An organic photoelectrochemical transistor biosensor for nucleic acid detection and a nucleic acid detection method
By forming a heterojunction of HOF and AIS QDs in an organic photoelectrochemical transistor biosensor and combining it with a multi-signal amplification strategy, the problems of insufficient photostability and sensitivity were solved, and highly sensitive detection of miRNA-21 was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHANGZHOU UNIV
- Filing Date
- 2026-03-20
- Publication Date
- 2026-06-12
Smart Images
Figure CN122193353A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biosensing technology, and specifically relates to an organic photoelectrochemical transistor biosensor for nucleic acid detection and a nucleic acid detection method. Background Technology
[0002] MicroRNAs are a class of endogenous non-coding small RNAs that play a crucial role in post-transcriptional gene regulation and are widely recognized as important biomarkers for various diseases, especially cancer. Therefore, achieving accurate and sensitive detection of specific miRNAs has become an important research direction. Currently used nucleic acid detection techniques include fluorescence, electrochemical, electrochemiluminescence, and surface-enhanced Raman spectroscopy. While these methods each have their advantages, they generally suffer from limitations such as insufficient sensitivity and lack of intrinsic signal amplification. In recent years, organic photoelectrochemical transistors (OPECTs) have shown significant potential as a next-generation biosensing platform for the sensitive detection of nucleic acids (including miRNAs). The working principle of OPECTs is based on photoinduced gate voltage modulation, which triggers ion injection into the bulk of the semiconductor channel and utilizes efficient ion-electron coupling to amplify the channel current, thereby providing intrinsic signal amplification and high gain conversion capabilities. However, the low abundance and small molecular size of miRNAs still pose challenges to achieving high detection sensitivity and selectivity. Therefore, integrating advanced signal amplification strategies into the OPECT system is crucial for improving miRNA detection performance. DNA cycling amplification techniques, such as catalytic hairpin assembly, hybridization chain reaction, and rolling circle amplification, have been successfully used to improve miRNA detection performance. These methods can generate a large number of DNA repetitive sequences based on trace amounts of target material, thereby significantly improving detection sensitivity and selectivity. For example, Chinese patent document (application number 202310647327.7) discloses the preparation and application of an organic photoelectrochemical transistor sensor based on MOF derivatives. A tetrahedral Ce-MOF is prepared by a solvothermal method, and Ce-MOF is derivatized into Ce-ZnIn2S4 using a one-pot method. Ce-ZnIn2S4 is then modified onto the surface of an FTO conductive glass using PAMAM. Y-DNA probes and double-stranded substrates are prepared: the Y-DNA probes are made by mixing equal amounts of single-stranded DNA, annealing, natural cooling, and cryogenic storage; the double-stranded substrates are prepared using the same method. The activated Y-DNA probes are used to modify the electrode surface, washed with buffer, and the unbound active sites are blocked with MEA solution. After washing, miRNA-25 is added for incubation, followed by washing again. The electrode is then immersed in a solution containing the hybridization substrate for incubation. Sensing detection is achieved using a nonlinear hybridization chain reaction, enabling sensitive detection of miRNA-25. Hydrogen-bonded organic frameworks (HOFs) have attracted widespread attention due to their tunable structure, inherent porosity, and good renewability, making them a promising material system for photoelectrochemical applications. However, their practical application in sensing is often limited by photostability issues under operating conditions, and they have not been effectively used in OPECT sensors. Summary of the Invention
[0003] To overcome the limitations of HOF applications in the OPECT biosensing platform and address the issues of HOF photostability and platform sensitivity, this invention provides an organic photoelectrochemical transistor biosensor for nucleic acid detection, specifically for ultrasensitive miRNA-21 analysis. By forming a heterojunction between HOF and AIS QDs (further, through electrostatic assembly deposition of PDDA (polydiallyldimethylammonium chloride), not only is photogenerated charge separation promoted and carrier recombination suppressed to enhance photocurrent response, but the photostability of the recombination interface is also enhanced under continuous operating conditions. Nucleic acid detection based on this sensor achieves a detection limit as low as 0.004 fM, enabling rapid, sensitive, accurate, and efficient detection of target analytes.
[0004] To achieve the above objectives, the technical solution of the present invention is as follows:
[0005] An organic photoelectrochemical transistor biosensor for nucleic acid detection uses an organic-inorganic HOF-101 / AIS QDs heterojunction material as the photosensitive gate material.
[0006] The nucleic acid detection method based on this sensor involves a rolling circle amplification initiated by a nucleic acid target (e.g., miRNA-21) through a catalytic hairpin assembly reaction, generating a DNA product rich in G bases. This product then induces a horseradish peroxidase-catalyzed precipitation reaction at the gate interface. The gradual formation of the insulating precipitation layer dynamically modulates the effective gate potential under illumination, thereby sensitively adjusting the channel response to achieve nucleic acid detection.
[0007] Furthermore, the gate electrode of the sensor includes an electrode substrate, a photosensitive gate material layer coated on the surface of the electrode substrate, and a biological probe layer coated on the photosensitive gate material layer. The biological probe layer is obtained by modifying H1 DNA, and the H1 DNA is complementary to the nucleic acid to be tested.
[0008] Furthermore, the method for forming the HOF-101 / AIS QDs heterojunction material includes: drop-coating HOF-101 nanorods onto an electrode substrate, and then loading AIS QDs via electrostatic assembly to obtain the HOF-101 / AIS QDs heterojunction material. More specifically, an aqueous suspension of HOF-101 nanorods at a concentration of 0.75–1.25 mg / ml is first prepared, then drop-coated onto the active region of a hydroxylated electrode substrate. After drying at room temperature, the suspension is immersed in a PDDA solution for at least 5 min, rinsed with deionized water, and then immersed in an AIS QDs solution to ensure complete adsorption and electrostatic assembly. The preferred concentration of the HOF-101 nanorod aqueous suspension is 1 mg / ml.
[0009] Furthermore, the crystal length of the HOF-101 nanorods ranges from 6 to 10 μm.
[0010] Taking miRNA-21 as an example, as a highly conserved endogenous non-coding small RNA, it is a key regulator in the post-transcriptional gene regulatory network, and its main functions involve basic biological processes such as cell proliferation, apoptosis inhibition, and immune response regulation. Simultaneously, miRNA-21 also plays an important role in maintaining tissue homeostasis and disease development. Studies have shown that various disease states, such as malignant tumors, cardiovascular diseases, fibrotic lesions, and inflammatory diseases, are closely related to abnormal miRNA-21 expression. In clinical diagnosis and treatment, accurate quantification of miRNA-21 expression levels has become an important basis for early disease screening, prognostic assessment, and treatment monitoring. Therefore, achieving highly sensitive detection of miRNA-21 in complex biological samples has significant clinical implications. Based on this, this invention selects miRNA-21 as a typical detection target, reflecting its representativeness in nucleic acid sensing research and highlighting the application prospects of this technology in clinical molecular diagnostics and precision medicine.
[0011] Another object of the present invention is to provide a method for preparing an organic photoelectrochemical transistor biosensor for nucleic acid detection.
[0012] To achieve the above objectives, the present invention adopts the following technical solution:
[0013] An organic photoelectrochemical transistor biosensor for nucleic acid detection, wherein the method for fabricating the sensing electrode includes the following steps:
[0014] (1) Preparation of ITO / HOF-101 / AIS QDs organic-inorganic heterojunction gate: HOF-101 nanorods were drop-coated on an indium-doped SnO2 conductive glass (ITO) electrode, and AIS QDs were deposited on the HOF-101 nanorods by electrostatic assembly of PDDA (polydiallyl dimethyl ammonium chloride) to obtain an ITO / HOF-101 / AIS QDs gate with high photocurrent signal response;
[0015] (2) Preparation of ITO / HOF-101 / AIS QDs / H1 DNA sensing electrode: Hairpin DNA1 (H1 DNA) was modified onto the gate of the ITO / HOF-101 / AIS QDs, and then bovine serum albumin (BSA) was dropped onto the electrode to obtain the ITO / HOF-101 / AIS QDs / H1 DNA sensing electrode.
[0016] The ITO / HOF-101 / AIS QDs / H1 DNA sensing electrode is incubated with the nucleic acid target (generally around 37 °C) to allow the H1 DNA to undergo a nucleic acid complementary reaction with the nucleic acid target. Then, hairpin DNA2 (H2 DNA) is added to initiate a triple signal amplification reaction, which is then combined with the channel of the organic photoelectrochemical transistor biosensor to achieve sensitive detection of the nucleic acid target.
[0017] By adopting the above technical solution, the beneficial effects of the present invention are as follows:
[0018] The preparation method disclosed in this invention is simple, easy to operate, and suitable for promotion and application.
[0019] Preferably, in step (1), AgNO3, In(NO3)3, and Na2S are used as Ag source, In source, and S source, respectively, and the molar ratio of Ag source, In source, and S source is fixed. The mixed solution is vigorously stirred under nitrogen protection, heated to reflux and held for a period of time, and then naturally cooled to room temperature to obtain the final AIS QDs. They are then fixed onto the ITO / HOF-101 electrode through electrostatic assembly.
[0020] More preferably, in the electrostatic assembly, the PDDA with a mass fraction of 1% is first soaked, and the assembly time is 5 minutes. Then, the AIS QDs are assembled, and the assembly time is 15 minutes.
[0021] It should be noted that ITO / HOF-101 / AIS QDs are environmentally friendly, have a simple preparation process, and exhibit significant current signal response and good stability.
[0022] Preferably, in step (2), 10 μL of H1 DNA is added to the ITO / HOF-101 / AIS QDs electrode and incubated at 4 °C for 12 h; after washing with tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (Tris–HCl) (10 mM, pH 7.4), 1.0% (w / v) bovine serum albumin (BSA) is added to the H1 DNA-modified electrode and incubated at room temperature for 30 min to block the active site and form a sensing electrode.
[0023] It should be noted that the above-mentioned sensor electrode preparation conditions are mild, the process is simple and rapid, and the amount of biological sample consumed is small.
[0024] Preferably, in step (3), the target compound miRNA-21 is added dropwise to the sensing electrode and incubated at room temperature for 10 min, followed by the addition of H2 DNA. Subsequently, a lock-lock DNA probe (PDNA) and T4 DNA ligase are added sequentially. Then, a mixture of phi29 DNA polymerase and dNTPs, along with heme, are added. The mixture is then placed in a solution of H2O2 and 4-chloro-1-naphthol and reacted at room temperature for 15 min to achieve the detection of miRNA-21.
[0025] It should be noted that the sensor is an organic photoelectrochemical transistor biosensor that utilizes an organic-inorganic HOF-101 / AIS QDs heterojunction gate combined with a triple signal amplification strategy. The detection of miRNA-21 is simple, highly sensitive, accurate, convenient, and fast. Attached Figure Description
[0026] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof.
[0027] Figure 1 This is an infrared variation diagram of the HOF-101 preparation process in Example 1 of the present invention.
[0028] Figure 2 This is a current signal response diagram of the ITO / HOF-101 electrode corresponding to different concentrations of HOF-101 in Example 1 of the present invention.
[0029] Figure 3 This is a scanning electron microscope image of the HOF-101 electrode in Embodiment 1 of the present invention.
[0030] Figure 4 This is a transmission electron microscope image of AIS QDs in Embodiment 2 of the present invention.
[0031] Figure 5 This is a transmission electron microscope image of the HOF-101 / AIS QDs electrode in Embodiment 3 of the present invention.
[0032] Figure 6 This is the X-ray photoelectron spectrum of HOF-101 / AIS QDs in Example 3 of the present invention.
[0033] Figure 7 This is a current signal response diagram of the DNA sensor fabrication process in Embodiment 4 of the present invention.
[0034] Figure 8 This is a standard curve of the DNA sensor detecting the target miRNA-21 in Example 4 of the present invention.
[0035] Figure 9 This is a graph showing experimental data of selective recognition by the DNA sensor in Embodiment 5 of the present invention.
[0036] Figure 10 This is a spike recovery experiment in the serum sample of Example 6. Detailed Implementation
[0037] The present invention will now be described in detail with reference to the embodiments.
[0038] In the experiments of this invention, the current signal response was tested on an organic photoelectrochemical transistor system, as detailed below:
[0039] An LED light source with an emission wavelength of 425 nm was used as the excitation source, and the electrolyte was 0.1 M phosphate-buffered saline (PBS, pH 7.4) containing 0.1 M ascorbic acid (AA). A switching cycle was performed every 10 s. An electrochemical workstation was used to record the channel current signal. When testing the channel current response, a modified 0.5 cm × 0.5 cm photoelectrode was used as the gate, and the source and drain of the transistor were connected to the corresponding electrodes. The system operated under zero gate voltage conditions without applying an external voltage. Conventional photoelectrochemical (PEC) measurements used a standard three-electrode system, employing a modified ITO electrode as the working electrode, a platinum wire as the counter electrode, and a saturated Ag / AgCl electrode as the reference electrode.
[0040] For the fabrication of the channel portion of the organic photoelectrochemical transistor, the glass substrate was ultrasonically cleaned with acetone, ethanol, and deionized water for 10 min each, and then dried at 60 °C. The substrate was then further treated with plasma. Subsequently, the substrate was shielded with a template with a channel length of 6.0 mm and a width of 0.2 mm, and coated with a 10 nm chromium and a 100 nm gold target by magnetron sputtering. Next, the obtained substrate was further cleaned with plasma, and then spin-coated onto the electrodes with a mixture of 1.5 mL of PEDOT:PSS solution and 7.5 μL of 5% (v / v) dimethyl sulfoxide at 3500 rpm to form a uniform PEDOT:PSS film. Finally, the substrate was annealed at 180 °C for 1 h in an inert atmosphere to ensure a more robust adhesion of the PEDOT:PSS to the electrodes, thus obtaining the channel portion of the OPECT device.
[0041] Example 1
[0042] Preparation of HOF-101: First, 20 mg of the precursor 1,3,6,8-tetratetra(4-carboxyphenyl)pyrene (H4TBAPy) was dispersed in 1 mL of dimethylformamide (DMF) and a homogeneous solution was obtained by sonication. Then, 12 mL of methanol was rapidly added and mixed with stirring for 12 h to ensure complete dissolution of the precursor and its self-assembly into a hydrogen-bonded organic framework. The resulting mixture was centrifuged, and the yellow precipitate was collected and washed several times with methanol to remove unreacted precursors and impurities, followed by rinsing with deionized water to remove residual solvent. Finally, the precipitate was freeze-dried to obtain the target product, HOF-101 powder.
[0043] The infrared spectrum of the HOF-101 preparation process is shown below. Figure 1 As shown in the figure, during the preparation process, the C=O stretching vibration peak of the carboxyl group (-COOH) (1695 cm⁻¹) can be observed. -1 The redshift occurred, possibly due to the formation of hydrogen bonds leading to a decrease in the stretching vibration energy of the C=O bond.
[0044] Optimization of detection conditions
[0045] HOF solution concentration
[0046] The magnitude of the current signal response of the ITO / HOF-101 photoelectrode has a significant impact on the detection sensitivity of the final organic photoelectrochemical transistor biosensor. Therefore, the fabrication process parameters of the ITO / HOF-101 photoelectrode were optimized as follows:
[0047] The concentrations of HOF-101 solutions were 0.5 mg / mL, 0.75 mg / mL, 1.00 mg / mL, 1.25 mg / mL, and 1.5 mg / mL. These solutions were coated onto an ITO electrode and dried at room temperature to obtain HOF-101 layers of varying thicknesses (0.06–0.1 mg / cm²). 2 ITO / HOF-101 electrode.
[0048] The results can be obtained by performing photocurrent characterization tests (i.e., the conventional PEC test mentioned above), as shown in the attached figure. Figure 2 As shown, the ITO / HOF-101 electrode exhibits the best current signal response when the HOF-101 concentration is 1.0 mg / mL. Therefore, a HOF-101 concentration of 1.0 mg / mL is selected as the optimal preparation process parameter.
[0049] Scanning electron microscope image as shown Figure 3As shown, HOF-101 consists of uniformly dispersed rod-shaped crystals with lengths ranging from 6 to 10 μm. These rod-shaped crystals exhibit a regular morphology and size distribution, indicating that crystal growth was relatively uniform during the synthesis of HOF-101. The crystal surfaces are smooth and no obvious aggregation was observed, indicating that the material has good dispersibility.
[0050] Example 2
[0051] Preparation of AIS QDs: A 60 mL aqueous solution containing 20 mM In(NO3)3 and 5 mM AgNO3 was transferred to a 100 mL three-necked flask. 100 μL of mercaptopropionic acid (MPA) was added to the solution, and the pH was adjusted to 10 using 1 M NaOH. The mixture was then purged with nitrogen under an inert atmosphere. Under a nitrogen atmosphere, the mixture was vigorously stirred, and 3.6 mL of 0.2 M Na2S solution was rapidly injected into the mixture. The solution was then heated to 100 °C and refluxed for 3 h to ensure sufficient growth of the AIS QDs. The resulting quantum dot dispersion (approximately 4.7 mM) was cooled to room temperature and stored at 4 °C for later use.
[0052] Transmission electron microscope image as shown Figure 4 As shown, the AIS QDs are approximately spherical and uniformly distributed, with a relatively uniform particle size distribution, indicating that the self-assembly process of the material is well controlled during synthesis, effectively avoiding particle aggregation.
[0053] Example 3
[0054] Fabrication of ITO / HOF-101 / AIS QDs gate
[0055] ITO substrate pretreatment: ITO conductive glass was sequentially immersed in acetone, anhydrous ethanol, and deionized water, and ultrasonically cleaned for 15 min in each solvent. After cleaning, it was dried with high-purity nitrogen and then subjected to plasma treatment for 5 min to improve the degree of hydroxylation and wettability of the substrate surface.
[0056] Preparation of the HOF-100 modified layer: First, 6 mg of HOF-101 powder was dispersed in 6 mL of deionized water and ultrasonically treated to obtain a uniform 1 mg / mL suspension. The pre-cleaned ITO electrode was shielded with opaque insulating tape to define the effective working area of the electrode, wherein the effective working area was 0.5 cm × 0.5 cm. Then, 20 μL of the HOF-101 suspension was dropped onto this area and allowed to air dry at room temperature to obtain a uniform HOF-101 thin film electrode.
[0057] Electrostatic assembly of the AIS QDs layer: The HOF-101 electrode was immersed in a 1.0% (w / w) PDDA solution for 5 min, rinsed with deionized water, and then immersed in the AIS QDs solution for 15 min. After rinsing to remove non-specific adsorbates, the HOF-101 / AIS QDs photoelectrode was obtained. This fabrication process ensures uniform film coverage, good interfacial adhesion, and a favorable composite interface to achieve efficient charge separation and transport.
[0058] Scanning electron microscope, such as Figure 5 As shown in the figure, after AIS QDs deposition, nanoscale particles are uniformly anchored on the HOF-101 rod-like structure, resulting in an increase in surface roughness.
[0059] X-ray photoelectron spectroscopy, such as Figure 6 As shown, the C 1s and O 1s characteristic peaks from HOF-101 are obvious, as are the Ag 3d, In 3d and S 2p characteristic peaks from AIS QDs, confirming the presence of the expected element and verifying the successful composite of HOF-101 / AIS QDs.
[0060] Example 4
[0061] Fabrication of ITO / HOF-101 / AIS QDs / H1 DNA Sensing Electrode
[0062] The carboxyl groups of AIS QDs on the HOF-101 / AIS QDs photoelectrode were activated with 10 mM EDC / NHS solution at room temperature for 30 min to promote covalent coupling. Prior to immobilization, all hairpin probes (H1 DNA and H2 DNA) were heated to 95 °C and incubated for 5 min, then gradually cooled to room temperature to restore their correct secondary structure. Subsequently, 10 μL of H1 DNA solution (1.0 μM) was added to the photoelectrode and incubated overnight at 4 °C to achieve covalent immobilization (i.e., covalent coupling) via the activated carboxyl groups. The electrode was rinsed with 10 mM Tris–HCl buffer (pH 7.4) to remove unbound probes, and residual active sites were blocked with 1.0% (w / v) bovine serum albumin (BSA).
[0063] The detection of the target miRNA-21 was performed by a combination of a triple signal amplification reaction: hairpin self-assembly (CHA), rolling circle amplification (RCA), and horseradish peroxidase-catalyzed precipitation.
[0064] For CHA, the ITO / HOF-101 / AIS QDs / H1 DNA sensing electrode was incubated with solutions containing different concentrations of miRNA-21 (10 μL) for 10 min, followed by the addition of 10 μL of H2 DNA (1.0 μM) and incubation at 37°C for 2 h. The target miRNA-21 opened the H1 DNA and complemented it with the H2 DNA to form an H1 / H2 DNA double-stranded structure, while simultaneously releasing miRNA-21 to participate in subsequent reactions.
[0065] For RCA, annealed pDNA (10 μL, 1.0 μM) was added to a pre-formed H1 / H2 double-stranded electrode, followed by 0.5 μL of T4 DNA ligase (5 U / μL). The ligation reaction was carried out at 37°C for 30 min, followed by enzyme inactivation at 65°C for 10 min. Then, 2 μL of a dNTP mixture (25 mM each) and 0.5 μL of phi29 DNA polymerase (10 U / μL) were added, and the mixture was incubated at 37°C for 50 min to perform the RCA reaction. This process generates a long single-stranded G-quadruplex DNA sequence containing repeating sequences complementary to the pDNA on the electrode surface. The electrode was washed with Tris–HCl buffer to remove unreacted reagents.
[0066] Finally, 5.0 μL of heme (6.0 μM) was added to the electrode surface and incubated for 60 min to allow it to intercalate into the G-quadruplex structure formed by the RCA product, generating a heme / G-quadruplex DNase with horseradish peroxidase-like activity. For catalytic precipitation, 5 μL of H₂O₂ (0.15 mM) and 5 μL of 1 mM 4-chloro-1-naphthol (4-CN) were added and incubated for 15 min. The heme / G-quadruplex DNase catalyzed the H₂O₂-mediated oxidation of 4-CN, forming an insoluble precipitate on the electrode surface, providing a stable and amplified signal for the detection of miRNA-21.
[0067] Nucleic acid sequence design and synthesis
[0068] All nucleic acid sequences used in this invention were synthesized by Shanghai Sangon Biotech Co., Ltd. Their design and function are as follows:
[0069] H1 DNA sequence (5'-3'): TCA ACA TCA GTC TGA TAA GCT ACC TCA CAC GAA TTGTAG CTT ATC AGA CTT-(CH2)6- NH2
[0070] H2 DNA sequence (5'-3'): TAA GCT ACA ATT CGT GTG AGG TAG CTT ATC AGA CTCTCA CAC GAA TTC ATC TAA TTT
[0071] Lock-lock DNA probe (pDNA) sequence (5'-3'): Phosphate- AAT TCG TGT GAG TTT CCC AACCCG CCC TAC CCT TTT CAA CAT CAG TCT GAT AAG CTA TTT TTT CCC AAC CCG CCC TACCCT TAA ATT AGA TG
[0072] Target miRNA-21 sequence (5'-3'): UAG CUU AUC AGA CUG AUG UUG A
[0073] miRNA-25 sequence (5'-3'): CAT TGC ACT TGT CTC GGT CTG AA
[0074] miRNA-10b sequence (5'-3'): UAC CCU GUA GAA CCG AAU UUG UG
[0075] miRNA-17 sequence (5'-3'): CAA AGU GCU UAC AGU GCA GGU AA
[0076] The current signal response diagram during the DNA sensor fabrication process is shown in the figure below. Figure 7 As shown. After H1 DNA assembly (2), BSA blocking (3) and CHA process (4) were performed sequentially, the channel current of ITO / HOF-101 / AIS QDs (1) gradually decreased. This was due to the modification of the insulating BSA layer and the gradual accumulation of negatively charged nucleic acids on the gate. After the RCA and enzyme-catalyzed precipitation reaction, the channel current decreased significantly (step 5).
[0077] Within the target miRNA-21 concentration range of 0.01 fM to 10.0 pM, the channel current change rate showed a linear relationship with the logarithm of the miRNA-21 concentration. The standard curve for the detection of the target miRNA-21 by the DNA sensor is shown below. Figure 8As shown, the linear fitting equation is ΔI / I0 = 1.97 + 0.12logC (M), with a linear correlation coefficient of 0.9949 and an experimental limit of detection of 0.004 fM. This indicates that the organic photoelectrochemical transistor biosensor based on the HOF-101 / AgInS2 heterojunction and triple signal amplification strategy has high sensitivity for miRNA-21 detection. Where I0 is the channel current value without miRNA-21, ΔI is the difference between the channel current (I) at the corresponding concentration of miRNA-21 and the channel current (I0) without miRNA-21, and C is the concentration of miRNA-21.
[0078] The detection of unknown concentrations of miRNA-21 was performed by a combination of a triple signal amplification reaction, namely, catalytic hairpin assembly reaction (CHA), rolling circle amplification reaction (RCA), and horseradish peroxidase-catalyzed precipitation.
[0079] For CHA, the ITO / HOF-101 / AIS QDs / H1 DNA sensing electrode was incubated with a solution containing an unknown concentration of miRNA-21 (10 μL) for 10 min, followed by the addition of 10 μL of H2 DNA (1.0 μM) and incubation at 37°C for 2 h. The target miRNA-21 opened the H1 DNA and complemented it with the H2 DNA to form an H1 / H2 DNA double-stranded structure, while simultaneously releasing miRNA-21 to participate in subsequent reactions.
[0080] For RCA, annealed pDNA (10 μL, 1.0 μM) was added to a pre-formed H1 / H2 double-stranded electrode, followed by 0.5 μL of T4 DNA ligase (5 U / μL). The ligation reaction was carried out at 37°C for 30 min, followed by enzyme inactivation at 65°C for 10 min. Then, 2 μL of a dNTP mixture (25 mM each) and 0.5 μL of phi29 DNA polymerase (10 U / μL) were added, and the reaction was incubated at 37°C for 50 min to perform the RCA reaction. This process generates a long single-stranded G-quadruplex DNA sequence containing repeating sequences complementary to the pDNA on the electrode surface. The electrode was washed with Tris–HCl buffer to remove unreacted reagents.
[0081] Finally, 5.0 μL of heme (6.0 μM) was added to the electrode surface and incubated for 60 min to allow it to intercalate into the G-quadruplex structure formed by the RCA product, generating a heme / G-quadruplex DNase with horseradish peroxidase-like activity. For catalytic precipitation, 5 μL of H₂O₂ (0.15 mM) and 5 μL of 1 mM 4-CN were added and incubated for 15 min. The heme / G-quadruplex DNase catalyzed the H₂O₂-mediated oxidation of 4-CN, forming an insoluble precipitate on the electrode surface, providing a stable and amplified signal for the detection of miRNA-21. Based on the obtained detection signal, the concentration of miRNA was obtained by substituting it into a linear fitting equation.
[0082] Example 5
[0083] Selectivity testing of organic photoelectrochemical transistor biosensors based on HOF-101 / AgInS2 organic-inorganic heterojunction and triple signal amplification strategy:
[0084] To demonstrate the excellent selectivity of the organic photochemical transistor biosensor based on the HOF-101 / AgInS2 organic-inorganic heterojunction and triple signal amplification strategy, three potential interfering agents (miRNA-25, miRNA-10b, and miRNA-17) were used for selectivity evaluation. The concentrations of the target and each interfering agent were 10 pM. The results are as follows: Figure 9 As shown, only the target miRNA-21 and its mixture caused significant signal changes, confirming the high selectivity of the sensor device.
[0085] Example 6
[0086] The practical application capability of the organic photochemical transistor biosensor based on the HOF-101 / AgInS2 organic-inorganic heterojunction and triple signal amplification strategy prepared in this invention was evaluated through a spiked recovery experiment in serum samples. The specific operation is as follows:
[0087] Serum was diluted 10-fold and divided into three groups. miRNA-21 standard samples at concentrations of 0.1 fM, 1 fM, and 10 fM were added to each group, respectively. Using the organic photoelectrochemical transistor biosensor based on the HOF-101 / AgInS2 heterojunction and triple signal amplification strategy prepared in this invention, 10 μL of serum spiked samples at different concentrations were incubated for 10 min at room temperature. After washing the electrode, 10 μL of H2 DNA (1.0 μM) was added and incubated at 37°C for 2 h. Then, 10 μL of pDNA (1.0 μM) was added to the pre-formed H1 / H2 DNA double-stranded electrode, followed by 0.5 μL of T4 DNA ligase (5 U / μL). The ligation reaction was carried out at 37°C for 30 min, followed by enzyme inactivation at 65°C for 10 min. Next, 2 μL of dNTP mixture (25 mM each) and 0.5 μL of phi29 DNA polymerase (10 U / μL) were added, and the mixture was incubated at 37 °C for 50 min. The electrode was rinsed to remove unreacted reagents. Finally, 5.0 μL of heme (6.0 μM) was added to the electrode surface and the mixture was incubated for 60 min. Finally, 5 μL of H2O2 (0.15 mM) and 5 μL of 1 mM 4-chloro-1-naphthol (4-CN) were added, and the mixture was incubated for 15 min. Photocurrent signal was measured in PBS buffer (pH 7.4, 0.1 M) containing 0.1 MAA of electron donor. Based on the obtained detection signal, the concentration of miRNA was obtained by substituting it into a linear fitting equation.
[0088] Spike recovery experiments in serum samples are attached. Figure 10 As shown in the figure, Added represents the added standard concentration, and Found represents the actual detected concentration. The recovery rate of the spiked sample is in the range of 95.3% to 102.5%, and the relative standard deviation of the test results is within 8%. This demonstrates the excellent application potential of the prepared organic photoelectrochemical transistor biosensor based on HOF-101 / AgInS2 heterojunction and triple signal amplification strategy in accurately detecting miRNA-21 in real complex biological samples, and can achieve rapid, sensitive, accurate and efficient detection of the target analyte.
[0089] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the technical scope disclosed in the present invention, based on the technical solution and concept of the present invention, should be covered within the scope of protection of the present invention. It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.
Claims
1. An organic photoelectrochemical transistor biosensor for nucleic acid detection, characterized in that, The HOF-101 / AIS QDs heterojunction material formed by HOF-101 nanorods and AgInS2 quantum dots (AIS QDs) was used as the photosensitive gate material.
2. The organic photoelectrochemical transistor biosensor for nucleic acid detection according to claim 1, characterized in that, The method for forming HOF-101 / AIS QDs heterojunction material includes: drop-coating HOF-101 nanorods on an electrode substrate, and then loading AIS QDs by electrostatic assembly to obtain HOF-101 / AIS QDs heterojunction material.
3. The organic photoelectrochemical transistor biosensor for nucleic acid detection according to claim 1, characterized in that, The method for forming HOF-101 / AIS QDs heterojunction materials also includes: first, preparing an aqueous suspension of HOF-101 nanorods at a concentration of 0.75~1.25 mg / ml, then drop-coating it onto the active region of a surface-hydroxylated electrode substrate, drying it at room temperature, immersing it in a PDDA solution for at least 5 min, rinsing it with deionized water, and then immersing it in an AIS QDs solution to fully adsorb and complete electrostatic assembly.
4. The organic photoelectrochemical transistor biosensor for nucleic acid detection according to claim 3, characterized in that, The crystal length of HOF-101 nanorods ranges from 6 to 10 μm; And / or, the amount of HOF-101 nanorods dropped onto the electrode substrate surface is 0.06~0.1 mg / cm³. 2 .
5. The organic photoelectrochemical transistor biosensor for nucleic acid detection according to claim 1, characterized in that, The concentration of the HOF-101 nanorod aqueous suspension was 1 mg / ml.
6. The organic photoelectrochemical transistor biosensor for nucleic acid detection according to claim 1, characterized in that, The sensor's sensing electrode includes an electrode substrate, a photosensitive gate material layer coated on the surface of the electrode substrate, and a biological probe layer coated on the photosensitive gate material layer. The biological probe layer is obtained by modifying H1 DNA.
7. The organic photoelectrochemical transistor biosensor for nucleic acid detection according to claim 6, characterized in that, The method for fabricating the sensing electrode of the sensor includes the following steps: (1) Preparation of ITO / HOF-101 / AIS QDs organic-inorganic heterojunction gate: HOF-101 nanorods are dropped onto an indium-doped SnO2 conductive glass ITO electrode, and AIS QDs are deposited on the HOF-101 nanorods by PDDA electrostatic assembly to obtain the gate. (2) Preparation of ITO / HOF-101 / AIS QDs / H1 DNA sensing electrode: Hairpin H1 DNA is modified onto the gate of the ITO / HOF-101 / AIS QDs, and then BSA is dropped onto the electrode to obtain the ITO / HOF-101 / AIS QDs / H1 DNA sensing electrode.
8. A nucleic acid detection method, characterized in that, Based on the organic photoelectrochemical transistor biosensor for nucleic acid detection according to any one of claims 1 to 7, the method comprises the following steps: in the presence of a nucleic acid target, a target-triggered catalytic hairpin assembly reaction initiates rolling circle amplification to generate a DNA product rich in G bases; the product then induces a horseradish peroxidase-catalyzed precipitation reaction at the gate interface.
9. The nucleic acid detection method according to claim 8, characterized in that, It also includes the following steps: Immerse the ITO / HOF-101 / AIS QDs / H1 DNA sensing electrode in miRNA-21 solution and incubate. Then add H2 DNA and incubate until miRNA-21 opens H1 DNA and complements H2 DNA to form H1 / H2 DNA double-stranded structure, while releasing miRNA-21. Then, annealed PDNA was added to the electrode, followed by the addition of T4 DNA ligase solution for ligation. After the reaction was completed, the enzyme was inactivated. Next, dNTP mixture and phi29 DNA polymerase were added, and the mixture was incubated at room temperature for the RCA reaction. Heme was then added and incubated to allow it to embed into the G-quadruplex structure formed by the RCA product, generating a heme / G-quadruplex DNase with horseradish peroxidase-like activity. During catalytic precipitation, H2O2 solution and 4-chloro-1-naphthol solution were added and incubated. The heme / G-quadruplex DNase catalyzed the H2O2-mediated oxidation reaction of 4-chloro-1-naphthol, forming an insoluble precipitate on the electrode surface. The channel current was measured and a linear equation was constructed between the rate of change of channel current and the logarithm of the miRNA-21 concentration. Finally, the ITO / HOF-101 / AIS QDs / H1 DNA sensing electrode was immersed in the miRNA-21 solution to be tested and incubated. The above steps were repeated, the channel current was measured, and the concentration of the miRNA-21 solution to be tested was calculated by substituting it into the linear equation.