PNA-based electrochemical-photo-thermal dual-mode biosensor and method for detecting SRB DsrC gene

Abstract

The invention belongs to the field of biosensor preparation methods, and particularly relates to a PNA-based electrochemical-photo-thermal dual-mode biosensor and a method for detecting an SRB DsrC gene. An electrode structure of the sensor is CHA / PNA / ITO, a PNA probe captures a CHA product triggered by a target gene, and MB and ICG are incubated respectively to serve as electrochemical and photo-thermal signal probes and used for detecting the SRB DsrC gene. Compared with a single-mode biosensor, the dual-mode biosensor has the advantages of rapidness, sensitivity, low background signal and strong anti-interference capability of electrochemical sensing and photo-thermal sensing, and the detection accuracy is improved through mutual verification of the two modes.

Description

Technical Field

[0001] This invention belongs to the field of biosensor preparation methods, specifically relating to an electrochemical-photothermal dual-mode biosensor based on PNA and a method for detecting the SRB DsrC gene. Background Technology

[0002] Marine industry and marine equipment have become indispensable pillar industries for economic development. However, the marine environment is an extremely harsh corrosive environment for metals and other materials used in marine engineering. Marine organisms and their metabolic products in the marine environment also synergistically affect the corrosion process of materials. Corrosion of metal materials caused by the presence or growth and metabolism of microorganisms, known as microbial corrosion, accounts for 70%-80% of marine material damage and is one of the key threats to marine and near-shore industries. There are many types of marine corrosive microorganisms, among which sulfate-reducing bacteria (SRBs) are frequently found in the corrosion of metals in marine engineering equipment. They are among the most widely studied and most corrosive microorganisms. Developing detection methods and technologies for SRBs is essential for timely monitoring of SRB population concentrations in the environment, as well as for studying corrosion mechanisms, identifying microbial corrosion, detecting bactericidal effects, and exploring related research.

[0003] Common methods for SRB detection include the maximum probable number (MPN) method, polymerase chain reaction (PCR), fluorescence assay, and immunoassay. However, these methods suffer from drawbacks such as cumbersome operation, long processing times, low sensitivity, and poor environmental adaptability. With the rapid development of nanotechnology and biosensing, biosensor technology utilizes SRB metabolites, cellular structures, and characteristic genetic material as identification targets, effectively improving detection efficiency and accuracy. However, current biosensing strategies are mostly limited to single targets or detection modes, making it difficult to meet the requirements of multiple validations and interference resistance in complex real-world environments. Therefore, there is an urgent need to develop more sensitive and specific SRB detection methods. Summary of the Invention

[0004] In view of the necessity of SRB detection and the limitations of traditional detection methods, the purpose of this invention is to provide a PNA-based electrochemical-photothermal dual-mode biosensor and a method for detecting the SRB DsrC gene, so as to achieve high sensitivity and high specificity detection of the SRB DsrC gene.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An electrochemical / photothermal dual-mode biosensor based on PNA is disclosed. The sensor electrode structure is CHA / PNA / ITO. PNA probes immobilized on the surface of Au / ITO electrodes capture CHA products induced by target genes and incubate MB and ICG as electrochemical and photothermal signal probes, respectively.

[0006] The PNA probe is fixed on the surface of the Au / ITO electrode.

[0007] An electrochemical / photothermal dual-mode biosensor based on PNA is used to detect the SRB DsrC gene.

[0008] The method for analyzing the SRB DsrC gene is as follows: Construct the aforementioned PNA-based electrochemical / photothermal dual-mode biosensor, and use the current value and system temperature change value measured by square wave voltammetry as the sensor signal output to perform electrochemical-photothermal dual-mode quantitative analysis of the SRB DsrC gene.

[0009] The main testing steps include the following: Electrochemical mode detection: The sensor electrode was incubated with MB solution at 20℃-25℃ for 1-1.5 hours; then the electrode was washed with PBS buffer to remove excess MB; and then the electrochemical signal was detected by square wave voltammetry, with a potential range of -0.4V to 0.1V.

[0010] Photothermal mode detection: The sensor electrode was incubated with ICG solution at 20℃-25℃ for 1-1.5 hours; then the electrode was washed with PBS buffer to remove excess ICG; then photothermal detection was performed using an 808nm near-infrared light source (1W) for 280-320s, and the temperature change of the system was recorded using a temperature sensor.

[0011] When measuring different concentrations of DsrC gene, the square wave volt-ampere current value (I) of the sensor is positively correlated with the DsrC gene concentration, and the temperature change value (ΔT) is also positively correlated with the DsrC gene concentration.

[0012] A method for fabricating the PNA-based electrochemical / photothermal dual-mode biosensor includes the following steps: Step 1: Au / ITO electrode preparation: Clean the ITO electrode in an ultrasonic bath for 10-15 minutes and gently dry it with nitrogen gas; then immerse the clean ITO in a solution of HAuCl4 and Na2SO4, and perform electrodeposition using both CV and current-time methods. The CV method is performed for 10 cycles within the potential range of 0 to +1.5V at a scan rate of 100 mV·s. -1 The current-time curve method was performed at a constant potential of -0.2V for 300s. Then it was dried. Step 2: CHA amplification: The target fragment is incubated with CHA hairpin probes H1 and H2 at a molar ratio of 1:1:1 at 20℃-25℃ for 2 hours to perform target cyclic CHA amplification; Step 3: Sensor incubation: The 0.5-2 μM thiol-modified PNA probe (SH-PNA) was treated with TCEP at 20℃-25℃ for 1 hour, then dropped onto the surface of the Au / ITO electrode obtained in Step 1, and incubated at 37℃ for 2 hours; then washed with PBS buffer, and the electrode was blocked with MCH for 30 minutes; then washed alternately with 60% ethanol and ultrapure water; finally, the sensor electrode was incubated with the CHA product obtained in Step 2 at 37℃ for 1 hour to obtain the CHA / PNA / ITO sensor, which can be used for sample detection.

[0013] The principle of the PNA-based electrochemical / photothermal dual-mode biosensor for detecting the SRB DsrC gene in this invention is as follows: PNA was used as a capture probe, immobilized on the Au / ITO electrode surface via Au-S bonds. When the target fragment DsrC gene was absent, the CHA cascade reaction could not occur, and the PNA probe would not capture the CHA products, thus failing to adsorb the electrochemical and photothermal probes MB and ICG, resulting in a small current signal and low temperature change. When the DsrC gene was present, the CHA cascade amplification occurred, and the PNA probe selectively captured the CHA products induced by the target fragment, forming numerous double-stranded structures. This increased the adsorption of MB and ICG, significantly increasing both the current and temperature change. Therefore, the square wave voltammetry (SWV) current (I) was positively correlated with the DsrC gene concentration, and the system temperature change (ΔT) was also positively correlated with the DsrC gene concentration. The DsrC gene content could be determined by the linear relationship between I and the logarithmic concentration of the DsrC gene; similarly, the DsrC gene content could be determined by the linear relationship between ΔT and the logarithmic concentration of the DsrC gene.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Compared with the single-mode biosensor, the dual-mode biosensor of the present invention has the advantages of both electrochemical sensing and photothermal sensing, such as speed, sensitivity, low background signal and strong anti-interference ability. The accuracy of detection is improved by mutual verification of the two modes. (2) The biosensor described in this invention uses electrically neutral PNA as a detection probe. PNA has high sensitivity and specificity when it binds to complementary DNA, and has good thermal stability, thereby reducing non-specific adsorption caused by electrostatic interaction and improving the selectivity and stability of detection. (3) The biosensor described in this invention utilizes the CHA cascade reaction to greatly promote the formation of the PNA-DNA H3WJ structure, improve the signal efficiency on the electrode surface, effectively expand the detection signal, and enhance the sensitivity and responsiveness of the biosensor. (4) The biosensor described in this invention utilizes the electroactivity of MB and the photothermal properties of ICG to generate electrochemical and photothermal signals. The materials are simple and readily available, and the incubation operation is simple, which improves the operability of the biosensor. (5) The biosensor described in this invention uses a dual-mode detection of the DsrC gene concentration in SRB using current value and temperature change value, which has the advantages of high selectivity, low cost, easy signal detection and reliable results. Attached Figure Description

[0015] Figure 1 A schematic diagram illustrating the assembly process of the dual-mode biosensor provided in an embodiment of the present invention; Figure 2 The SWV response (A) and temperature response (B) of the dual-mode biosensor with and without a target provided in this embodiment of the invention are shown in the following figures: Figure 3 Standard curves (A) of SWV value versus DsrC concentration and (B) of temperature change value versus DsrC concentration for the dual-mode biosensor provided in the embodiments of the present invention. Figure 4 Detection selectivity diagram of the dual-mode biosensor provided in the embodiments of the present invention: electrochemical mode (A), photothermal mode (B); Figure 5 The detection stability diagram of the dual-mode biosensor provided in the embodiments of the present invention: electrochemical mode (A), photothermal mode (B); Figure 6 The detection repeatability diagram of the dual-mode biosensor provided in the embodiments of the present invention: electrochemical mode (A), photothermal mode (B). Detailed Implementation

[0016] The present invention will be further illustrated by specific embodiments below, which will help those skilled in the art to have a more comprehensive understanding of the present invention, but will not limit the present invention in any way.

[0017] The main instruments used in the embodiments of this invention, and other experimental conditions not specifically specified, were performed according to standard procedures or the instrument manufacturer's recommendations. DNA sequences were purchased from Qingdao Ruiboxingke Biotechnology Co., Ltd., and PNA sequences were purchased from Hangzhou Taihe Biotechnology Co., Ltd.

[0018] This invention utilizes a PNA probe immobilized on the surface of an Au / ITO electrode to capture CHA products induced by a target gene, and then incubates MB and ICG as electrochemical and photothermal signal probes, respectively. The current value measured by square-wave voltammetry and the system temperature change are used as sensor signal outputs for electrochemical-photothermal dual-mode quantitative analysis of the SRB DsrC gene. The method of this invention achieves highly selective, stable, and accurate dual-mode detection of the SRB DsrC gene.

[0019] Example 1 Construction of a PNA-based electrochemical / photothermal dual-mode biosensor: Step 1: Au / ITO electrode preparation: The ITO electrode was cleaned in an ultrasonic bath for 15 min, gently dried with nitrogen, and then the clean ITO was immersed in a 1 mM HAuCl4 and 0.2 M Na2SO4 solution. Electrodeposition was performed sequentially using the CV method and the current-time curve method. The CV method was performed for 10 cycles in the potential range of 0 to +1.5 V at a scan rate of 100 mV·s. -1 The current-time curve method was performed at a constant potential of -0.2V for 300s, followed by drying; Step 2: CHA amplification: The target fragment and CHA hairpin probes H1 and H2 are incubated at 25°C for 2 hours in a molar ratio of 1:1:1 to perform target cyclic CHA amplification; Step 3: Sensor Incubation: 1 μM thiol-modified PNA probe (SH-PNA) was treated with 1 mM TCEP at 25 °C for 1 hour; then, 15 μL of the resulting solution was added to the surface of the Au / ITO electrode obtained in Step 1 and incubated at 37 °C for 2 hours; then, the electrode was washed with PBS buffer and blocked with 2 mM MCH for 30 minutes; then, it was washed alternately with 60% ethanol and ultrapure water; finally, the sensor electrode was incubated with 15 μL of the CHA product obtained in Step 2 at 37 °C for 1 hour to obtain the CHA / PNA / ITO sensor, as shown below. Figure 1 As shown, it can be used for sample testing.

[0020] Example 2 The SRB DsrC gene was detected using the PNA-based electrochemical / photothermal dual-mode biosensor electrode described in Example 1. Electrochemical mode detection: such as Figure 1 As shown, the sensor electrode was incubated with 200 μL of MB solution (100 μM) at 25 °C for 1 h. The electrode was then washed with PBS buffer to remove excess MB. Electrochemical signal detection was then performed by square wave voltammetry (SWV) with a potential range of -0.4 V to 0.1 V.

[0021] Photothermal mode detection: such as Figure 1As shown, the sensor was incubated with 200 μL of ICG solution (600 μM) at 25 °C for 1 h. The electrode was then washed with PBS buffer to remove excess ICG, followed by photothermal detection using an 808 nm near-infrared light source (1 W). The light-on time was 300 s, and temperature changes were recorded using a temperature sensor.

[0022] Feasibility characterization of PNA-based electrochemical / photothermal dual-mode biosensor: Using electrochemical detection methods, such as Figure 2 As shown in Figure A, there is essentially no peak current in the absence of a target (black line a); when the target is introduced, CHA occurs, forming a TWJ structure with PNA, adsorbing a large amount of MB, and a significant increase in current response can be observed (red line c); in the control experiment using random sequence DNA, the observed current response (blue line b) is also significantly lower than that of the experimental group, indicating the feasibility of this biosensor for electrochemical detection of the target.

[0023] Using photothermal testing methods, such as Figure 2 As shown in B, without the target, the system showed virtually no temperature change (black line a); when the target was introduced, CHA occurred, forming a TWJ structure with PNA, adsorbing a large amount of ICG, and a significant increase in temperature was observed (red line c); the temperature change observed in the control experiment using random sequence DNA (blue line b) was also significantly lower than that in the experimental group, indicating the feasibility of the proposed dual-mode biosensor for photothermal detection of the target.

[0024] Example 3 Constructing the SRB DsrC gene standard curve: In the electrochemical mode, the SWV current value (I) of the system was measured without the introduction of the target DsrC gene, and the I value of the system containing different concentrations of DsrC gene was measured when the target DsrC gene was introduced. In the photothermal mode, the temperature change (ΔT) of the system was recorded when the target DsrC gene was not introduced; when the target DsrC gene was introduced, the ΔT values ​​of the systems containing different concentrations of DsrC gene were recorded. The electrolyte for electrochemical testing was 10 mM PBS, with a potential range of -0.4 V to 0.1 V. The background solution for photothermal testing was 10 mM PBS, with an on-time of 300 s, and temperature changes were recorded using a temperature sensor.

[0025] Plot the standard curves of I value versus DsrC log concentration and ΔT value versus DsrC log concentration.

[0026] Characterization of the detection performance of a PNA-based electrochemical / photothermal dual-mode biosensor: Electrochemical and photothermal tests were performed on systems with different concentrations of the DsrC gene. When the target fragment DsrC gene was absent, the CHA cascade reaction could not occur, the PNA probe did not capture the CHA product, and therefore could not adsorb the two electrochemical and photothermal probes, MB and ICG, resulting in low current and temperature changes. When the DsrC gene was present, the CHA cascade amplification occurred, and the PNA probe selectively captured the CHA product initiated by the target fragment, forming numerous double-stranded structures. This led to increased adsorption of MB and ICG, and significantly increased current and temperature changes.

[0027] like Figure 3 A. Within the range of 1 fM to 1 nM, there is a strong linear correlation between the target DsrC gene concentration on a logarithmic scale and the I value, with a linear regression equation of I = 6.84 log c +110.51 (R) 2 =0.981), LOD is 0.17fM (S / N=3).

[0028] like Figure 3 B, ΔT showed a good linear association with the logarithmic concentration of the DsrC gene in the range of 1 fM to 1 nM, with a linear regression equation of ΔT = 1.58 log c +29.63 (R) 2 =0.992), LOD is 0.66fM (S / N=3).

[0029] DsrC fragment sequence: 5'-ACGAACATCAGAAAGTTCTCGA-3' PNA sequence: 5'-SH-C3-PEG-ACACATCTCGAATA-3' Random DNA sequence: 5'-TCATCGAACCCGGCATGCTCAT-3' Hairpin DNA-1 sequence: 5'-TATTCGAGAACTTTCTGATGTTCGTGGATGTGTAGAACGAACATCAGAAAGAT-3' Hairpin DNA-2 sequence: 5'-TGTTCGTTCTACACATCCACGAACATCAGAAAGTTCGATGTGTAGA-3'.

[0030] Example 4 Validation of the selectivity, stability, and reproducibility of the PNA-based electrochemical / photothermal dual-mode biosensor in Example 1: like Figure 4As shown in A and 4B, at the same concentration, the current and temperature signal values ​​of the interfering fragments, single-base mismatched sequences (SMT), triple-base mismatched sequences (TMT), and completely mismatched sequences (NCS), are all smaller than those of the target sequence (TD) and the sequence mixture. This is attributed to their inability to initiate the CHA reaction, thus preventing the formation of the PNA-DNA H3WJ structure. This indicates that both modes of the biosensor exhibit strong selectivity for the target fragment.

[0031] The sensor prepared in Example 1 was stored at 4°C for 1, 2, and 3 weeks before electrochemical and photothermal tests were performed. Figure 5 As shown in Figures A and 5B, the signals from both modes were well preserved. After 3 weeks, the current and temperature change signal values ​​remained at 95.5% and 91.2%, respectively, indicating that the biosensors of both modes exhibited good stability during the 3-week storage period.

[0032] Three biosensors were fabricated in parallel to detect the target substance using SWV and photothermal methods. The results are as follows: Figure 6 As shown in A and 6B, the RSDs of the I and ΔT values ​​of the parallel-prepared biosensors were 3.33% and 3.24%, respectively, indicating reliable reproducibility of the two detection modes.

[0033] Single base mismatch sequence (SMT): 5'-ACTAACATCAGAAAGTTCTCGA-3' Triple mismatch sequence (TMT): 5'-ACTAACATCAGCAAGTTCTAGA-3' Completely mismatched sequence (NCS): 5'-GATGCAGCTCTGCGTCCAGTAG-3'.

[0034] Application examples The detection was performed using actual cultured SRB strains. Target DNA was extracted from 2 mL of bacterial culture for PCR. The diluted PCR product was placed in a centrifuge tube, heated at 95°C for 2 minutes, and then rapidly cooled to obtain denatured ssDNA.

[0035] PCR primer 1 sequence: 5'-CCCTGAGTGGGTTGAGTACG-3' PCR primer 2 sequence: 5'-GGCCATCTTACAAGCACCCT-3' Using the methods in Examples 1 and 2, different concentrations of ssDNA were subjected to CHA, followed by hybridization with sensor electrodes and incubation using ICG and MB, respectively. The resulting electrodes were subjected to photothermal and electrochemical tests, with temperature and current as output signals. Simultaneously, qPCR was performed on actual samples, and the dual-mode results were compared with the qPCR method to verify its reliability. All tests were repeated three times.

[0036] Using the PNA-based electrochemical / photothermal dual-mode bioassay for the DsrC gene described in Example 1, the concentration of target DNA was calculated using the obtained I and ΔT values ​​via a standard curve, as shown in Table 1. The RSD range for the electrochemical mode was 2.12% to 4.77%, with recoveries ranging from 92.2% to 104.7%. The RSD range for the photothermal mode was 2.38% to 3.12%, with recoveries ranging from 94.36% to 104.03%.

[0037] As shown in Table 1, the results of the dual-mode detection method of the present invention are close to those of the qPCR method, thus proving the reliability of the dual-mode detection method of the present invention. At the same time, the method of the present invention is more miniaturized and lower in cost than qPCR technology. Therefore, the dual-mode detection method of the present invention can be used for the quantification of DsrC gene in actual samples.

[0038] Table 1. Comparison of dual-mode sensor and qPCR in DsrC gene assay.

[0039] As described above, this invention constructs a PNA-based electrochemical / photothermal dual-mode biosensor for efficient detection of the DsrC gene in SRB. The sensor combines electrochemical and photothermal detection modes and utilizes the excellent signal amplification capability of the CHA cascade amplification to achieve high sensitivity and specificity. Compared to single-mode biosensors, this dual-mode biosensor possesses the inherent properties of each mode and can mutually verify the results of the two modes, correcting errors caused by human operation and external conditions. The constructed dual-mode biosensor not only exhibits a lower LOD and a wider detection range than qPCR, but its accuracy and reproducibility for detecting the DsrC gene in real samples are also superior to the qPCR method.

[0040] The above are merely exemplary embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A PNA-based electrochemical / photothermal dual-mode biosensor, characterized in that: The sensor electrode structure is CHA / PNA / ITO. The PNA probe captures the CHA product initiated by the target gene and incubates MB and ICG as electrochemical and photothermal signal probes, respectively.

2. The PNA-based electrochemical / photothermal dual-mode biosensor according to claim 1, characterized in that: The PNA probe is fixed on the surface of the Au / ITO electrode.

3. A PNA-based electrochemical / photothermal dual-mode biosensor as described in claim 1 for detecting the SRB DsrC gene.

4. The application according to claim 3, characterized in that: The method for detecting the SRB DsrC gene is as follows: Construct the aforementioned PNA-based electrochemical / photothermal dual-mode biosensor, and use the current value and system temperature change value measured by square wave voltammetry as the sensor signal output, respectively, to perform electrochemical-photothermal dual-mode quantitative analysis of the SRB DsrC gene.

5. The application according to claim 4, characterized in that: The electrochemical mode detection method of the PNA-based electrochemical / photothermal dual-mode biosensor is as follows: The PNA-based electrochemical / photothermal dual-mode biosensor electrode was incubated with MB solution at 20℃-25℃. The electrode was then washed with PBS buffer to remove excess MB. Electrochemical signals were then detected using square wave voltammetry, with a potential range of -0.4V to 0.1V.

6. The application according to claim 4, characterized in that: The photothermal mode detection method of the PNA-based electrochemical / photothermal dual-mode biosensor is as follows: The PNA-based electrochemical / photothermal dual-mode biosensor electrode was incubated with ICG solution at 20℃-25℃; then the electrode was washed with PBS buffer to remove excess ICG; subsequently, photothermal detection was performed using an 808nm near-infrared light source (1W) for 300s, and temperature changes were recorded using a temperature sensor.

7. The detection method according to claim 5 or 6, characterized in that: When measuring different concentrations of DsrC gene, the square wave volt-ampere current value (I) of the sensor is positively correlated with the DsrC gene concentration, and the temperature change value (ΔT) is also positively correlated with the DsrC gene concentration.

8. A method for fabricating a PNA-based electrochemical / photothermal dual-mode biosensor as described in claim 1, characterized in that, Includes the following steps: Step 1: Au / ITO electrode preparation: Clean the ITO electrode and dry it with nitrogen; then immerse the ITO in a solution of HAuCl4 and Na2SO4, and perform electrodeposition using the CV method and current-time curve method, followed by drying; Step 2: CHA amplification: Incubate the target fragment with CHA hairpin probes H1 and H2 to perform target cyclic CHA amplification; Step 3: Sensor incubation: The PNA probe is dropped onto the surface of the Au / ITO electrode obtained in step 1 and incubated; then it is washed with PBS buffer and the electrode is blocked with MCH; then it is washed alternately with ethanol and ultrapure water; finally, the sensor electrode is incubated with the CHA product obtained in step 2 to obtain the CHA / PNA / ITO sensor.

9. The preparation method according to claim 8, characterized in that, In step 1, the CV method is performed for 10 cycles within the potential range of 0 to +1.5V, with a scan rate of 100mV·s. -1 The current-time curve method was performed for 300 seconds at a constant potential of -0.2V.

10. The preparation method according to claim 8, characterized in that, Step 3: The PNA probe (SH-PNA) is modified with thiol and treated with TCEP at 20℃-25℃ for 1 hour; the PNA probe concentration is 0.5-2μM.

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