A photoelectrochemical biosensor based on ZnIn2S4 quantum dots and BiOI microflowers and its detection application

Abstract

The present invention discloses a photoelectrochemical biosensing platform based on ZnIn2S4 quantum dots and BiOI microflowers. The technical scheme of the present invention is to use the target microRNA-21 to trigger a cyclic amplification reaction, thereby generating a large number of tripod DNA walkers, combining ZnIn2S4 quantum dots with TiO2 nanoparticles to form a heterojunction signal amplification probe, which is introduced into the electrode through DNA-specific hybridization to enhance the BiOI signal, thereby preparing a simple and practical photoelectrochemical (PEC) sensing platform, realizing ultra-sensitive detection of microRNA-21, and the research has great application potential in clinical diagnosis, gene therapy, environmental monitoring, food safety and other fields.

Description

Technical field:

[0001] The invention relates to a photoelectrochemical biosensor based on ZnIn2S4 quantum dots and BiOI microflowers, as well as a preparation method of the sensor and an analytical application thereof for detecting microRNA-21. Background technology:

[0002] MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs that are important markers for cancer and autoimmune diseases. However, accurate detection and quantification of miRNAs are challenging due to their small size, low expression, and easy degradation. Traditional methods such as polymerase chain reaction and microarray analysis have been used to detect miRNAs, but their application is limited by high cost, complex operation, poor sensitivity, and false positive results. Among the many detection methods, photoelectrochemical bioanalysis has the characteristics of high sensitivity and fast reaction speed, and is of great value for the detection and research of tumor markers [Lin, QY; Huang, X. et, al. Biosens. Bioelectron. 2022, 216, 114679]. BiOI microflowers have excellent photoelectric properties, simple synthesis steps, and narrow band gap, which is conducive to the generation of electron-hole pairs by photoexcitation, making them an ideal substrate for photoelectrochemical biosensors [Fan, LF; Liang, GF et, al. J. Hazard. Mater. 2021, 409, 124894]. ZnIn2S4 quantum dots are composed of ternary mixtures and have the advantages of low toxicity, narrow band gap and simple synthesis. More importantly, ZnIn2S4 quantum dots can form heterojunctions with BiOI on the electrode, with perfect band gap matching, greatly enhancing the photoelectric signal of BiOI [Huang, C.; Liu, YQet, al.ACSAppl.Mater.Interfaces 2021, 13, 35389-35396].

[0003] In this paper, BiOI microflowers were used as substrates and ZnIn2S4 QDs as sensitizing substances. HP4 DNA-ZnIn2S4 QDs-TiO2NPs signal probes were prepared by compounding with TiO2, HP4 DNA and barcode DNA. At the same time, Target-microRNA-21 was cyclically amplified to produce a large number of tripod DNA walkers, and then the HP4DNA-ZnIn2S4 QDs-TiO2 signal probes were successfully modified onto the electrode through the capture chain HP5 DNA on the electrode, realizing the sensitization of PEC signals and ultra-sensitive detection of the target microRNA-21. Summary of the invention:

[0004] One of the objectives of the present invention is to use BiOI microflowers with narrow bandgap width for photoelectrochemical biosensors.

[0005] The specific steps include:

[0006] Synthesis of BiOI microflowers. 30 mL of 0.10 M Bi(NO3)3·5H2O dissolved in ethylene glycol was slowly added dropwise to 30 mL of 0.10 M KI. After stirring at room temperature for 1 hour, the resulting mixture was transferred to a polytetrafluoroethylene-lined autoclave (100 mL) and reacted at 160°C for 12 hours. The final product was washed three times with double distilled water (DDW) and ethanol and vacuum dried at 60°C for 4 hours. Finally, BiOI microflowers with good dispersion were obtained.

[0007] The second purpose of the present invention is to construct a signal-sensitizing photoelectric biological probe with ZnIn2S4 QDs as the core.

[0008] The specific steps include:

[0009] Step 1. Synthesis of ZnIn2S4 QDs: 0.186 g Zn(OAC)2·2H2O, 0.496 g In(NO3)34.5H2O and 0.244 g thioacetamide were dissolved in 35 mL of DDW. After complete dissolution, the mixture was stirred vigorously for another 30 minutes, then placed in a polytetrafluoroethylene-lined autoclave and heated to 110 °C for 4 hours. After the reaction was completed, the mixture was taken out and cooled. After washing with ethanol and DDW, it was vacuum dried at 80 °C to finally obtain ZnIn2S4 QDs.

[0010] Step 2. Synthesis of TiO2NPs: An appropriate amount (1 g) of titanium dioxide P25 was dispersed in 20 mL of ethanol and stirred for 30 minutes. Under continuous stirring, a mixture of ethanol and water in a volume ratio of 1:1 was added to the above suspension and stirred for 30 minutes. The resulting suspension was then heated to 180°C in a polytetrafluoroethylene-lined autoclave for 6 hours. The resulting product was centrifuged, washed, and dried to obtain white TiO2NPs.

[0011] Step 3. Synthesis of probe (HP4 DNA-ZnIn2S4 QDs-TiO2NPs): 8 μL EDC was added to 100 μL of the prepared ZnIn2S4 QDs solution, shaken at room temperature for 40 minutes to activate the carboxyl group, and then 20 μL NHS stabilization solution was added. Then 100 μL of the amination TiO2NPs solution was added and shaken at 37 °C overnight. Finally, ZnIn2S4 QDs-TiO2 NPs were obtained. Then, the prepared ZnIn2S4 QDs-TiO2 NPs were placed in a centrifuge tube, 8 μL EDC was added, shaken at room temperature for 40 minutes, and then 20 μL NHS was added to stabilize the solution. Different concentrations of amination HP4 DNA and barcode DNA were added in a ratio of 3:7 and shaken at 37 °C overnight. Then, the unattached DNA was separated by centrifugation and redispersed in the same volume of DDW to form the HP4 DNA-ZnIn2S4 QDs-TiO2NPs signal probe. The DNA sequence used is:

[0012] HP4 DNA:5'-H2N-(CH2)6-ACA CTG CCG AAA CGC GGC AGT GA-3'

[0013] Barcode DNA: 5'-H2N-(CH2)6-TGG GTG TGC ACG CTA-3'

[0014] The third purpose of the present invention is to construct a photoelectrochemical biosensor based on ZnIn2S4 quantum dots and BiOI microflowers to detect targets.

[0015] The specific steps include:

[0016] Step 1. Target cyclic amplification: First, the DNA sequence used was treated at 95°C for 5 minutes, and then slowly cooled to room temperature. Then, 10 μL of target miRNA-21 of different concentrations and 15 μL of hairpin DNA of corresponding concentrations (HP1, HP2 and HP3 were 5 μL) were reacted in a constant temperature shaker at 37°C for 6 hours to produce a large number of tripod DNA walkers. The DNA sequence used was:

[0017] MiRNA-21: 5'-UAGCUUAUCAGACUGAUGUUGA-3'

[0018] HP1: 5'-TCG TTT CAA CAT CAG TCT GAT AAG CTA TGA TCA GAT CGT TAG CTTATC AGA CTG CGC GGC AGT GAT TAT A-3'

[0019] HP2: 5'-CAG TCT GAT AAG CTA ACG ATC TGA TCA CTG ATG TTG AAA TGA TCAGAT CGT TAG CGC GGC AGT GAT TAT A-3'

[0020] HP3: 5'-CTA ACG ATC TGA TCG TTT CAA CAT CAG TAG CTT ATC AGA CTG ATGTTG AAA CGA CGC GGC AGT GAT TAT A-3'

[0021] Step 2. Preparation of signal-enhanced photoelectric sensor: The ITO electrode was ultrasonicated in acetone, ethanol and double distilled water for 10 min, and then dried with N2. Next, a small rectangular ITO glass was glued to a 0.25 cm 2 (0.5cm×0.5cm) area.

[0022] Before use, all DNA strands were heated to 95 °C for 5 min and then slowly cooled to room temperature. After BiOI was aminated with (3-aminopropyl)triethoxysilane (APTES), 20 μL of BiOI solution was dropped onto the ITO surface and allowed to dry naturally. Then 20 mg mL -1 EDC and 10 mg mL -1 HP5 DNA was activated by NHS for 40 minutes, and the activated HP5 DNA (20 μL) was added to the BiOI modified electrode and incubated at 37 ° C for 2 hours to form ITO / BiOI / HP5. After slight washing, 20 μL of the tripod DNA walker was dropped on the electrode and incubated at 37 ° C for 2 hours to form ITO / BiOI / HP5 / Walker. After slight washing, 20 μL of the signal probe was dropped on the surface of the ITO / BiOI / HP5 / Walker modified electrode, reacted in a humid environment for 3 hours, and then lightly washed with TE buffer to remove the unconnected HP4 DNA signal probe. Finally, it was dried at room temperature to obtain a photoelectric biosensor, and the photoelectric signal was measured to detect the target miRNA-21. The DNA sequence used is:

[0023] HP5: 5'-COOH-CCG CCT ATA ATC ACT GCC GCG TTT CGG CAG TGT TCC CC-3'

[0024] Step 3. PEC detection: PEC detection was carried out in Tris HCl containing 10mM Amm ascorbic acid (AA) using a CHI660e electrochemical workstation and blue light as the excitation light source. A three-electrode system was used in this experiment: ITO as the working electrode, Pt wire as the counter electrode, and Ag / AgCl electrode as the reference electrode. Description of the drawings:

[0025] Figure 1 Principle of photoelectrochemical biosensor for detecting miRNA-21 based on ZnIn2S4 QDs-TiO2 NPs-BiOI composite.

[0026] Figure 2 Characterization of ZnIn2S4 quantum dots and TiO2 nanoparticles: (A) TEM image of ZnIn2S4 quantum dots, (B) UV-visible absorption image of ZnIn2S4 quantum dots, (C) band gap width image of ZnIn2S4 quantum dots, (D) TEM image of TiO2 NPs, (E) FT-IR image of TiO2 NPs.

[0027] Figure 3 Characterization of HP4 DNA-ZnIn2S4 QDs-TiO2 NPs and BiOI: (A) (B) TEM images of HP4 DNA-ZnIn2S4 QDs-TiO2 NPs, (C) UV-visible absorption spectrum of HP4 DNA-ZnIn2S4 QDs-TiO2NPs, (D) photoelectric signals of (a) TiO2NPs, (b) ZnIn2S4QDs, (c) ZnIn2S4QDs-TiO2 NPs, (E) SEM image of BiOI, (F) XRD pattern of BiOI.

[0028] Figure 4 Polyacrylamide gel electrophoresis (PAGE) and photoelectrochemical enhancement mechanism characterization: (A) PAGE analysis of the tripod DNA walker, (B) photocurrent intensity of BiOI, (C) photocurrent intensity of BiOI+signal probe, (D) photoelectrochemical enhancement mechanism.

[0029] Figure 5 Characterization of the photoelectrochemical biosensor preparation process: (A) photocurrent intensity characterization, (B) EIS characterization, (C) CV characterization.

[0030] Figure 6Analytical performance of photoelectrochemical biosensor for miRNA-21: (A) Photocurrent signal of biosensor for different concentrations of miRNA-21: 0M, 0.01fM, 0.1fM, 1fM, 0.01pM, 0.1pM, 1pM, 0.01nM, 0.1nM, 1nM, 0.01μM, 0.1μM, 1μM. (am)(B) Linear relationship between photocurrent change value ΔI and miRNA-21 concentration. (C) Selectivity of PEC biosensor for different targets. (D) Photocurrent stability for detecting target miRNA-21. Specific implementation method:

[0031] Example 1. An optoelectronic sensing detection platform based on ZnIn2S4 quantum dots and BiOI microflowers is used to achieve ultrasensitive detection of miRNA-21.

[0032] 8 μL EDC was added to 100 μL of the prepared ZnIn2S4 QDs solution, shaken at room temperature for 40 minutes to activate the carboxyl group, and then 20 μL NHS stabilization solution was added. Then 100 μL of the amination TiO2NPs solution was added and shaken at 37 °C overnight. Finally, ZnIn2S4 QDs-TiO2NPs were obtained. Then, the prepared ZnIn2S4 QDs-TiO2 NPs were placed in a centrifuge tube, 8 μL EDC was added, shaken at room temperature for 40 minutes, and then 20 μL NHS was added to stabilize the solution. Different concentrations of amination HP4 DNA and barcode DNA were added in a ratio of 3:7 and shaken at 37 °C overnight. Then, the unattached DNA was separated by centrifugation and redispersed in the same volume of DDW to form the HP4 DNA-ZnIn2S4 QDs-TiO2 NPs signal probe.

[0033] The DNA sequence was first treated at 95°C for 5 minutes and then slowly cooled to room temperature. Then, 10 μL of target miRNA-21 of different concentrations and 15 μL of hairpin DNA of corresponding concentrations (HP1, HP2 and HP3 were 5 μL) were reacted in a constant temperature shaker at 37°C for 6 hours to produce a large number of tripod DNA walkers.

[0034] After BiOI was aminated with (3-aminopropyl)triethoxysilane (APTES), 20 μL of BiOI solution was dropped onto the ITO surface and allowed to dry naturally. -1 EDC and 10 mg mL -1HP5 DNA was activated by NHS for 40 minutes, and the activated HP5 DNA (20 μL) was added to the BiOI modified electrode and incubated at 37 °C for 2 hours to form ITO / BiOI / HP5. After slight washing, 20 μL of the tripod DNA walker was dropped on the electrode and incubated at 37 °C for 2 hours to form ITO / BiOI / HP5 / Walker. After slight washing, 20 μL of the signal probe was dropped on the surface of the ITO / BiOI / HP5 / Walker modified electrode, reacted in a humid environment for 3 hours, and then lightly washed with TE buffer to remove the unconnected HP4 DNA signal probe. Finally, it was dried at room temperature to obtain the photoelectric biosensor, and the photoelectric signal was measured to detect the target miRNA-21.

[0035] PEC detection was carried out in Tris HCl containing 10mMAAmM ascorbic acid (AA) using a CHI660e electrochemical workstation and blue light as the excitation light source. A three-electrode system was used in this experiment: ITO as the working electrode, Pt wire as the counter electrode, and Ag / AgCl electrode as the reference electrode.

[0036] Example 2. An optoelectronic sensing detection platform based on ZnIn2S4 quantum dots and BiOI microflowers is used to achieve ultrasensitive detection of miRNA-21.

[0037] The phrase "adding activated HP5 DNA (20 μL) to the BiOI modified electrode and incubating at 37°C for 2 hours" was changed to "adding activated HP5 DNA (20 μL) to the BiOI modified electrode and incubating at 37°C for 3 hours". The other preparation conditions were the same as those in Example 1, and a biosensor with morphology and properties similar to those in Example 1 was obtained. The detection results of miRNA-21 were the same as those in Example 1.

[0038] Example 3. An optoelectronic sensing detection platform based on ZnIn2S4 quantum dots and BiOI microflowers is used to achieve ultrasensitive detection of miRNA-21.

[0039] The sentence “After slight washing, 20 μL of the tripod DNA walker was dropped on the electrode and incubated at 37°C for 2 hours” was changed to “After slight washing, 20 μL of the tripod DNA walker was dropped on the electrode and incubated at 37°C for 3 hours”. The other preparation conditions were the same as those in Example 1, and a biosensor with morphology and properties similar to those in Example 1 was obtained. The detection results of miRNA-21 were the same as those in Example 1.

[0040] Example 4. An optoelectronic sensing detection platform based on ZnIn2S4 quantum dots and BiOI microflowers is used to achieve ultrasensitive detection of miRNA-21.

[0041] The sentence “drop 20 μL of the signal probe on the surface of the ITO / BiOI / HP5 / Walker modified electrode and react in a humid environment for 3 h” was changed to “drop 20 μL of the signal probe on the surface of the ITO / BiOI / HP5 / Walker modified electrode and react in a humid environment for 2.5 h”. The other preparation conditions were the same as those in Example 1, and a biosensor with morphology and properties similar to those in Example 1 was obtained. The detection results of miRNA-21 were the same as those in Example 1.

[0042] Example 5. An optoelectronic sensing detection platform based on ZnIn2S4 quantum dots and BiOI microflowers is used to achieve ultrasensitive detection of miRNA-21.

[0043] The phrase "Finally, drying at room temperature to obtain a photoelectric biosensor" was changed to "Finally, drying at 37°C to obtain a photoelectric biosensor". The other preparation conditions were the same as those in Example 1, and a biosensor having a morphology and properties similar to those in Example 1 was obtained. The detection results of miRNA-21 were the same as those in Example 1.

Claims

1. A photoelectrochemical biosensor based on ZnIn2S4 quantum dots and BiOI microflowers and detection applications, characterized by: The surface of the ITO electrode was modified with BiOI microflowers through electrostatic binding force to generate a stable PEC photocurrent signal. A ZnIn2S4 QDs-TiO2 NPs sensitization probe for heterojunction signal amplification was constructed by loading a large number of ZnIn2S4 quantum dots on TiO2 nanoparticles. A large number of tripod DNA walkers were induced by the target microRNA-21, and the ZnIn2S4 QDs-TiO2 NPs probe was modified onto the electrode through DNA-specific hybridization to enhance the PEC signal and achieve ultra-sensitive detection of the target microRNA-21. The biosensor system has high sensitivity and practical applicability and can be used for various biological detection and early disease diagnosis. The characteristic method consists of the following steps: Step 1. Synthesis of BiOI microflowers: 30 mL of 0.10 M Bi(NO3)3·5H2O dissolved in ethylene glycol was slowly added dropwise to 30 mL of 0.10 M KI; after stirring at room temperature for 1 hour, the resulting mixture was transferred to a polytetrafluoroethylene-lined autoclave (100 mL) and reacted at 160°C for 12 hours; the final product was washed three times with double distilled water and ethanol, and vacuum dried at 60°C for 4 hours to finally obtain BiOI microflowers with good dispersion; Step 2. Synthesis of probe HP4 DNA-ZnIn2S4 QDs-TiO2NPs: 8 μL EDC was added to 100 μL ZnIn2S4QDs solution, shaken at room temperature for 40 minutes to activate the carboxyl group, and then 20 μL NHS stabilization solution was added; then 100 μL amination TiO2NPs solution was added, and shaken at 37°C overnight; finally, ZnIn2S4 QDs-TiO2 NPs were obtained; then, the prepared ZnIn2S4QDs-TiO2 NPs were placed in a centrifuge tube, 8 μL EDC was added, and shaken at room temperature for 40 minutes, and then 20 μL NHS was added to stabilize the solution; different concentrations of amination HP4 DNA and barcode DNA were added in a ratio of 3:7, and shaken at 37°C overnight; Then, the unattached DNA was separated by centrifugation and redispersed in the same volume of water to form the HP4DNA-ZnIn2S4 QDs-TiO2NPs signal probe; Step 3. Target cyclic amplification: First, the DNA sequence was treated at 95°C for 5 minutes, and then slowly cooled to room temperature; then, 10 μL of target miRNA-21 of different concentrations and 15 μL of hairpin DNA of corresponding concentrations, of which HP1, HP2 and HP3 were 5 μL, were reacted in a constant temperature shaker at 37°C for 6 hours to produce a large number of tripod DNA walkers; Step 2. Preparation of photoelectric sensor: After BiOI was aminated with APTES, 20 μL BiOI solution was added to the ITO surface and dried naturally; then 20 mg mL -1 EDC and 10 mg mL -1 HP5 DNA was activated by NHS for 40 minutes, and 20 μL of activated HP5 DNA was added to the BiOI modified electrode and incubated at 37°C for 2 hours to form ITO / BiOI / HP5; after slight washing, 20 μL of the tripod DNA walker was dropped on the electrode and incubated at 37°C for 2 hours to form ITO / BiOI / HP5 / Walker; after slight washing, 20 μL of the signal probe was dropped on the surface of the ITO / BiOI / HP5 / Walker modified electrode, reacted in a humid environment for 3 hours, and then lightly washed with TE buffer to remove the unconnected HP4 DNA signal probe; finally, dried at room temperature to obtain a photoelectric biosensor, and the photoelectric signal was measured to detect the target miRNA-21; Step 3. PEC detection: PEC detection was carried out in Tris HCl containing 10 mM ascorbic acid. The photocurrent was measured using a CHI660e electrochemical workstation with blue light as the excitation light source. A three-electrode system was used: ITO as the working electrode, Pt wire as the counter electrode, and Ag / AgCl electrode as the reference electrode.

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