wash it with sterilized distilled water, absorb the excess water with sterilized blotting paper, and dry the electrode in an oven at 90 °C. At this time, the electrode with CdTe attached is obtained. Repeat this process four times to obtain the TiO2:C-NPs / CdTe electrode.

[0082] 1.5 Preparation of Au / SH-pRNA-NH2

[0083] Activate 280 μL of 10 μM pRNA probe (SH-pRNA-NH2) with 10 μL of 10 mM TCEP for 1 h to break the disulfide bonds between the thiol-functionalized pRNAs. Then, add 1000 μL of purified Au NPs solution to the pRNA probe solution and shake and incubate it in the dark for 20 h. To prevent the non-specific adsorption of Au NPs, inject 50 μL of 0.1 mM MCH into the above solution and continue to shake and incubate for 2 h. After centrifuging the obtained mixture several times, the required Au NPs-pRNA conjugate is obtained.

[0084] 1.6 Construction of the aptasensor

[0085] The electrode modified with CdTe QDs was activated by dropping 25 μL of a mixture containing 20 mM EDC and 10 mM NHS at room temperature. After 30 min, it was slowly rinsed with deionized water, then rinsed with Tris-HC1 buffer (10 mM, pH 7.4) to remove the excess water, and dried in an oven at 60 °C for 30 min. Then, 20 μL of 1 μM NH2-RNA-SH-Au NP was dropped onto the electrode surface and incubated overnight at 4 °C. The electrode was rinsed with Tris-HCl buffer to remove the unbound NH2-RNA-SH-AuNP. Then, the electrode was blocked with 20 μL of 1 mM MEA at room temperature for 1 h and thoroughly rinsed with Tris-HCl buffer. Next, the obtained electrode was used as a photoelectrochemical aptasensor and incubated with 20 μL of different concentrations of the virus at 37 °C for 1 h. Then, the electrode was slowly rinsed with Tris-HCl buffer in a petri dish. At this time, it was visible to the naked eye that there were dense attachments on the electrode, and then the detection of photocurrent could be carried out immediately.

[0086] 1.7 Measurement of photocurrent

[0087] Ascorbic acid (AA) was used as the electron donor in all detection solution systems for the detection of photocurrent signals. The ascorbic acid solution was deoxygenated with argon before use. Light source: light with a wavelength of 460 nm, the light source was turned on and off at intervals of 10 s each time. The external voltage of the photoelectrochemical system was 0.0 V.

[0088] 2 Results

[0089] 2.1 Working mechanism of the energy transfer type sensor

[0090] The aptasensor used in this experiment took rabies virus as the target analyte, and as shown in Figure 1 A, the constructed aptasensing platform was elaborated in detail. First, CdTe QDs were assembled on the surface of the carbon-doped TiO2 nanoparticle (TiO2:C-NPs) electrode by electrostatic adsorption. NH2-RNA-SH / Au NPs were immobilized on the TiO2:C-NP / CdTe electrode through the classical EDC coupling reaction between the carbonyl group on CdTe QDs and the amino group of the probe, thus constructing the aptasensor TiO2:C-NP / CdTe / AuNPs / PRNA. Immediately afterwards, monoethanolamine (MFA) was used to block the unbound sites on the electrode surface to avoid false positives caused by non-specific binding. Thus, the aptasensor for detecting rabies virus was constructed.

[0091] The photoelectrochemical mechanism of the aptasensor is as shown in Figure 1As shown in B. Under light illumination, TiO2:C-NPs and CdTe QDs are excited by light to generate electron-hole pairs (also called excitons). The photo-generated electrons jump to the conduction band (BD), while the holes are in the valence band (VB). At this time, the photo-generated electrons of CdTe QDs have two flow directions. The first direction ( Figure 1 , process 1), forms an electron flow to TiO2:C-NPs, thereby enhancing the photocurrent intensity of the sensor; the other direction ( Figure 1 , process 2), the electrons flow back to the holes again, promoting the recombination of electron-hole pairs, and thus weakening the photocurrent intensity of the sensor. The two processes are in a competitive relationship. The doping of C provides low-energy impurity levels required for electron transition in TiO2 NPs, promotes the separation of photo-generated electron-hole pairs, thereby increasing the generation of electron flow in TiO2 NPs, and thus promotes the transfer of the electron flow of CdTe QDs to process 1. Since the photoluminescence (PL) of CdTe QDs will excite AuNPs to generate SPR absorption, thereby causing energy transfer. At this time, a local electric field is generated around AuNPs, increasing the recombination probability of electron-hole pairs in CdTe QDs ( Figure 1 , process 2), and reducing the photocurrent of the sensor. Therefore, the exciton energy transfer effect can be used to control the direction of electron transfer, thereby realizing the regulation of photocurrent intensity. That is, when the sensor does not encounter the target analyte - rabies virus, rapid and efficient exciton energy transfer occurs between CdTe QDs and AuNPs, that is, the electron-hole pairs are mainly recombined (process 2), and the electron transfer (process 1) is largely inhibited, resulting in a rapid decrease in the current of the photocurrent sensor. When the electrode is incubated with rabies virus, the aptamer will specifically bind to the rabies virus, and Au / SH-RNA is released from the electrode surface, and the exciton energy transfer effect is destroyed, mainly with electron transfer (process 1), thereby significantly increasing the photocurrent intensity. Based on this principle, we established a photoelectrochemical sensor to specifically detect the nucleic acid of rabies virus.

[0092] 2.2 Characterization of CdTe QDs and Au NPs

[0093] To verify the successful synthesis of CdTe QDs and Au NPs, high-resolution transmission electron microscopy (HRTEM) characterizations were carried out on them respectively, as shown in Figure 2 A and 2C. In the HRTEM images, the lattice fringes of CdTe QDs and Au NPs can be clearly seen, and their average particle sizes are 2.93±0.53nm ( Figure 2B) and 3.59 ± 0.62 nm( Figure 2 D). Consistent with the empirical formula for ultraviolet-visible absorption derived by Peng et al., the particle sizes of CdSe QDs and Au NPs were calculated to be the same. The ultraviolet-visible absorption peak of CdSe QDs was 510 nm( Figure 2 E), and that of Au NPs was 538 nm( Figure 2 F (red curve)). For efficient energy transfer, the emission spectrum generated by CdTe QDs must be absorbed by Au NPs. From Figure 2 Figure F is the emission spectrum curve of CdTe QDs, and its emission peak is at 560 nm (blue curve); while the red curve is the light absorption curve of Au NPs, and the absorption peak is reached at 538 nm. There is a relatively wide overlapping region between the blue curve and the red curve, meeting the necessary conditions for efficient energy transfer. The second condition for the sensor to have efficient energy transfer is that the distance between the energy transfer donor CdTe QDs and the acceptor Au NPs is less than 10 nm. The actual distance between the energy transfer donor and the acceptor in this experiment is the length of NH2-RNA-SH-Au NPs. This chain has 38 bases, and its length is calculated to be approximately 5 nm. All the necessary conditions for energy transfer are met. Therefore, the constructed energy transfer type sensor must be able to be used for subsequent detection and analysis.

[0094] 2.3 Photoelectrochemical properties of TiO2:C-NPs / CdTe electrodes

[0095] The biorecognition element (photoelectroactive material) and the biorecognition probe are two essential devices for constructing a current-type photoelectrochemical sensor. In the constructed device, the biorecognition probe is basically the same in all types of biosensors, usually protein or nucleic acid molecules. Therefore, to improve the performance of the sensor, researchers have conducted various studies on the photoelectroactive material to improve its performance. TiO2-NPs are often used as the substrate material for sensor electrodes due to their high stability, non-toxicity, and high biosafety. To make it have better photoelectric response performance, we modified it by doping C element into TiO2-NPs. The doping of C element formed new electron energy states between the band gaps of TiO2, which can not only promote the flow of photogenerated electrons to the electrode but also promote the transfer of photogenerated holes in the energy donor CdTe QDs to the VO electron state in TiO2( Figure 1 , process 3), effectively reducing the probability of recombination of electron-hole pairs in CdTe QDs. From the scanning electron micrograph of TiO2:C-NPs( Figure 3 A), it can be seen that the average inner diameter of TiO2:C-NPs is about 50 nm. Figure 3B corresponds to the XPS spectrum of TiO2:C-NPs, and it can be determined that C element has been successfully doped into the TiO2-NPs electrode. In this experiment, the TiO2:C-NPs with successful C doping was used as the substrate material of the sensor electrode to construct a photoelectrochemical aptasensor as the substrate.

[0096] 2.4 Measurement of photocurrent during the sensor preparation process

[0097] The aptasensor constructed this time is an energy transfer type sensor, and the detection signal of the sensor is reflected by the photocurrent values of each component ( Figure 4 ). TiO2:C-NPs is used as the substrate electrode of the sensor, and photocurrent is generated on the electrode (the average photocurrent intensity is 15.3963 μA, curve a), and the current value is the lowest component in the whole sensor, because it can only absorb part of ultraviolet light and the photoelectric conversion efficiency is low. After attaching CdTe QDs to the TiO2:C-NPs electrode, the photocurrent intensity is greatly improved, about 5 times that of the TiO2:C-NPs electrode (the average photocurrent intensity is 78.3229 μA, curve f). This is mainly because: 1. CdTe QDs, as the donor of energy transfer, has a strong electron transfer ability, and the existence of TiO2:C-NPs can load more CdTe QDs with a larger specific surface area, significantly increasing the absorption of the electrode to the energy of the excitation light source; 2. The doping of C element in the TiO2-NPs electrode greatly inhibits the recombination of electron-hole pairs in CdTe QDs. Since there are some small organic molecules after connecting pRNA, there is a small part of electron transfer, so the current value drops slightly (the average photocurrent intensity is 73.0621 μA, such as curve e). When RNA-Au-SH is fixed to the electrode, the current drops sharply (the average photocurrent intensity is 18.1393 μA, curve c), which is exactly because the exciton energy transfers from CdTe QDs to Au NPs. When the electrode is blocked with MEA, the current drops slightly compared with that of RNA-Au-SH (curve c) (the average photocurrent intensity is 16.1631 μA, curve b).

[0098] When the sensing electrode is incubated with 20 μL of 2.16 ffu / mL RABV, the photocurrent intensity increases significantly compared with that after RNA-Au-SH is fixed to the electrode (curve c) (the average photocurrent intensity is 55.1642 μA, curve d). This is because after the target rabies virus to be detected specifically binds to the biorecognition probe of the sensor, it promotes the energy receptor RNA-Au / SH to be released from the electrode surface and can no longer accept the energy generated by excitation. Therefore, the energy transfers to the electrode of the sensor again, increasing the photocurrent value of the electrode. In this experiment, it is proved that the designed aptasensor can be successfully used for the detection of rabies virus by measuring the photocurrent values of each component of the sensor.

[0099] 2.5 Photoelectrochemical Detection of Rabies Virus Nucleic Acid

[0100] In the above experiment, it was demonstrated that the energy transfer type sensor composed of the energy donor CdTe QDs and the energy acceptor Au NPs had good photocurrent signal differences and could be used for the detection of rabies virus. In this experiment, in order to verify the detection sensitivity of the constructed sensor to the analyte, rabies virus was used as the target analyte and detected with this photoelectrochemical sensing platform. The experimental results showed that from 2.16×10 0 ffu / mL to 2.16×10 7 ffu / mL, the photocurrent intensities were: 55.16 μA, 57.191 μA, 59.49 μA, 63.17 μA, 66.10 μA, 67.59 μA, 66.65 μA, 66.51 μA( Figure 5 A), indicating that with the increase of concentration, the photocurrent response gradually increased. In the range of 2.6×10 0 ffu / mL to 2.6×10 5 ffu / mL, the magnitude of the photocurrent was linearly related to the logarithm of the concentration of the target analyte. The linear regression equation was y = 2.4651 + 54.6749logC, and the linear correlation coefficient R 2 was 0.9951( Figure 5 B). The lowest detection concentration limit for RABV was approximately 2.6×10 0 ffu / mL, which was 1 - 3 orders of magnitude higher than the detection sensitivity reported recently for RABV. It can be seen that the sensor designed by us has extremely high sensitivity for the specific detection of rabies, and the detection limit concentration shown for rabies detection has greater superiority than other detection platforms.

[0101] 2.6 Selectivity and Repeatability of the Sensor

[0102] To verify whether the sensor constructed in this experiment was specific, nucleic acids of some representative pathogens similar to the target analyte RABV were selected, such as: CSFV, PRRSV - 5, PRRSV - 96, PCV, and VSV as interfering substances for interference tests. The concentrations of all analytes to be detected were 2.16×10 5 flu / mL. The results showed that the photocurrent intensity of CSFV( Figure 6 d) was 19.95 μA, that of PRRSV - 5( Figure 6 e) was 19.31 μA, that of PRRSV - 96( Figure 6 f) was 19.28 μA, that of PCV( Figure 6 g) was 20.94 μA, and that of VSV(Figure 6 h) The photocurrent intensity is 19.53 μA, and for the three strains of rabies virus, rRC-HL( Figure 6 a) The photocurrent intensity is 66.29 μA, CVS-11( Figure 6 b) The photocurrent intensity is 66.64 μA, GX07( Figure 6 c) The photocurrent intensity is 66.98 μA. Thus, it can be seen that the photocurrent intensities of all interfering source virus nucleic acids are small, while the response to rabies virus is basically more than 3 times that of theirs. The probe we designed is a universal probe for rabies, which can detect rabies regardless of the strain, but has no reaction to other viruses, indicating that the sensor we designed has good specificity.

[0103] 2.7 Detection of living tissues with PBA sensor

[0104] We used PBA to evaluate brain and lung samples of rabid dogs and a rabies rodent model. We simultaneously measured the same samples by MIT and RT-PCR. The results showed that the sensitivity of PBA was far better than that of RT-PCR. Positive brain samples detected by RT-PCR could also be detected as positive by PBA: DB1 (45.37 ± 2.61 μA), DB2 (42.17 ± 1.83 μA), DB3 (49.17 ± 4.15 μA), and DB4 (40.54 ± 2.62 μA). However, three lung tissue samples were negative by RT-PCR but positive by an increase in current: dog lung sample (DL1: 26.55 ± 1.23 μA) and two mouse lung samples (ML1: 26.53 ± 2.12 μA, ML2: 26.53 ± 2.12 μA)( Figure 7 ).

[0105] For the sensor constructed by energy transfer, the optoelectroactive material on the sensor, under the excitation of light, the energy generated by the excitation of the energy donor is transferred to the adjacent energy acceptor. This is the basic process of energy transfer. Two key words that need to be noted in this process are: one is the existence of an energy donor and an energy acceptor, and the relationship between the two is that one can emit and the other can absorb; the other is the positional relationship between the two is adjacent. Generally speaking, the generation of energy transfer of this type of sensor needs to meet these two conditions simultaneously: one is that the distance from the energy donor to the energy acceptor is between 10 ± 2 nm; the other is that the emission spectrum of the energy donor overlaps with the absorption spectrum of the energy acceptor.

[0106] In this experiment, an energy transfer-based sensor was constructed by using CdTe QDs as the energy transfer donor and Au NCs as the energy transfer acceptor. As an energy donor, CdTe QDs have good emission spectrum regulation ability, and the range of its emission spectrum can be regulated by adjusting the ratio of Cd / Te. In this adjustment, Cd∶Te = 1∶0.1 (molar ratio), and the particle size of CdTe QDs obtained is 2.93 ± 0.53 nm, the ultraviolet-visible light absorption peak is 510 nm, and the emission peak of the emission spectrum is 560 nm. The absorption peak of the absorption spectrum of the energy acceptor Au NCs is 538 nm. In this experiment, an energy transfer-based sensor was constructed for the detection of rabies virus. Through a complete construction strategy, starting from the sensor substrate, CdTe AQDs with a powerful energy donor were adsorbed onto the surface of the substrate layer of carbon-doped titanium dioxide nanomaterials (TiO2:C-NPs) electrodes by the layer-by-layer assembly method. Subsequently, the biorecognition probe NH2-RNA-SH-AuNP was immobilized on the surface of the TiO2:C-NPs / CdTe electrode through the EDC / NHS coupling reaction between the amino group and the carboxyl group. After blocking the electrode active sites with ethanolamine (MEA), the sensor electrode was successfully constructed. In this case, the excitons of CdTe QDs and the SPR effect of Au NPs were simultaneously excited, resulting in rapid and efficient energy transfer. If the electrode is independently incubated with rabies virus cell lysate to specifically bind the electrode to rabies virus nucleic acid, the energy acceptor RNA-Au / SH is released from the electrode surface and can no longer accept the energy generated by excitation. The sensor can determine whether the target substance exists in the test substance by the difference in the current signal before and after detecting the target test substance.

[0107] In this experiment, since the designed electrode aptamer is a universal probe for rabies virus and can detect rabies virus regardless of the strain, in the next step, we will apply this platform to establish the detection of different strains of rabies virus through reasonable design of the probe. This platform can detect RABV infection in brain and lung tissue samples or cell culture samples. Compared with samples without RABV infection, the photocurrent from brain samples increased by more than twice. Some lung tissue samples that were negative by PCR detection were detected as positive by PBA, and the photocurrent increased. The successful construction of this diagnostic platform broadens the detection methods available for bioanalysis of various samples and provides a theoretical and practical basis for the establishment of other bioanalysis detection methods.

[0108] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. An optoelectrochemical aptasensor for detecting rabies virus, characterized in that: It is prepared through the following steps: CdTe QDs are assembled on the surface of a carbon-doped TiO2 nanoparticle electrode by electrostatic adsorption; NH2-RNA-SH / Au NPs are immobilized on the TiO2:C-NPs / CdTe electrode through a classical EDC coupling reaction between the carbonyl group on CdTe QDs and the amino group of the probe, thereby constructing an aptasensor TiO2:C-NPs / CdTe / AuNPs / PRNA; immediately afterwards, unbound sites on the electrode surface are blocked with monoethanolamine to avoid false positives caused by non-specific binding. Specifically, it includes the following steps: S1. Synthesis of CdTe QDs First, add 120 mL of ultrapure water to a 250 mL three-necked flask. Then add 0.137 g of CdCl2 and 89 μL of MPA. Pass argon gas and stir. Dropwise add 1.0 M NaOH to make the pH reach 11.

8. Continue to pass argon gas to completely remove oxygen. Add 120 mg of NaBH4 and 0.0133 g of Na2TeO3. At this time, the molar ratio of Cd 2+ : Te 2- : MPA = 1:0.1:1.7; The obtained solution is refluxed in an oil bath at 100 °C for 3 h under argon protection, centrifuged at 12000 rpm for 10 min, and purified 3 times. The final solution is stored in a 4 °C refrigerator after deoxygenation; S2. Pretreatment of ITO conductive glass The ITO conductive glass is ultrasonically treated in an acetone solution for 15 min, then placed in a NaOH solution and ultrasonically treated for 15 min to thoroughly remove organic substances. Then it is placed in deionized water and ultrasonically treated for 15 min, washed 5 times with deionized water, ultrasonically treated with deionized water for 15 min again, and washed 5 times with deionized water; dried in an argon atmosphere at 90 °C for 6 hours, cooled, and reserved for use. S3. Preparation of TiO2:C-NPs / CdTe electrode 4 mg of TiO2:C-NPs powder is ultrasonically dispersed in 2 mL of deionized water to prepare a TiO2:C-NPs suspension with a concentration of 2.0 mg / mL. Using a pipette, 20 μL of the TiO2:C-NPs suspension is quantitatively aspirated and dropped from one end of the ITO glass, and slightly tilted and mixed evenly to allow the TiO2:C-NPs suspension to be evenly coated on the 0.5 cm×0.5 cm ITO electrode. After drying at room temperature, it is calcined in a muffle furnace at 450 °C for 30 min, and then left to stand on a clean bench to slowly cool to room temperature to obtain the required TiO2:C-NPs / ITO electrode. Then, the TiO2:C-NPs electrode is immersed in a 1 wt% PDDA solution for 2 min, and then slowly immersed and washed with sterilized distilled water; then, a petri dish is used to hold CdTe QDs, and the electrode is placed in it and immersed for 10 min, slowly immersed and washed with sterilized distilled water, and the excess water is blotted off with sterilized absorbent paper. The electrode is dried in a drying oven at 90 °C. At this time, an electrode with CdTe attached is obtained. Repeating this process four times can obtain the TiO2:C-NPs / CdTe electrode. S4. Preparation of Au / SH-pRNA-NH2 280 μL of a 10 μM pRNA probe is activated with 10 μL of 10 mM TCEP for 1 h to break the disulfide bonds between the thiol-functionalized pRNAs; then, 1000 μL of a purified Au NPs solution is added to the pRNA probe solution, and shaken and incubated in the dark for 20 h; afterwards, 50 μL of 0.1 mM MCH is injected into the above solution, and shaking and incubation are continued for 2 h. After centrifuging several times, the required Au NPs-pRNA conjugate is obtained. S5. Construction of aptasensor At room temperature, 25 μL of a mixed solution containing 20 mM EDC and 10 mM NHS was dropped onto the CdTe QDs-modified electrode for activation. After 30 min, it was slowly rinsed with deionized water, then rinsed with 10 mM Tris-HCl buffer solution with a pH of 7.4 to remove the excess water, and dried in an oven at 60 °C for 30 min; 20 μL of 1 μM NH2-RNA-SH-Au NP was dropped onto the electrode surface and incubated overnight at 4 °C. Then the electrode was rinsed with Tris-HCl buffer solution to remove the unbound NPs; The electrode was blocked with 20 μL of 1 mM MEA at room temperature for 1 h and thoroughly rinsed with Tris-HCl buffer solution.

2. Application of a photoelectrochemical aptasensor for detecting rabies virus according to claim 1, characterized in that: It can be used to detect rabies virus.

3. The application of an optoelectrochemical aptasensor for detecting rabies virus according to claim 2, characterized in that: The obtained photoelectrochemical aptasensor was incubated with 20 μL of viruses at different concentrations at 37 °C for 1 h. Then, the electrode was slowly rinsed with Tris-HCl buffer solution in a petri dish. At this time, it was visible to the naked eye that there were dense attachments on the electrode, and then the photocurrent detection could be carried out immediately; During the photocurrent detection, ascorbic acid was used as the electron donor of the photocurrent signal detection system. The ascorbic acid solution was deoxygenated by argon. Light source: light with a wavelength of 460 nm, the light source was turned on / off at intervals of 10 s / time, and the external voltage of the photoelectrochemical system was 0.0 V.

4. A method for detecting rabies virus using an optoelectrochemical aptasensor for detecting rabies virus as described in claim 1, characterized in that: Using semiconductor CdTe quantum dots as the energy donor and AuNPs as the energy transfer acceptor, an enhanced photoelectrochemical aptasensor platform for rabies virus detection was constructed. Its emission spectrum was regulated by controlling the ratio of Cd and Te and the reaction process conditions. AuNPs, as the energy transfer acceptor, its absorption spectrum was regulated by controlling the dosage of HAuCl4 dropped and the ratio with trisodium citrate and NaBH4; The enhanced photoelectrochemical aptasensor platform realizes the detection of rabies virus based on the aptasensor. The aptasensor was prepared through the following steps: CdTe QDs were assembled on the surface of the carbon-doped TiO2 nanoparticle electrode by electrostatic adsorption; NH2-RNA-SH / Au NPs were fixed on the TiO2:C-NPs / CdTe electrode through the classical EDC coupling reaction between the carbonyl group on CdTe QDs and the amino group of the probe, thus constructing the aptasensor TiO2:C-NPs / CdTe / AuNPs / PRNA; then the unbound sites on the electrode surface were blocked with monoethanolamine to avoid false positives caused by non-specific binding.

5. The rabies virus detection method according to claim 4, wherein: Including the following steps: S1. Synthesis of CdTe QDs First, add 120 mL of ultrapure water to a 250 mL three-necked flask. Then, add 0.137 g of CdCl2 and 89 μL of MPA. Pass argon gas and stir. Dropwise add 1.0 M NaOH to make the pH reach 11.

8. Continue to pass argon gas to completely remove oxygen. Add 120 mg of NaBH4 and 0.0133 g of Na2TeO3. At this time, the molar ratio of Cd 2+ : Te 2- : MPA = 1:0.1:1.7; The resulting solution is refluxed in an oil bath at 100 °C for 3 h under argon protection, centrifuged at 12000 rpm for 10 min, and purified 3 times. The final solution is stored in a 4 °C refrigerator after deoxygenation; S2. Pretreatment of ITO conductive glass The ITO conductive glass was ultrasonically treated in an acetone solution for 15 min, then placed in an NaOH solution and ultrasonically treated for 15 min to completely remove the organic matter. Then it was ultrasonically treated in deionized water for 15 min, washed with deionized water 5 times, ultrasonically treated in deionized water for 15 min again, and washed with deionized water 5 times; dried in an argon atmosphere at 90 °C for 6 h, cooled, and reserved; S3. Preparation of TiO2:C-NPs / CdTe Electrode Disperse 4 mg of TiO2:C-NPs powder in 2 mL of deionized water by ultrasonic treatment to prepare a TiO2:C-NPs suspension with a concentration of 2.0 mg / mL; Quantitatively pipette 20 μL of the TiO2:C-NPs suspension and drop it from one end of the ITO glass. Tilt it slightly and mix well to evenly coat the TiO2:C-NPs suspension on the 0.5 cm×0.5 cm ITO electrode. After drying at room temperature, calcine it in a muffle furnace at 450 °C for 30 min, and then let it stand on a clean bench and slowly cool to room temperature to obtain the required TiO2:C-NPs / ITO electrode; Then, immerse the TiO2:C-NPs electrode in a 1 wt % PDDA solution for 2 min, and then slowly immerse and wash it with sterilized distilled water; then place the CdTe QDs in a petri dish and immerse the electrode in it for 10 min, slowly immerse and wash it with sterilized distilled water, and use sterilized blotting paper to absorb the excess water. Dry the electrode in a drying oven at 90 °C. At this time, the electrode with CdTe attached is obtained. Repeat this process four times to obtain the TiO2:C-NPs / CdTe electrode; S4. Preparation of Au / SH-pRNA-NH2 Activate 280 μL of 10 μM pRNA probe with 10 μL of 10 mM TCEP for 1 h to break the disulfide bonds between the thiol-functionalized pRNAs; then add 1000 μL of purified Au NPs solution to the pRNA probe solution and shake and incubate in the dark for 20 h; then inject 50 μL of 0.1 mM MCH into the above solution and continue to shake and incubate for 2 h. After centrifuging several times, the required Au NPs-pRNA conjugate is obtained; S5. Construction of the Aptamer Sensor Activate the electrode modified with CdTe QDs by dropping 25 μL of a mixed solution containing 20 mM EDC and 10 mM NHS at room temperature. After 30 min, slowly rinse it with deionized water, and then rinse it with 10 mM Tris-HCl buffer solution with a pH value of 7.4 to remove the excess water, and dry it in an oven at 60 °C for 30 min; Drop 20 μL of 1 μM NH2-RNA-SH-Au NP onto the electrode surface and incubate it overnight at 4 °C, and then rinse the electrode with Tris-HCl buffer solution to remove the unbound NH2-RNA-SH-Au NP; Block the electrode with 20 μL of 1 mM MEA at room temperature for 1 h and rinse it thoroughly with Tris-HCl buffer solution; S6. Incubate the obtained photoelectrochemical aptamer sensor with 20 μL of viruses with different concentrations at 37 °C for 1 h. Then, slowly rinse the electrode with Tris-HCl buffer solution in a petri dish. At this time, it can be seen with the naked eye that there are dense attachments on the electrode, and then the photocurrent can be detected immediately; When detecting photocurrent, ascorbic acid is used as the electron donor in the photocurrent signal detection system. The ascorbic acid solution is deoxygenated with argon before use. Light source: light with a wavelength of 460 nm, the light source is turned on and off at intervals of 10 s each time, and the external voltage of the photoelectrochemical system is 0.0 V.

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