A preparation method of a dual-mode detection beta thalassemia gene CD122 self-powered biosensor

Abstract

The application discloses a preparation method of a dual-mode CD122 gene thalassemia detection self-powered biosensor, successfully applies an enzyme biological fuel cell (EBFC) to dual-mode detection of CD122 gene thalassemia, realizes quantitative analysis through changes of EBFC output voltage and changes of electrolyte color (RGB Blue value), and mainly concentrates on a cathode of the EBFC in the self-powered design. When CD122 exists, an electrode starts catalytic hairpin self-assembly (CHA) amplification, realizes recycling of CD122, and provides a binding site for subsequent hybridization chain reaction (HCR) amplification, at this time, methylene blue (MB) adsorbed by a biological cathode increases, and an electric signal is enhanced, and meanwhile, electrolyte color becomes lighter (RGB Blue value becomes larger). The self-powered CD122 dual-mode biosensor based on the EBFC disclosed by the application can realize simple, rapid, sensitive and efficient detection of a target.

Description

Technical Field

[0001] This invention relates to the field of biosensor fabrication methods, specifically to a self-powered biosensor based on an enzyme biofuel cell and its ultrasensitive dual-mode detection method for the thalassemia gene CD122. Background Technology

[0002] Enzyme biofuel cells (EBFCs), also known as bioelectrochemical fuel cells, are hybrid systems combining electrochemical energy conversion with biocatalysis, directly converting chemical energy into electrical energy. Traditional electrochemical sensors require an external power source, making rapid on-site detection of target analytes difficult. Self-powered biosensors based on EBFCs are sensors that use the battery's output signal as the analytical detection signal; this signal is proportional to the concentration of the analyte. Due to their advantages such as requiring no external power source, ease of operation, high sensitivity, and strong anti-interference capabilities, self-powered biosensors based on EBFCs hold promise as a novel detection technology combining high sensitivity, high selectivity, and high accuracy. During detection, expensive and complex electrochemical workstations and amplifiers can be eliminated, making them energy generators for implantable and portable biomedical devices. The signal can be detected using a simple voltmeter, enabling real-time monitoring.

[0003] Thalassemia, or thalassemia for short, is a genetic disorder controlled by a pair of alleles. It is one of the most prevalent single-gene genetic diseases worldwide, with nearly 7% of the global population being carriers. It is mainly distributed in Mediterranean countries, Southeast Asia, and Africa, with high incidence in southern my country, particularly in Guangxi, Guangdong, Hainan, Yunnan, Guizhou, and Sichuan provinces. Among thalassemia patients, those carrying the mutant thalassemia gene exhibit more severe clinical symptoms and anemia than those lacking the α-globin gene. Therefore, methodological research targeting mutant thalassemia genes is of practical significance. Common methods for detecting thalassemia genes include reverse dot blot hybridization (PCR-RDB), cross-breakpoint polymerase chain reaction (Gap-PCR), and reverse transcription real-time quantitative PCR (RT-qPCR). PCR-RDB has drawbacks such as long processing time, cumbersome operation, poor stability, and low automation. Gap-PCR is limited to detecting types involving clearly defined deletion regions and breakpoints, and requires interpretation via agarose gel electrophoresis, which can lead to nucleic acid dye contamination. Although RT-qPCR technology has high sensitivity, it requires sophisticated primer design and specialized detection instruments, which limits its application. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a self-powered dual-mode biosensor based on EBFC, which enables ultra-sensitive and highly selective detection of CD122 and helps to monitor abnormal expression of CD122.

[0005] The objective of this invention is achieved as follows:

[0006] A method for preparing a self-powered biosensor for dual-mode detection of the thalassemia gene CD122 is described, which constructs the anode and cathode of the self-powered biosensor based on carbon-coated molybdenum disulfide / gold nanoparticle composite (AuNPs / MoS2@C), carbon cloth, DNA strands, and biological enzymes.

[0007] The preparation method of AuNPs / MoS2@C consists of the following steps: Step 1. MoO3 template synthesis: Under magnetic stirring, 1~2 g of (NH4)6Mo7O 24 The powder was added to a solution containing deionized water (30–70 ml) and concentrated nitric acid (5–20 ml, 65% wt%). The mixture was then heat-treated in a 100 ml PTFE-lined autoclave at 150–250 °C. After cooling, the mixture was centrifuged, filtered, washed, and dried to obtain rod-shaped MoO3 templates.

[0008] Step 2. Synthesis of MoS2@C: 50–80 mg of MoO3 powder was ultrasonically dispersed in 60–100 ml of deionized water. Then, the MoO3 suspension was stirred with 100–200 mg of glucose and 100–200 mg of thiourea for 20–60 min. The mixture was then heated in a 100 ml autoclave at 100–200 °C for 15–25 h. After the autoclave cooled naturally, the resulting black precipitate was centrifuged and washed six times, and then vacuum dried overnight at 60 °C. The carbonized black powder was then heated at 500–800 °C under a nitrogen atmosphere for 1–3 h. Finally, the product was purified by elution with concentrated ammonia to remove the MoO3 template.

[0009] Step 3. Preparation of AuNPs / MoS2@C: 16 mg of MoS2@C was sonicated for 20-40 min and dissolved in 5-10 ml of PDDA (1%, 0.02 M NaCl). The residue of PDDA was removed by centrifugation with ultrapure water. Then, 5-10 ml of AuNPs activated by EDC / NHS was added, and the mixture was gently shaken overnight at room temperature. The free AuNPs were removed by centrifugation, and the precipitate was dispersed into 1 mg / ml AuNPs / MoS2@C using ultrapure water as the solvent.

[0010] The preparation method of bioanodes AuNPs / MoS2@C / GOD / BSA consists of the following steps:

[0011] 1 mg / mL, 30–70 μL of AuNPs / MoS2@C was drop-coated onto the surface of a carbon cloth electrode (1 cm × 1 cm). After vacuum drying at 37 °C for 2–4 h, 5 mg / mL, 30–70 μL of glucose oxidase (GOD) was drop-coated onto the electrode surface. After incubation at 4 °C for 10–15 h, the electrode was washed with ultrapure water. Subsequently, 1 mM, 30–70 μL of bovine serum albumin (BSA) was drop-coated onto the electrode. After incubation at room temperature for 20–50 min, the electrode was washed with ultrapure water to obtain the bioanolyte AuNPs / MoS2@C / GOD / BSA, which was stored at 4 °C for later use.

[0012] The preparation method of the biocathode AuNPs / MoS2@C / tetrahedral DNA (T-DNA) / CD122 / H1 / H2 / H3 / H4 consists of the following steps:

[0013] Step 1. Preparation of tetrahedral DNA (T-DNA): Dissolve 20 μL of each of the four ssDNAs (A, B, C, D) at 100 μM in 480 μL of 10 mM Tris-HCl buffer solution (10 mM TCEP, 50 mM MgCl2, pH 8.0) to form 4 μM solutions of the four ssDNAs. Mix 40–70 μL of each of the four 4 μM solutions and heat in a water bath at 95 °C for 2 min. Cool the mixture to 4 °C in an ice-water bath to obtain tetrahedral DNA (T-DNA).

[0014] Step 2. Preparation of catalytic hairpin self-assembly products: Mix 40-70 μL of CD122 at different concentrations with 2 μM H1 and H2, and incubate at 37 °C for 1-3 h to obtain catalytic hairpin self-assembly products CD122 / H1 / H2.

[0015] Step 3. Preparation of hybridization chain reaction (HCR) products: Mix 40-70 μL each of 0.5-2 μM H3 and H4, and incubate at 37 °C for 1-3 h to obtain hybridization chain reaction (HCR) products H3 / H4.

[0016] Step 4. Take 1 mg / mL, 30~70 μL of AuNPs / MoS2@C and drop it onto the surface of a carbon cloth electrode (1cm×1cm). After vacuum drying at 37 ℃ for 2~4 h, drop 20~50 μL of the product from step 1 onto the electrode surface. Incubate at 4 °C for 10~20 h and then wash with ultrapure water.

[0017] Step 5. Based on Step 4, add 1 mM, 20-50 μL of hexamercaptohexanol (MCH), let stand at room temperature for 20-40 min to block non-specific binding sites, and wash with ultrapure water.

[0018] Step 6. Add 20-50 μL of the product from Step 2, incubate at 37 °C for 30-100 min, and wash with ultrapure water.

[0019] Step 7. Add 20-70 μL of the product from Step 3, incubate at 37 °C for 30-100 min, and wash with ultrapure water. This yields the biocathode AuNPs / MoS2@C / tetrahedral DNA (T-DNA) / CD122 / H1 / H2 / H3 / H4.

[0020] The construction and measurement of the self-powered CD122 dual-mode biosensor consist of the following steps:

[0021] Step 1. Measure the sensor's... without introducing the target CD122. E OCV And the RGB Blue value of the electrolyte;

[0022] Step 2. When introducing the target CD122, measure the concentration of CD122 in the biosensor. E OCV And the RGB Blue value of the electrolyte;

[0023] The supporting electrolyte for the membrane-free glucose / methylene blue (MB) sensor constructed above was a 0.01 M PBS buffer system containing 5 mM glucose, 500 μM methylene blue (MB), and pH 7.4.

[0024] The principle of dual-mode ultrasensitive detection of CD122 by a self-powered biosensor based on EBFC:

[0025] When CD122 is absent, only tetrahedral DNA (T-DNA) is incubated on the electrode, preventing catalytic hairpin self-assembly (CHA) and hybridization chain reaction (HCR) amplification. In this state, the biocathode adsorbs less methylene blue (MB), resulting in a weaker electrical signal and a darker electrolyte color (lower RGB Blue value). When CD122 is present, catalytic hairpin self-assembly (CHA) amplification is initiated, enabling CD122 recycling and providing binding sites for subsequent HCR amplification. This leads to increased methylene blue (MB) adsorption on the biocathode, a stronger electrical signal, and a lighter electrolyte color (increased RGB Blue value). The open-circuit voltage and CD122 concentration are positively correlated, as are the electrolyte color (RGB Blue value), thus enabling quantitative detection of CD122. The CD122 content is determined by the correlation between the increase in open-circuit voltage and the target CD122 concentration; similarly, the CD122 content is determined by the correlation between the increase in electrolyte color (RGB Blue value) and the target CD122 concentration.

[0026] Positive and beneficial effects: This invention provides a self-powered CD122 sensor based on EBFC, enabling simple, convenient, fast, sensitive, and highly selective dual-mode detection of CD122. Compared with existing CD122 detection methods, it has the following characteristics:

[0027] (1) The self-powered biosensor described in this invention has a detection process that is different from the traditional electrochemical detection three-electrode system. The self-powered biosensor only needs two electrodes, namely the anode and cathode of EBFC, to achieve detection.

[0028] (2) The self-powered biosensor testing system described in this invention does not require an additional power source, which can effectively prevent the electroactive substances that are prone to oxidation and reduction from reacting on the electrode surface, thereby improving the sensor's anti-interference capability.

[0029] (3) The self-powered biosensor described in this invention uses open-circuit voltage and color change (RGB Blue value) to perform dual-mode detection of the thalassemia gene CD122, which not only has extremely high selectivity, but also has the advantages of low cost and simple operation.

[0030] (4) The self-powered biosensor described in the invention uses tetrahedral DNA (T-DNA), catalytic hairpin self-assembly (CHA), and hybridization chain reaction (HCR) to amplify the signal, thereby increasing the loading of the cathode electron acceptor methylene blue (MB) and effectively expanding the electrical signal.

[0031] (5) In the self-powered biosensor described in this invention, AuNPs / MoS2@C has the advantages of high electrocatalytic activity, large specific surface area and good biocompatibility. As a substrate material, it gains and loses electrons at the EBFC cathode, realizing ultrasensitive detection of the target CD122, which greatly improves the detection sensitivity.

[0032] (6) The self-powered biosensor described in this invention does not require an external power source, and can eliminate the need for expensive and complex electrochemical workstations and amplifiers. It is an energy generator for implantable and portable biomedical devices, and can realize the miniaturization, portability and integration of CD122 detection. Attached Figure Description

[0033] Figure 1 Schematic diagram of the anode and cathode assembly process of the self-powered CD122 biosensor;

[0034] Figure 2 Schematic diagram of the self-powered CD122 biosensor detection device;

[0035] Figure 3 A schematic diagram illustrating the principle of dual-mode ultrasensitive detection of CD122 by a self-powered biosensor based on EBFC;

[0036] Figure 4 CV (A) and LSV (B) graphs of the presence or absence of glucose at the anode of the self-powered biosensor;

[0037] Figure 5 CV (A) and LSV (B) diagrams of the presence or absence of a target on the cathode of a self-powered biosensor;

[0038] Figure 6 Standard curves of open-circuit voltage versus CD122 concentration for self-powered biosensors (A) and standard curves of RGB Blue value versus CD122 concentration (B). Implementation

[0039] A method for preparing a self-powered biosensor for dual-mode detection of the thalassemia gene CD122 is described, which constructs the anode and cathode of the self-powered biosensor based on carbon-coated molybdenum disulfide / gold nanoparticle composite (AuNPs / MoS2@C), carbon cloth, DNA strands, and biological enzymes.

[0040] The preparation method of AuNPs / MoS2@C consists of the following steps: Step 1. MoO3 template synthesis: Under magnetic stirring, 1~2 g of (NH4)6Mo7O 24The powder was added to a solution containing deionized water (30–70 ml) and concentrated nitric acid (5–20 ml, 65% wt%). The mixture was then heat-treated in a 100 ml PTFE-lined autoclave at 150–250 °C. After cooling, the mixture was centrifuged, filtered, washed, and dried to obtain rod-shaped MoO3 templates.

[0041] Step 2. Synthesis of MoS2@C: 50–80 mg of MoO3 powder was ultrasonically dispersed in 60–100 ml of deionized water. Then, the MoO3 suspension was stirred with 100–200 mg of glucose and 100–200 mg of thiourea for 20–60 min. The mixture was then heated in a 100 ml autoclave at 100–200 °C for 15–25 h. After the autoclave cooled naturally, the resulting black precipitate was centrifuged and washed six times, and then vacuum dried overnight at 60 °C. The carbonized black powder was then heated at 500–800 °C under a nitrogen atmosphere for 1–3 h. Finally, the product was purified by elution with concentrated ammonia to remove the MoO3 template.

[0042] Step 3. Preparation of AuNPs / MoS2@C: 16 mg of MoS2@C was sonicated for 20-40 min and dissolved in 5-10 ml of PDDA (1%, 0.02 M NaCl). The residue of PDDA was removed by centrifugation with ultrapure water. Then, 5-10 ml of AuNPs activated by EDC / NHS was added, and the mixture was gently shaken overnight at room temperature. The free AuNPs were removed by centrifugation, and the precipitate was dispersed into 1 mg / ml AuNPs / MoS2@C using ultrapure water as the solvent.

[0043] The preparation method of bioanodes AuNPs / MoS2@C / GOD / BSA consists of the following steps:

[0044] 1 mg / mL, 30–70 μL of AuNPs / MoS2@C was drop-coated onto the surface of a carbon cloth electrode (1 cm × 1 cm). After vacuum drying at 37 °C for 2–4 h, 5 mg / mL, 30–70 μL of glucose oxidase (GOD) was drop-coated onto the electrode surface. After incubation at 4 °C for 10–15 h, the electrode was washed with ultrapure water. Subsequently, 1 mM, 30–70 μL of bovine serum albumin (BSA) was drop-coated onto the electrode. After incubation at room temperature for 20–50 min, the electrode was washed with ultrapure water to obtain the bioanolyte AuNPs / MoS2@C / GOD / BSA, which was stored at 4 °C for later use. The assembly process is as follows. Figure 1 .

[0045] The preparation method of the biocathode AuNPs / MoS2@C / tetrahedral DNA (T-DNA) / CD122 / H1 / H2 / H3 / H4 consists of the following steps:

[0046] Step 1. Preparation of tetrahedral DNA (T-DNA): Dissolve 20 μL of each of the four ssDNAs (A, B, C, D) at 100 μM in 480 μL of 10 mM Tris-HCl buffer solution (10 mM TCEP, 50 mM MgCl2, pH 8.0) to form 4 μM solutions of the four ssDNAs. Mix 40–70 μL of each of the four 4 μM solutions and heat in a water bath at 95 °C for 2 min. Cool the mixture to 4 °C in an ice-water bath to obtain tetrahedral DNA (T-DNA).

[0047] Step 2. Preparation of catalytic hairpin self-assembly products: Mix 40-70 μL of CD122 at different concentrations with 2 μM H1 and H2, and incubate at 37 °C for 1-3 h to obtain catalytic hairpin self-assembly products CD122 / H1 / H2.

[0048] Step 3. Preparation of hybridization chain reaction (HCR) products: Mix 40-70 μL each of 0.5-2 μM H3 and H4, and incubate at 37 °C for 1-3 h to obtain hybridization chain reaction (HCR) products H3 / H4.

[0049] Step 4. Take 1 mg / mL, 30~70 μL of AuNPs / MoS2@C and drop it onto the surface of a carbon cloth electrode (1cm×1cm). After vacuum drying at 37 ℃ for 2~4 h, drop 20~50 μL of the product from step 1 onto the electrode surface. Incubate at 4 °C for 10~20 h and then wash with ultrapure water.

[0050] Step 5. Based on Step 4, add 1 mM, 20-50 μL of hexamercaptohexanol (MCH), let stand at room temperature for 20-40 min to block non-specific binding sites, and wash with ultrapure water.

[0051] Step 6. Add 20-50 μL of the product from Step 2, incubate at 37 °C for 30-100 min, and wash with ultrapure water.

[0052] Step 7. Add 20–70 μL of the product from Step 3, incubate at 37 °C for 30–100 min, and wash with ultrapure water. The biocathode AuNPs / MoS2@C / tetrahedral DNA (T-DNA) / CD122 / H1 / H2 / H3 / H4 is prepared, and its assembly process is as follows: Figure 1 .

[0053] The construction and measurement of the self-powered CD122 dual-mode biosensor consist of the following steps:

[0054] Step 1. Measure the sensor's... without introducing the target CD122. E OCV And the RGB Blue value of the electrolyte;

[0055] Step 2. When introducing the target CD122, measure the concentration of CD122 in the biosensor. E OCV And the RGB Blue value of the electrolyte;

[0056] The supporting electrolyte for the membrane-free glucose / methylene blue (MB) sensor described above was a 0.01 M PBS buffer system containing 5 mM glucose, 500 μM methylene blue (MB), and pH 7.4. The measuring device is as follows: Figure 2 .

[0057] The principle of dual-mode ultrasensitive detection of CD122 by a self-powered biosensor based on EBFC is as follows: Figure 3 As shown:

[0058] When CD122 is absent, only tetrahedral DNA (T-DNA) is incubated on the electrode, preventing catalytic hairpin self-assembly (CHA) and hybridization chain reaction (HCR) amplification. In this state, the biocathode adsorbs less methylene blue (MB), resulting in a weaker electrical signal and a darker electrolyte color (lower RGB Blue value). When CD122 is present, catalytic hairpin self-assembly (CHA) amplification is initiated, enabling CD122 recycling and providing binding sites for subsequent HCR amplification. This leads to increased methylene blue (MB) adsorption on the biocathode, a stronger electrical signal, and a lighter electrolyte color (increased RGB Blue value). The open-circuit voltage and CD122 concentration are positively correlated, as are the electrolyte color (RGB Blue value), thus enabling quantitative detection of CD122. The CD122 content is determined by the correlation between the increase in open-circuit voltage and the target CD122 concentration; similarly, the CD122 content is determined by the correlation between the increase in electrolyte color (RGB Blue value) and the target CD122 concentration. Example

[0059] (1) Synthesis of MoO3 template: Under magnetic stirring, 1.4 g of (NH4)6Mo7O 24The powder was added to a solution containing deionized water (50 ml) and concentrated nitric acid (10 ml, 65% wt%). The mixture was then heat-treated in a 100 ml PTFE-lined autoclave at 200 °C. After cooling, the mixture was centrifuged, filtered, washed, and dried to obtain rod-shaped MoO3 templates.

[0060] (2) Synthesis of MoS2@C: 70 mg of MoO3 powder was ultrasonically dispersed in 80 ml of deionized water. Then, the MoO3 suspension was stirred with 150 mg of glucose and 150 mg of thiourea for 0.5 h. Then, it was heated in a 100 ml autoclave at 180 °C for 20 h. After the autoclave cooled naturally, the resulting black precipitate was centrifuged and washed six times, and then vacuum dried overnight at 60 °C. Then, the carbon black powder was heated at 600 °C and kept under a nitrogen flow in the furnace for 2 h. Finally, the product was purified by elution with concentrated ammonia to remove the MoO3 template.

[0061] (3) Preparation of AuNPs / MoS2@C: 16 mg of MoS2@C was sonicated for 30 min and dissolved in 8 ml of PDDA (1%, 0.02 M NaCl). The residual PDDA was removed by centrifugation with ultrapure water. Then, 6 ml of AuNPs activated by EDC / NHS was added, and the mixture was gently shaken overnight at room temperature. The free AuNPs were removed by centrifugation, and the precipitate was dispersed into 1 mg / ml AuNPs / MoS2@C using ultrapure water as the solvent.

[0062] (4) Preparation method of bioanod AuNPs / MoS2@C / GOD / BSA: 1 mg / mL, 50 μL of AuNPs / MoS2@C was drop-coated onto the surface of a carbon cloth electrode (1 cm × 1 cm). After vacuum drying at 37 ℃ for 2 h, 5 mg / mL, 50 μL of glucose oxidase (GOD) was drop-coated onto the electrode surface. After incubation at 4 ℃ for 12 h, it was washed with ultrapure water. Subsequently, 1 mM, 40 μL of bovine serum albumin (BSA) was drop-coated onto the electrode. After standing at room temperature for 30 min, it was washed with ultrapure water to obtain the bioanod AuNPs / MoS2@C / GOD / BSA, which was stored at 4 ℃ for later use.

[0063] (5) Preparation of tetrahedral DNA (T-DNA): 100 μM of each of the four ssDNAs (A, B, C, D) were dissolved in 480 μL of 10 mM Tris-HCl buffer solution (10 mM TCEP, 50 mM MgCl2, pH 8.0) to form 4 μM solutions of the four ssDNAs. 50 μM solutions of the four ssDNAs were mixed and heated in a water bath at 95 °C for 2 min. The mixture was then cooled to 4 °C in an ice bath to obtain tetrahedral DNA (T-DNA).

[0064] (6) Preparation of catalytic hairpin self-assembly (CHA) products: 50 μL of CD122 at different concentrations was mixed with 50 μL of 2 μM H1 and H2, and incubated at 37 °C for 2 h to obtain catalytic hairpin self-assembly products CD122 / H1 / H2.

[0065] (7) Preparation of hybridization chain reaction (HCR) products: Mix 50 μL of 1 μM H3 and H4 and incubate at 37 °C for 70 min to obtain hybridization chain reaction (HCR) products H3 / H4.

[0066] (8) Preparation method of biocathode AuNPs / MoS2@C / tetrahedral DNA (T-DNA) / CD122 / H1 / H2 / H3 / H4: Take 1 mg / mL, 50 μL of AuNPs / MoS2@C and drop it onto the surface of carbon cloth electrode (1cm×1cm). After vacuum drying at 37 °C for 2 h, drop 25 μL of the product from step (5) onto the electrode surface. After incubation at 4 °C for 12 h, wash with ultrapure water. Then add 1 mM, 30 μL of hexamethylenetetramine alcohol (MCH), and place at room temperature for 30 min to block non-specific binding sites. Wash with ultrapure water. Then add 30 μL of the product from step (6), incubate at 37 °C for 60 min, and wash with ultrapure water. Then add 50 μL of the product from step (7), incubate at 37 °C for 70 min, and wash with ultrapure water. A biological cathode was prepared using AuNPs / MoS2@C / tetrahedral DNA / CD122 / H1 / H2 / H3 / H4.

[0067] (8) Construction and measurement of a self-powered CD122 biosensor: In electrochemical mode, without introducing the target CD122, the sensor's... E OCV After introducing the target CD122, measurements were taken on biosensors containing different concentrations of CD122. E OCVIn colorimetric mode, without introducing the target CD122, the dual-electrode system was placed in the electrolyte for 20 min, and the color of the electrolyte (RGB Blue value) was recorded; after introducing the target CD122, the dual-electrode system was placed in the electrolyte for 20 min, and the color of the electrolyte (RGB Blue value) was recorded.

[0068] (9) Plot the standard curve of open circuit voltage versus CD122 concentration and the standard curve of RGB Blue value versus CD122 concentration to complete the determination of CD122.

[0069] (10) Electrochemical characterization of self-powered biosensors

[0070] (11) To verify the bioanode reactivity of the sensor, CV and LSV tests were performed on the bioanode of the sensor. For example... Figure 4 As shown in Figure A, when the electrolyte does not contain glucose (curve a), a pair of weak redox characteristic peaks appear near -0.5V, which are characteristic peaks of glucose oxidase; when the electrolyte contains glucose (curve b), the peak potential of the bioanode increases, and the LSV ( Figure 4 B) The test results are consistent with the CV results, indicating the successful assembly of the bioanode.

[0071] (12) To verify the biocathode reactivity of the sensor, CV and LSV tests were performed on the biocathode. For example... Figure 5 As shown in Figure A, when the biocathode does not contain the target material (curve a), the peak potential of the biocathode is relatively small; when the biocathode contains the target material (curve b), the peak potential of the biocathode is much larger than that without the target material, and the LSV ( Figure 5 B) The test results are consistent with the CV results, indicating the successful assembly of the biocathode.

[0072] (13) Performance characterization of self-powered biosensors

[0073] (14) such as Figure 6 A. Open-circuit voltage tests were performed on different concentrations of the target compound (CD122). When the target compound was absent, only tetrahedral DNA remained on the electrode substrate. Catalytic hairpin self-assembly amplification could not occur, hybridization chain reaction amplification could not be fixed on the electrode surface, and the biocathode received fewer electrons. In this case, the EBFC... E OCV Smaller; as the concentration of the target compound increases, the amount of DNA strands loaded on the electrode increases, and the EBFC... E OCV And it is gradually increasing. E OCV The values ​​showed a good linear relationship with CD122 concentration in the range of 0.1 fM–100 pM, and the linear equation was: EOCV = 0.0377 log c + 0.727 (R) 2 =0.994), and the detection limit was 78.7 aM (S / N=3).

[0074] (15) such as Figure 6 B. Color change tests were performed on different concentrations of the target analyte (CD122). When the target analyte was absent, only tetrahedral DNA remained on the electrode substrate. Catalytic hairpin self-assembly amplification could not occur, hybridization chain reaction amplification could not be fixed on the electrode surface, and the biocathode adsorbed less MB. Therefore, fewer electrons were conducted in the EBFC system, resulting in less MB reduction and a darker electrolyte color with a lower RGB Blue value. As the target analyte concentration increased, the biocathode adsorbed more MB, the amount of MB reduced increased, the electrolyte color gradually lightened, and the corresponding RGB Blue value gradually increased. The RGB Blue value showed a good linear relationship with the CD122 concentration in the range of 0.1 fM-100 pM, with the linear equation being RGB Blue = 10.830 log c + 325.166 (R²). 2 =0.998), and the detection limit was 40.47 aM (S / N=3).

[0075] The supporting electrolyte for the membrane-free glucose / methylene blue (MB) sensor constructed above was a 0.01 M PBS buffer system containing 5 mM glucose, 500 μM methylene blue (MB), and pH 7.4.

[0076] CD122 (C→G) mutant sequence: 5'- GCC GAG TTC ACC CCT GCG GGT GCA GGC CTC CCTG-3'

[0077] H1 sequence: 5'- CAG GGA GGC CTG CAC CCG CAG GAC ACA ACT GTA GAT GTA ACCGTG CAG-3'

[0078] H2 sequence: 5'- TAC ATC TAC AGT TGT GTC CTG CGG GTG CAG CGC TGC CAA ACAACT-3'

[0079] H3 sequence: 5'-ATG TGT TTG GCA GCG GAA GTG CGC TGC CAA-3'

[0080] H4 sequence: 5'- CGC TGC CAA ACA ACT TTG GCA GCG CAC TTC-3'

[0081] A sequence: 5'- SH-C6-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTGTAT TGG ACC CTC GCA T-3'

[0082] B sequence: 5'- SH-C6-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACG TGG GAA TCTACT ATG GCG GCT CTT C-3'

[0083] C sequence: 5'- SH-C6-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGATGC GAG GGT CCA ATA C-3'

[0084] D sequence: 5'- ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGCCGC CAT AGT ATT TTT TTT TTT CTG CAC GGT-3'

[0085] This invention addresses the problems existing in prior art by constructing a self-powered biosensor based on EBFC and detecting the CD122 (C→G) mutant thalassemia gene (CD122) in a dual-mode manner. EBFC assembly is the core of the self-powered biosensor design, and the self-powered design of this invention mainly focuses on the cathode of the EBFC. When CD122 is present, the electrode initiates catalytic hairpin self-assembly (CHA) amplification, realizing the recycling of CD122 and providing binding sites for subsequent hybridization chain reaction (HCR) amplification. At this time, the adsorption of methylene blue (MB) on the biocathode increases, the electrical signal is enhanced, and the electrolyte color becomes lighter (RGB Blue value increases). The open-circuit voltage is positively correlated with the CD122 concentration, and the electrolyte color (RGB Blue value) is also positively correlated with the CD122 concentration, thereby achieving quantitative detection of CD122. The self-powered CD122 dual-mode biosensor based on EBFC designed in this invention can achieve simple, rapid, sensitive, and efficient detection of the target analyte. This invention provides a self-powered CD122 sensor based on EBFC, enabling simple, convenient, fast, sensitive, and highly selective dual-mode detection of CD122. Compared with existing CD122 detection methods, it has the following characteristics:

[0086] (1) The self-powered biosensor described in this invention has a detection process that is different from the traditional electrochemical detection three-electrode system. The self-powered biosensor only needs two electrodes, namely the anode and cathode of EBFC, to achieve detection.

[0087] (2) The self-powered biosensor testing system described in this invention does not require an additional power source, which can effectively prevent the electroactive substances that are prone to oxidation and reduction from reacting on the electrode surface, thereby improving the sensor's anti-interference capability.

[0088] (3) The self-powered biosensor described in this invention uses open-circuit voltage and color change (RGB Blue value) to perform dual-mode detection of the thalassemia gene CD122, which not only has extremely high selectivity, but also has the advantages of low cost and simple operation.

[0089] (4) The self-powered biosensor described in the invention uses tetrahedral DNA (T-DNA), catalytic hairpin self-assembly (CHA), and hybridization chain reaction (HCR) to amplify the signal, thereby increasing the loading of the cathode electron acceptor methylene blue (MB) and effectively expanding the electrical signal.

[0090] (5) In the self-powered biosensor described in this invention, AuNPs / MoS2@C has the advantages of high electrocatalytic activity, large specific surface area and good biocompatibility. As a substrate material, it gains and loses electrons at the EBFC cathode, realizing ultrasensitive detection of the target CD122, which greatly improves the detection sensitivity.

[0091] (6) The self-powered biosensor described in this invention does not require an external power source, and can eliminate the need for expensive and complex electrochemical workstations and amplifiers. It is an energy generator for implantable and portable biomedical devices, and can realize the miniaturization, portability and integration of CD122 detection.

Claims

1. A method for preparing a dual-mode self-powered biosensor for detecting the thalassemia gene CD122, characterized in that: The anode and cathode of a self-powered biosensor are constructed based on carbon-coated molybdenum disulfide / gold nanoparticle composite AuNPs / MoS2@C, carbon cloth electrodes, DNA strands, and biological enzymes. The assembly steps for the anode of the self-powered biosensor are as follows: Take 30~50 AuNPs / MoS2@C and drop it onto the surface of the carbon cloth electrode. Dry it at 37 ℃ for 2~4 h. Then drop it onto 20~50 μL of glucose oxidase GOD at 3~5 mg / mL and incubate for 12~24 h. After washing with ultrapure water, add 1 mmol / L of 20~50 μL of bovine serum albumin (BSA). After standing at room temperature for 30 min, wash it with ultrapure water and store it at 4 ℃. The assembly steps for the self-powered biosensor cathode AuNPs / MoS2@C / tetrahedral DNA / CD122 / H1 / H2 / H3 / H4 are as follows: Step 1. Drop 30-50 μL of AuNPs / MoS2@C onto the surface of a carbon cloth electrode, dry at 37 °C for 2-4 h, immerse it in 1-5 mg / mL EDC and NHS solution for 0.5-1 h, rinse with ultrapure water, then drop 10-30 μL of tetrahedral DNA onto it, place at 4 °C for 10-15 h, add 20-50 μL of 1 mM hexamethylene hexanol (MCH) and react for 0.5-1 h, then wash to remove excess MCH. Preparation of the tetrahedral DNA: 20 μL of each of four ssDNAs (A, B, C, and D) at 100 μM were dissolved in 480 μL of 10 mM Tris-HCl buffer solution (10 mM TCEP, 50 mM MgCl2, pH 8.0) to form 4 μM solutions of the four ssDNAs. 40-70 μL of each of the four 4 μM ssDNAs were mixed and heated in a water bath at 95 °C for 1-5 min. The mixture was then cooled to 4 °C in an ice-water bath to obtain the tetrahedral DNA. Step 2. Drop 20-50 μL of the catalytic hairpin self-assembled CHA product CD122 / H1 / H2 onto the electrode surface and incubate at 37 °C for 1-2 h, then wash with ultrapure water; finally, drop 40-70 μL of the hybridization chain reaction (HCR) product H3 / H4 onto the electrode surface and incubate at 37 °C for 1-2 h, then wash with 0.01 M sodium-based PBS buffer to obtain the biocathode, which is then stored at 4 °C for later use.

2. The method for preparing a dual-mode detection self-powered biosensor for the thalassemia gene CD122 according to claim 1, characterized in that, The preparation method of AuNPs / MoS2@C comprises the following steps: Step 1. MoO3 template synthesis: Under magnetic stirring, 1~2 g of (NH4)6Mo7O 24 The powder was added to a solution containing 30-70 ml of deionized water and 5-20 ml of 65%wt% concentrated nitric acid; then, the above mixed solution was heat-treated in a 100 ml polytetrafluoroethylene-lined autoclave at 150-250 °C, and after cooling, the white precipitate was centrifuged, filtered, washed and dried to obtain rod-shaped MoO3 templates. Step 2. Synthesis of MoS2@C: 50-80 mg of MoO3 powder was ultrasonically dispersed in 60-100 ml of deionized water; then, the MoO3 suspension was stirred with 100-200 mg of glucose and 100-200 mg of thiourea for 20-60 min; then, it was heated in a 100 ml autoclave at 100-200 °C for 15-25 h; after the autoclave cooled naturally, the resulting black precipitate was centrifuged and washed six times, and then vacuum dried overnight at 60 °C; then, the carbon black powder was heated at 500-800 °C and kept under a nitrogen flow in the furnace for 1-3 h; finally, the product was purified by elution with concentrated ammonia to remove the MoO3 template. Step 3. Preparation of AuNPs / MoS2@C: 16 mg of MoS2@C was sonicated for 20-40 min and dissolved in 5-10 ml of 0.02 M NaClPDDA containing 1% NaCl. The residue of PDDA was removed by centrifugation with ultrapure water. Then, 5-10 ml of AuNPs activated by EDC and NHS was added, and the mixture was gently shaken overnight at room temperature. The free AuNPs were removed by centrifugation, and the precipitate was dispersed into 1 mg / ml AuNPs / MoS2@C using ultrapure water as the solvent.

3. The method for preparing a dual-mode self-powered biosensor for detecting the thalassemia gene CD122 according to claim 1, characterized in that, The preparation method of the biocathode AuNPs / MoS2@C / tetrahedral DNA / CD122 / H1 / H2 / H3 / H4 consists of the following steps: Step 1). Preparation of tetrahedral DNA: Dissolve 20 μL of each of the four ssDNAs (A, B, C, and D) at 100 μM in 480 μL of 10 mM Tris-HCl buffer solution (10 mM TCEP, 50 mM MgCl2, pH 8.0) to form 4 μM solutions of the four ssDNAs. Mix 40-70 μL of each of the four 4 μM solutions and heat in a water bath at 95 °C for 1-5 min. Cool the mixture to 4 °C in an ice-water bath to obtain tetrahedral DNA. Step 2). Preparation of catalytic hairpin self-assembled CHA product: 40-70 μL of CD122 at different concentrations was mixed with 2 μM H1 and H2, 40-70 μL each, and incubated at 37 °C for 1-3 h to obtain the catalytic hairpin self-assembled product CD122 / H1 / H2. Step 3). Preparation of hybridization chain reaction (HCR) products: Mix 40-70 μL each of 0.5-2 μM H3 and H4, and incubate at 37°C for 1-3 h to obtain hybridization chain reaction (HCR) products H3 and H4; Step 4). Take 1 mg / mL, 30~70 μL of AuNPs / MoS2@C and drop it onto the surface of a 1cm×1cm carbon cloth electrode. After vacuum drying at 37 ℃ for 2~4 h, drop 20~50 μL of the product from step 1) onto the electrode surface. Incubate at 4 °C for 10~20 h and then wash with ultrapure water. Step 5). Based on step 4), add 1 mM, 20-50 μL of hexamercaptohexanol (MCH), and let stand at room temperature for 20-40 min to block non-specific binding sites, then wash with ultrapure water. Step 6). Add 20-50 μL of the product from Step 2), incubate at 37 °C for 30-100 min, and wash with ultrapure water; Step 7). Add 20-70 μL of the product from Step 3), incubate at 37 °C for 30-100 min, and wash with ultrapure water.

4. A method for non-disease diagnostic purposes using a dual-mode detection biosensor for the CD122 self-powered thalassemia gene prepared by the preparation method according to any one of claims 1-3, characterized in that: When the system does not contain the thalassemia gene CD122, the open-circuit voltage of the sensor is measured. E OCV After introducing the thalassemia gene CD122, the open-circuit voltage of the sensor was measured at different concentrations of the thalassemia gene CD122. E OCV ; The supporting electrolyte for the biosensor is a 0.01 MPBS buffer system at pH 7.4 containing 5 mM glucose and 500 μM methylene blue MB.

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