Electrochemical sensor for detecting total number of colonies in water body and preparation method and application thereof

Abstract

The electrochemical sensor for detecting the total number of water bacteria colonies and its preparation method and application belong to the technical field of water detection. In order to solve the technical problems of long time consumption, inability to detect VBNC bacteria, lack of general target for detection, etc. in the existing water bacteria colony number detection method, the present application constructs an ultra-sensitive electrochemical sensor based on a triple signal amplification strategy by screening a general nucleic acid target fragment for water bacteria colony number detection, designing a corresponding hairpin probe and a hairpin structure capture probe. The sensor can realize rapid determination of the total number of water bacteria colonies, has good anti-interference, reproducibility and stability, can realize high-sensitivity detection of bacterial 16S rRNA gene, the linear range is 0.1 fM to 100 pM, the detection limit is as low as 1.0 aM; at the same time, it can realize actual detection of bacteria as low as 1 CFU / mL, and the detection result is highly consistent with the plate counting method, which is the "gold standard" for water bacteria colony number detection.

Description

Technical Field

[0001] This invention belongs to the field of water body detection technology, specifically an electrochemical sensor for detecting total bacterial count in water, its preparation method, and its application. Background Technology

[0002] Aquatic bacterial communities are not dominated by a single species, but rather form a dynamic, balanced ecological network through interactions such as competition, symbiosis, and mutualism, exhibiting high biodiversity. Bacterial contamination can directly or indirectly cause various diseases, posing a serious threat to public health and safety. Total bacterial count, as a key parameter in water quality testing, directly reflects the degree of risk of bacterial contamination in water bodies. Therefore, developing accurate and effective broad-spectrum bacterial detection methods for total bacterial count detection and safety assessment in water bodies is of significant practical importance.

[0003] Currently, the determination of total bacterial count in water bodies mainly relies on the "plate count method," which is considered the gold standard. However, this method has significant inherent limitations: on the one hand, the culture period is long (at least 48 hours), which cannot meet the needs of rapid on-site early warning; on the other hand, this method is based on the principle of live bacteria culture, making it difficult to effectively detect "living but unculturable" (VBNC) bacteria that are widely present in actual water environments, leading to test results that often underestimate the actual biosafety risks.

[0004] To overcome these limitations, molecular diagnostic techniques based on polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) have emerged. However, PCR technology is expensive, cumbersome to operate, and prone to false positives due to aerosol contamination; ELISA technology faces problems such as insufficient sensitivity, frequent cross-reactions, and limited detection targets, making it difficult to cope with the complex diversity of bacterial communities in water bodies.

[0005] In recent years, electrochemical nucleic acid sensors have shown great potential in the field of bacterial detection due to their advantages such as fast response speed, low cost, and ease of miniaturization. However, existing technologies still face several technical bottlenecks in practical applications, limiting their use in broad-spectrum, rapid detection: First, there is a lack of broad-spectrum detection targets. Most electrochemical nucleic acid sensors rely on specific recognition elements for specific bacterial species (such as specific aptamers). For example, existing technologies (Cai, Ming, et al. "Detection of Escherichiacoli O157: H7 in food based on magnetic separation and hybridization chainreaction." Food Control 177 (2025): 111446.) can achieve specific detection of different pathogens (such as Escherichia coli O157:H7, Staphylococcus aureus, and Vibrio parahaemolyticus) by changing the aptamer. However, due to the lack of universal bacterial nucleic acid targets, they cannot achieve total detection of multiple mixed bacterial communities in water, that is, they do not have the ability to determine the total number of colonies.

[0006] Second, signal amplification strategies rely on expensive and unstable natural enzymes. To improve detection sensitivity, existing technologies often incorporate nucleic acid amplification techniques (such as PCR) or natural enzyme-mediated signal amplification strategies. For example, existing technologies (Li, Meixing, et al. "Label-free homogeneous electrochemical sensing strategy for microRNA detection." Microchemical Journal 183 (2022): 108097.) utilize exonuclease III (Exo III) to assist in signal amplification. Although this improves performance, natural enzymes suffer from poor stability, easy inactivation, demanding storage conditions, and high costs. This not only increases detection costs but also significantly limits the field deployment of sensors in non-laboratory environments.

[0007] Third, the detection process is complex and difficult to apply on-site. Whether based on PCR amplification or natural enzyme catalysis, existing technologies often rely on sophisticated thermal cyclers or complex temperature control equipment, and the operation steps are cumbersome, requiring professional technicians to perform, making it difficult to meet the actual needs of rapid and convenient on-site detection.

[0008] To address the aforementioned issues, there is an urgent need for a novel technological solution that can rapidly and sensitively detect the total bacterial count in water bodies without relying on natural nucleic acid hydrolases or complex temperature control equipment, and based on universal targets. Summary of the Invention

[0009] To address the technical challenges of existing methods for detecting total bacterial count in water bodies, such as long processing times, inability to detect VBNC bacteria, lack of broad-spectrum detection targets, and high costs and complex operations due to reliance on nucleic acid amplification or natural nucleases, this invention constructs an ultrasensitive electrochemical nucleic acid sensor capable of rapid determination of total bacterial count in water bodies. The sensor's construction method and detection mechanism are as follows: First, using the National Center for Biotechnology Information (NCBI) database as a reference, universal nucleic acid target fragments for detecting total bacterial count in water bodies were screened from 20 strains of 10 common aquatic bacteria. Based on this, corresponding hairpin-structured capture probes were designed and coupled to carboxyl-functionalized polystyrene magnetic particles (MPs). The coupled MPs specifically recognize the conserved region of the 16S rRNA gene of aquatic bacteria, opening the hairpin structure of the capture probe, and then undergoing a hybridization chain reaction (HCR) with the designed hairpin DNA probes H1 and H2, achieving nucleic acid chain amplification and the first stage of signal amplification. Subsequently, under the influence of potassium ions, a G-quadruplex spatial structure is formed, allowing more electrochemical signal molecules, methylene blue (MB), to be embedded, resulting in a second amplification of the signal. After magnetic separation and washing, the artificial nuclease 1MIA-Zr is used to hydrolyze the phosphodiester bonds, cleaving the nucleic acid chain and releasing the electrochemical signal molecule MB, thus triggering and amplifying the signal a third time. Quantification of the total bacterial count in water can be achieved by detecting the electrochemical signal molecule MB. The sensor provided by this invention exhibits excellent anti-interference, reproducibility, and stability, enabling highly sensitive detection of bacterial 16S rRNA genes, with a linear range of 0.1 fM to 100 pM and a detection limit as low as 1.0 aM. When used for total bacterial count detection in environmental water, it can achieve bacterial detection down to 1 CFU / mL, with results highly consistent with the "gold standard" plate counting method for total bacterial count in water.

[0010] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide an electrochemical sensor for detecting total bacterial count in water, the electrochemical sensor comprising target DNA for detecting total bacterial count in water, CMPs formed by coupling an amino-modified hairpin-structured capture probe with carboxyl-modified polystyrene magnetic particles, a hairpin probe, potassium ions, methylene blue, and an artificial nuclease 1MIA-Zr; the nucleotide sequence of the target DNA is shown in SEQ ID NO.21, the nucleotide sequence of the capture probe is shown in SEQ ID NO.22, the hairpin probe is composed of H1 and H2, and the nucleotide sequences of H1 and H2 are shown in SEQ ID NO.23 and SEQ ID NO.24, respectively; When bacteria are present in the water, CMPs can specifically recognize the conserved region of the 16S rRNA gene of aquatic bacteria, open the hairpin structure of the capture probe, and then undergo a hybridization chain reaction with the designed hairpin probes H1 and H2 to achieve nucleic acid chain amplification, resulting in the first amplification of the signal. Subsequently, under the action of potassium ions, a G-quadruplex spatial structure is formed to embed more electrochemical signal molecules, methylene blue, resulting in the second amplification of the signal. After magnetic separation and washing, the artificial nucleic acid hydrolase 1MIA-Zr is used to hydrolyze the phosphodiester bond, cut the nucleic acid chain, and release the electrochemical signal molecule MB, achieving the third amplification of the signal. By detecting the electrochemical signal molecule MB, the total number of bacterial colonies in the water can be quantified.

[0011] A second objective of this invention is to provide a method for preparing the aforementioned electrochemical sensor, the method comprising the following steps: 1) Prepare a Tris-HCl buffer solution containing H1, H2 and KCl; 2) Prepare a Tris-HCl buffer solution containing methylene blue; 3) Prepare Tris-HCl buffer containing artificial nuclease 1MIA-Zr; 4) Preparation of CMP suspension: After magnetic separation and washing, the carboxyl-modified polystyrene magnetic particle solution was placed in imidazole-hydrochloric acid buffer containing NHS and EDC and activated at room temperature in the dark for 30 min; an amino-modified hairpin-structured capture probe was added and the reaction was carried out overnight with gentle shaking at 37°C; after the reaction, after magnetic separation and washing, the solution was placed in PBS buffer containing BSA and Tween-20 and blocked at 37°C for 1 h; after blocking, the solution was washed with PBS buffer, and finally the CMP product was resuspended in 100 μL Tris-HCl buffer.

[0012] In one embodiment of the present invention, the concentrations of H1, H2, and KCl in the Tris-HCl buffer in step 1) are 1 μM, 1 μM, and 100 mM, respectively, the concentration of Tris-HCl in the Tris-HCl buffer is 0.01 M, and the pH of the Tris-HCl buffer is 7.4.

[0013] In one embodiment of the present invention, the concentration of methylene blue in the Tris-HCl buffer in step 2) is 10-25 mM, the concentration of Tris-HCl in the Tris-HCl buffer is 0.1 M, and the pH of the Tris-HCl buffer is 7.4.

[0014] In one embodiment of the present invention, the concentration of the artificial nuclease 1MIA-Zr in step 3) in the Tris-HCl buffer is 2 mg / mL, the concentration of Tris-HCl in the Tris-HCl buffer is 0.1 M, and the pH of the Tris-HCl buffer is 7.4.

[0015] In one embodiment of the present invention, the specific method of step 4) is as follows: Take 50 μL of a carboxyl-modified polystyrene magnetic particle solution with a concentration of 5 mg / mL, perform magnetic separation, wash three times with 200 μL of imidazole-hydrochloric acid buffer containing 0.02% Tween-20, place it in 50 μL of imidazole-hydrochloric acid buffer containing 10 mg / mL NHS and 20 mg / mL EDC, and activate at room temperature in the dark for 30 min; after activation, add 50 μL of 1 μM amino-modified capture probe, and react with gentle shaking at 37°C overnight; after the reaction, perform magnetic separation, wash three times with imidazole-hydrochloric acid buffer, and then place it in 100 μL of PBS buffer containing 3% BSA and 0.02% Tween-20, and block at 37°C for 1 h; after blocking, wash three times with PBS buffer, and finally resuspend the product CMPs in 100 μL of Tris-HCl buffer to prepare a suspension of 2.5 mg / mL.

[0016] In one embodiment of the present invention, the concentration of the imidazole-hydrochloric acid buffer is 0.1 M and the pH is 7.4; the concentration of the PBS buffer is 0.1 M and the pH is 7.4; and the concentration of the Tris-HCl buffer is 0.01 M and the pH is 7.4.

[0017] The third objective of this invention is to provide a method for detecting the total bacterial count in water using the aforementioned electrochemical sensor. The method comprises the following steps: collecting a water sample of known volume that meets the requirements for subsequent detection; filtering the sample through a 0.22 μm filter membrane to remove environmental DNA; collecting bacteria enriched on the filter membrane; extracting the 16S rRNA gene using a boiling extraction method to obtain the sample to be tested; mixing the sample with a CMP suspension at a concentration of 2.5 mg / mL and incubating for 1 h; washing three times with PBS buffer after magnetic separation, followed by adding a Tris-HCl buffer containing 1 μM H1, 1 μM H2, and 100 mM KCl, and performing a hybridization chain reaction at 37°C for 0.5-4 h; adding a Tris-HCl buffer containing 10-25 mM methylene blue after magnetic separation, and washing multiple times until no residual MB is detected in the supernatant; adding a Tris-HCl buffer containing 2 mg / mL 1MIA-Zr and performing a hydrolysis reaction at 37°C for 0.5-6 h. h, obtain the hydrolysate; using a screen-printed carbon electrode as a three-electrode system, take the supernatant of the hydrolysate after vortex mixing and magnetic separation, and drop it onto the three-electrode region of the screen-printed carbon electrode (or immerse the three-electrode region of the screen-printed carbon electrode in the solution), and detect it using square wave voltammetry.

[0018] In one embodiment of the present invention, water samples of known volume that meet the requirements for subsequent detection are collected because the types of water to be tested include surface water, tap water, and drinking water, among which the background concentration of target microorganisms in clean water bodies such as drinking water is extremely low. If conventional small-volume sampling is used, the total amount of analytes cannot meet the detection limit requirements of the subsequent detection method. Therefore, a sufficiently large volume of water sample must be collected and its accurate volume recorded. The target microorganisms are then concentrated to a detectable level through enrichment operations, thereby ensuring the sensitivity and reliability of the detection results.

[0019] Preferably, the hybridization chain reaction takes 2 hours.

[0020] Preferably, a Tris-HCl buffer containing 20 mM methylene blue is added.

[0021] Preferably, the hydrolysis reaction takes 4 hours.

[0022] The boiling extraction method was chosen for extracting bacterial 16S rRNA genes primarily because of its advantages of being convenient, quick, and requiring simple equipment. However, other methods that achieve the same extraction results (such as microwave heating, column extraction, or rapid nucleic acid release) can also be used to extract bacterial 16S rRNA genes.

[0023] In one embodiment of the present invention, the working electrode and the counter electrode in the three-electrode system are both carbon electrodes, and the reference electrode is an Ag / AgCl electrode.

[0024] In one embodiment of the present invention, the square wave voltammetry scanning parameters are set as follows: potential range -0.5-0V; step potential 0.005V; amplitude 20mV; frequency 25Hz; resting time 2s.

[0025] In one embodiment of the present invention, a screen-printed electrode is selected as the three-electrode system, primarily based on its ease of integration, simple operation, and suitability for rapid on-site detection. Under the condition of proportionally increasing the electrolyte volume of the reaction system, the detection method of the present invention is also applicable to other conventionally configured three-electrode systems. For example, an ITO electrode or other carbon material electrode can be used as the working electrode, combined with an Ag / AgCl reference electrode and a Pt wire electrode to form a detection system, achieving the same detection effect.

[0026] In one embodiment of the present invention, square wave voltammetry (SWV) is used for electrochemical signal acquisition. It should be noted that the signal of this detection system originates from the electrochemical response of methylene blue, and detection techniques for such electroactive substances are already quite mature. Therefore, other types of electrochemical detection techniques (such as cyclic voltammetry, differential pulse voltammetry, etc.) can be used as substitutes without departing from the principles of the present invention.

[0027] The beneficial effects of this invention are: The present invention has the following advantages over the prior art: (1) The electrochemical sensor provided by the present invention can achieve highly sensitive detection of total bacterial count in water and can achieve broad-spectrum detection of bacteria, not limited to the detection of a single species, and can also detect unknown bacteria in actual water bodies that are "living but not culturable".

[0028] (2) The electrochemical sensor provided by this invention has the versatility of detecting multiple targets. It can not only detect bacteria, but also perform highly specific identification of nucleic acid targets. By reasonably changing the target nucleic acid sequence and adjusting the design of the capture probe and hairpin probe accordingly, it can be further extended to the detection of other nucleic acid targets.

[0029] (3) The method for detecting total bacterial count in water using an electrochemical sensor provided by this invention has high sensitivity and low detection limit. In terms of target DNA detection, the detection limit (LOD) reaches 1.0 aM; in terms of bacterial detection, it can achieve actual detection as low as 1 CFU / mL for both laboratory-cultured bacteria and actual total bacterial count in water.

[0030] (4) The method for detecting total bacterial count in water using an electrochemical sensor provided by this invention has a short detection cycle. Compared with the "gold standard" plate counting method (at least 48 hours) for detecting total bacterial count in water, this electrochemical sensor detection method can achieve detection within 7 hours, greatly improving detection efficiency.

[0031] (5) The electrochemical sensor detection method provided by the present invention is simple to operate and low in cost. This method does not rely on large-scale special instruments such as PCR instruments, avoids the use of natural nucleases with poor stability and high cost, and greatly reduces the difficulty of operation and cost. Attached Figure Description

[0032] Figure 1 A schematic diagram of the detection mechanism of the electrochemical sensor provided by the present invention; Figure 2 The figure shows the characterization results of the electrochemical sensor provided by this invention; where a is the non-denaturing polyacrylamide gel electrophoresis analysis results of different DNA samples ("+" and "-" indicate the presence and absence of the corresponding nucleic acid or metal ions, respectively), and lane 1 contains only target DNA and K. + Lane 2 contains only the capture probe and K + Lane 3 contains only hairpin probes H1 and K. + Lane 4 contains only hairpin probes H2 and K. + Lane 5 contains only hairpin probes H1, H2, and K. + Lane 6 contains only the capture probe, hairpin probes H1, H2, and K. + Lane 7 contains only target DNA, capture probe, and K. + Lane 8 contains only target DNA, capture probe, hairpin probe H1, and K. + Lane 9 contains target DNA, capture probes, hairpin probes H1, H2, and K. + Lane 10 contains only target DNA, capture probe, hairpin probes H1 and H2. b is the circular dichroism detection result of HCR product with and without metal cations. c is the UV-Vis absorption spectrum of methylene blue (MB) interacting with different DNA structures. d is the circular dichroism detection result of HCR product containing G-quadruplex structure after incubation with MB. Figure 3 The figure shows the electrochemical feasibility verification results of the triple signal amplification strategy for the electrochemical sensor provided by this invention; where a is the square wave voltammogram before and after hydrolysis with the addition of 1MIA-Zr, b is the square wave voltammogram before and after HCR, and c is the K... + The square wave volt-ampere curves before and after the addition are shown, where d is the peak current after each signal amplification (solid circles and hollow circles represent presence and absence, respectively). Figure 4The graph shows the evaluation results of the electrochemical sensor provided by the present invention for the detection capability of target DNA; where a is a square wave voltammetry curve of target DNA concentration in the range of 0-100 pM, and b is a linear fitting curve between the peak current after background subtraction and the logarithm of target DNA concentration. Figure 5 The graphs show the detection results of the electrochemical sensor provided by this invention on laboratory water samples containing different concentrations of Escherichia coli and different concentrations of Staphylococcus aureus; wherein, a is the detection result graph of the electrochemical sensor on laboratory water samples containing different concentrations of Escherichia coli, and b is the detection result graph of the electrochemical sensor on laboratory water samples containing different concentrations of Staphylococcus aureus. Figure 6 The graphs show the evaluation results of the anti-interference ability, reproducibility, and stability of the electrochemical sensor provided by the present invention; wherein, a is the evaluation result of the anti-interference ability of the electrochemical sensor against anionic interfering substances, b is the evaluation result of the anti-interference ability of the electrochemical sensor against organic compound interfering substances, c is the evaluation result of the reproducibility of the electrochemical sensor, and d is the evaluation result of the stability of the electrochemical sensor. Figure 7 The graphs show the detection results of the electrochemical sensor provided by this invention on actual water samples from three different sources. Among them, a is a square wave voltammogram of surface water with different bacterial colony concentrations, b is a linear fitting curve between the peak current after background subtraction and the logarithm of the total number of bacterial colonies in surface water, c is a square wave voltammogram of tap water with different bacterial colony concentrations, d is a linear fitting curve between the peak current after background subtraction and the logarithm of the total number of bacterial colonies in tap water, e is a square wave voltammogram of drinking water with different bacterial colony concentrations, and f is a linear fitting curve between the peak current after background subtraction and the logarithm of the total number of bacterial colonies in drinking water. Figure 8 The graphs show the correlation analysis results of detecting the total number of colonies in water samples from different sources using the electrochemical sensor provided by this invention and the traditional plate counting method; where a is the correlation analysis result of detecting the total number of colonies in surface water, b is the correlation analysis result of detecting the total number of colonies in tap water, and c is the correlation analysis result of detecting the total number of colonies in drinking water. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. In the art, any embodiments obtained by those skilled in the art without creative effort are protected by this invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0035] The artificial nuclease 1MIA-Zr used in this invention is disclosed in the following literature: Yu, Zhixuan, et al. "Harnessing infinite coordination polymers to eliminate harmful environmental DNA for water purification" Analytical Chemistry 97 (2025): 5546 5553 was prepared using the following method: a solvothermal synthesis. Specifically, 0.75 mmol of zirconium chloride (ZrCl4) and 0.75 mmol of 1-methyl-1H-imidazolium-4,5-dicarboxylic acid (1MIA) were added to 50 mL of N,N-dimethylformamide (DMF). After stirring for 5 min, the mixture was transferred to a 100 mL stainless steel reactor with a polytetrafluoroethylene liner and reacted at 150°C for 72 h. After the reaction was completed, the material was collected by centrifugation, washed three times each with DMF and ethanol, and then vacuum dried overnight at 60°C.

[0036] The Escherichia coli ATCC 25922 and Staphylococcus aureus CMCC(B) 26003 used in this invention were both purchased from the Beijing Biotechnology Preservation Center.

[0037] Example 1: Preparation method of electrochemical sensor for total bacterial count detection in water This embodiment aims to prepare an electrochemical sensor for detecting total bacterial count in water. The detection mechanism of this electrochemical sensor is as follows: Figure 1As shown: First, an amino-modified hairpin-structured capture probe is covalently coupled to the surface of carboxyl-functionalized polystyrene magnetic particles (MPs) via an amidation reaction. When the 16S rRNA gene released by bacterial lysis is present in the system, it specifically recognizes the capture probe, opening its hairpin structure. Subsequently, it undergoes a hybridization chain reaction (HCR) with hairpin probes H1 and H2, extending on the MPs surface to form a long double-stranded DNA (dsDNA) nanostructure. Simultaneously, in K... + With the assistance of [unclear], the dsDNA strand undergoes self-assembly on both sides to form a G-quadruplex spatial structure, providing numerous intercalation sites for the electrochemical signal molecule methylene blue (MB). Further introduction of the artificial nuclease 1MIA-Zr, which can specifically hydrolyze phosphodiester bonds, disrupts the DNA nanostructure on the surface of MPs, releasing the embedded MB molecules and generating a detectable electrochemical signal.

[0038] 1. Screening of target DNA sequences for total bacterial count detection in water bodies This invention compares the different 16S rRNA gene sequences of 20 strains of 10 bacteria (Pseudomonas aeruginosa, Escherichia coli, Enterobacter aerogenes, Enterococcus faecalis, Staphylococcus aureus, Vibrio cholerae, Salmonella typhi, Shigella dysenteriae, Aeromonas hydrophila, and Enterobacter cloacae) and selects suitable conserved regions as target DNA. The sequence alignment results are shown in Table 1.

[0039] Table 1. Results of 16S rRNA gene sequence alignment of common bacterial strains in water bodies

[0040] Note: The sequence in bold is the universal detection target sequence designed in this invention; the sequence in underlined areas indicates areas that have been matched. 2. Design of capture probe and hairpin probe Combining the hybridization chain reaction and the G-quadruplex formation mechanism, a series of capture probes and hairpin probes (H1, H2) with partially complementary hairpin structures were designed. H1 and H2 each contain splitting (3:1) G-quadruplex sequences at both ends (H1 has one G-quadruplex at the 5' end and three G-quadruplexes at the 3' end; H2 is the opposite). The specific sequences of the capture probes and hairpin probes are shown in Table 2.

[0041] Table 2. Sequence information of the target, trapping probe, and hairpin probe used in the electrochemical sensor provided by this invention.

[0042] Note: The bolded region sequence is the G-quadruplex formation sequence. 3. Preparation of capture probe coupled with carboxyl-modified polystyrene magnetic particles (CMPs): Take 50 μL of MPs solution with a concentration of 5 mg / mL, and after magnetic separation, wash three times with 200 μL of imidazole-hydrochloric acid buffer (0.1 M, pH 7.4) containing 0.02% Tween-20. Then, place the MPs in 50 μL of imidazole-hydrochloric acid buffer (0.1 M, pH 7.4) containing 10 mg / mL NHS and 20 mg / mL EDC, and activate at room temperature in the dark for 30 min. After activation, add 50 μL of 1 μM amino-modified capture probe to the MPs solution, and react overnight at 37°C with gentle shaking. After the reaction, after magnetic separation, wash three times with imidazole-hydrochloric acid buffer (0.1 M, pH 7.4), and then place in 100 μL of PBS buffer (0.1 M, pH 7.4) containing 3% BSA and 0.02% Tween-20, and block at 37°C for 1 h to reduce nonspecific adsorption. After blocking, the product CMPs were washed three times with PBS buffer (0.1 M, pH 7.4), and finally resuspended in 100 μL Tris-HCl buffer (0.01 M, pH 7.4) to prepare a suspension of 2.5 mg / mL.

[0043] 4. Characterization of electrochemical sensors This experiment aims to characterize the successful construction of the electrochemical sensor. The specific experimental methods are as follows: (1) Successful characterization of HCR by polyacrylamide gel electrophoresis (PAGE): Ultrapure water, 10×TBE buffer (pH 7.4), 30% acrylamide solution, TEMED, and 10% APS were mixed in appropriate proportions and polymerized to obtain a 12% concentration of natural polyacrylamide gel. 5 μL of different DNA samples with a concentration of 1 μM (e.g., ...) were then used. Figure 2 (a) Add an equal volume of loading buffer, mix thoroughly, and then add the mixture to the sample wells of the gel. Use 1×TBE buffer as the electrophoresis buffer and perform electrophoresis at a constant voltage of 90 V for 90 minutes. After electrophoresis, stain the gel with GelRed nucleic acid dye for 30 minutes, and then use an iBright FL1000 imaging system to acquire images of the gel.

[0044] (2) Circular dichroism (CD) characterization of G-tetrachain nanostructures: CD spectra were acquired in the wavelength range of 220 nm to 340 nm, with a response time of 1 s, a bandwidth of 1.0 nm, and a data point interval of 1.0 nm.

[0045] The hybridization and amplification of nucleic acid chains during the construction of this electrochemical sensor were verified by polyacrylamide gel electrophoresis. Figure 2 As shown in Figure a, lanes 1-4 contain target DNA and three probes, respectively. Compared to the probes loaded individually, the mixture of H1 and H2 (lane 5) and the mixture of the three probes (lane 6) only show clear bands corresponding to each individual probe, with almost no non-specific hybridization, indicating that the hairpin structure of the three probes is relatively stable in the absence of target DNA. When the target DNA is mixed with the capture probe (lane 7) and H1 is further added (lane 8), a new band with slower migration appears, indicating that the target DNA breaks open the capture probe of the hairpin structure, exposes the trigger region, and can partially hybridize with H1. Lanes 9 and 10 correspond to the presence of target DNA, respectively, in the presence or absence of K. + HCR results under the given conditions. The results clearly show that, under K... + In the presence of these components, an intramolecular G-quadruplex structure was formed during the HCR process, and its migration rate was significantly faster than that of linear double-stranded DNA. These findings clearly demonstrate the successful establishment of HCR and the formation of G-quadruplexes within the sensor.

[0046] Subsequently, circular dichroism spectroscopy was used to characterize the formation of G-tetramers in the electrochemical sensor. Loading metal cations within the G-tetramer plane can promote the formation of G-tetramers and enhance their stability, where K... + Than Na + It possesses stronger capabilities. For example... Figure 2 As shown in b, in K + In the presence of these peaks, the HCR product exhibited the strongest characteristic peaks at 245 nm and 263 nm, confirming the successful formation of the G-quadruplex and laying the foundation for the subsequent embedding of electroactive signaling molecules.

[0047] We further investigated the effect of MB intercalation into HCR products using ultraviolet-visible absorption spectroscopy (UV-Vis). Figure 2 As shown in c, free MB exhibits two characteristic absorption peaks at 610 nm and 664 nm, corresponding to the 0-1 band vibrational transition of the MB dimer and the 0-0 band vibrational transition of the monomer, respectively. The peak intensity decreases after the addition of the capture probe and hairpin probe, which is due to the MB embedding into the minor groove of the double strand of the hairpin structure through electrostatic adsorption. Subsequently, target DNA and K were added. + The HCR process and G-quadruplex formation were initiated, leading to a more pronounced hypochromic effect and redshift, indicating that MB is more deeply embedded in the G-quadruplex structure. Simultaneously, the interaction between MB and the G-quadruplex was characterized by CD spectroscopy. Figure 2(d) The characteristic peak ellipticity of the mixture is higher than that of the individual HCR products containing the G-quadruplex structure. This is because the positively charged MB has a large π-aromatic plane, which acts as a cationic ligand to stabilize the G-quadruplex structure. These results indicate that MB can be efficiently intercalated into nucleic acid chains and G-quadruplexes immobilized on CMPs.

[0048] 5. Electrochemical detection of target DNA: Take 50 μL of the CMPs prepared in step 3 and mix them with 50 μL of the test sample containing different concentrations of target DNA, and incubate for 1 h. After magnetic separation, wash three times with PBS buffer (0.1 M, pH 7.4), then add 50 μL of Tris-HCl buffer (0.01 M, pH 7.4, containing 100 mM KCl to promote G-quadruplex formation) containing 1 μM H1 and H2, and perform hybridization chain reaction at 37°C for 0.5-4 h (the optimal reaction time in this example is 2 h). After magnetic separation, add 100 μL of 10-25 mM (the optimal concentration of 20 mM in this example) methylene blue solution prepared with Tris-HCl buffer (0.1 M, pH 7.4). After magnetic separation, wash several times until no residual MB is detected in the supernatant. Hydrolysis was performed by adding 100 μL of a 2 mg / mL 1MIA-Zr solution prepared with Tris-HCl buffer (0.1 M, pH 7.4) at 37°C for 0.5–6 h (4 h was used in this example as the optimal reaction time) to obtain the hydrolysis product. A screen-printed carbon electrode (SPCE) was used as the three-electrode system, where the working electrode and counter electrode were both carbon electrodes, and the reference electrode was an Ag / AgCl electrode. 60 μL (the detection volume can be increased as needed) of the supernatant of the hydrolysis product after vortex mixing and magnetic separation was added dropwise to the three-electrode region of the SPCE (or the three-electrode region of the SPCE was immersed in the solution), and square wave voltammetry (SWV) was used for detection. The square wave voltammetry scanning parameters were set as follows: potential range -0.5–0 V; step potential 0.005 V; amplitude 20 mV; frequency 25 Hz; and settling time 2 s.

[0049] This embodiment verifies the electrochemical feasibility of the triple signal amplification strategy. The verification results are shown in [link to documentation]. Figure 3 .like Figure 3 As shown in Figure a, before the addition of 1MIA-Zr for hydrolysis, the current signal was essentially zero; however, after the addition of 1MIA-Zr catalyzes DNA hydrolysis, MB is released, thereby generating a significant current signal, achieving signal triggering and amplification. Figure 3As shown in b, introducing H1 and H2 in the presence of the target can induce HCR, forming long dsDNA nanostructures, thereby embedding more MB molecules. After hydrolysis, the current signal is significantly enhanced, completing the signal amplification again. Figure 3 As shown in c, K is introduced during the HCR reaction. + It can induce the formation of G-quadruplexes, providing more potential embedding sites for MB and further enhancing the current signal after hydrolysis. Figure 3 The 'd' in the diagram illustrates that the current intensity increases sequentially as the three-stage signal amplification occurs.

[0050] This embodiment also evaluated the detection capability of the electrochemical sensor for target DNA; the evaluation results are shown in [see figure]. Figure 4 . Figure 4 The value of 'a' in the figure indicates that as the concentration of artificially synthesized target DNA increases, the peak current of the corresponding SWV curve also increases. Figure 4 Figure b indicates that, within the range of 0.1 fM–100 pM, the change in the sensor peak current (ΔI = I - I0, where I represents the peak current and I0 represents the background signal current) exhibits a linear relationship with the logarithm of the target DNA concentration. The regression equation for the fitted curve is ΔI = 0.473 lgC. 靶标DNA +8.623, correlation coefficient R 2 =0.999, detection limit is 1.0 aM (S / N=3).

[0051] Example 2: Application of electrochemical sensors in detecting total bacterial count in laboratory water samples To verify that the electrochemical sensor provided by this invention has the ability to detect both Gram-positive and Gram-negative bacteria, this embodiment first prepared bacterial suspensions containing Escherichia coli and Staphylococcus aureus, respectively. Then, the bacterial concentration in the bacterial suspensions was determined by plate counting method, and finally, the electrochemical sensor was used to detect the bacterial concentration in the bacterial suspensions. The specific experimental method is as follows: Laboratory-preserved bacterial strains (Escherichia coli ATCC 25922 and Staphylococcus aureus CMCC(B) 26003) were inoculated into LB liquid medium and cultured overnight at 37°C with shaking at 220 rpm. The bacterial cells were collected by centrifugation, the supernatant was discarded, and the cells were resuspended in 0.01 M Tris-HCl buffer (pH 7.4). Bacterial counts were determined by serial dilution and incubation at 37°C for 18 h in solid LB medium, expressed as colony forming units (CFU). 16S rRNA gene extraction was performed using a boiling extraction method: the bacterial suspension was incubated at 100°C for 20 min, centrifuged at 6000 rpm for 5 min at 4°C, and the supernatant was collected. The supernatant was detected using the electrochemical sensor provided in Example 1.

[0052] Figure 5 This demonstrates the detection capability of the electrochemical sensor provided in Example 1 for the two bacterial suspensions described above. The results indicate that when the bacterial concentration in the suspension is between 1 and 10... 4 Within the CFU / mL range, there was a good linear relationship between the peak current and the logarithm of the concentrations of both bacteria, and the actual detected bacterial concentration was as low as 1 CFU / mL. Figure 5 The 'a' in the figure indicates that for Escherichia coli (E. coli) E. coli For example, the regression equation for the fitted curve is ΔI = 0.947 lgC. E. coli +3.095, correlation coefficient R 2 =0.996; Figure 5 b in the text indicates that for Staphylococcus aureus ( S. aureus For example, the regression equation for the fitted curve is ΔI = 0.680 lgC. S. aureus +3.320, correlation coefficient R 2 =0.997. The above results demonstrate that the sensor provided in Example 1 has the ability to detect both Gram-negative and Gram-positive bacteria.

[0053] Example 3: Evaluation of the anti-interference, reproducibility and stability of the electrochemical sensor 1. Evaluation of anti-interference performance Select common water disturbances, such as F - Cl - NO2 - NO3 - and CO3 2- As anionic interferences, 4-nitrophenol (4-NP), carbendazim (CBZ), ascorbic acid (AA), uric acid (UA), and glucose (Glu) were used as organic compound interferences. 1 nM of each of these interferences was added to a Tris-HCl solution (0.01 M, pH 7.4) containing 10 pM of the target, and the peak current of the SWV curve was recorded. The results of the anti-interference evaluation are as follows: Figure 6 As shown in a and b, even when the concentration of interfering substances is 100 times higher than that of the target, the impact on the sensor response is still negligible, indicating that the sensor has excellent anti-interference performance.

[0054] 2. Evaluation of reproducibility The current response of five identical sensors was tested at the same target concentration (three parallel experiments were performed for each sensor group). The reproducibility evaluation results are as follows: Figure 6 As shown in c, the relative standard deviation (RSD) of the current intensity measured by the five identical sensors is 2.11%, indicating that the sensor has good reproducibility.

[0055] 3. Evaluation of stability The sensor's stock solution was stored at 4°C for one week, and SWV tests were performed daily to record changes in peak current. The stability evaluation results are as follows: Figure 6 As shown in d, after the sensor's storage liquid was placed at 4°C for seven days, the sensor's current response still maintained 95.36% of the initial value, further proving its high stability.

[0056] Example 4: Application of electrochemical sensors in detecting total bacterial count in actual water samples To verify that the electrochemical sensor provided by this invention has the ability to detect bacteria in various actual water samples, this embodiment first enriches the collected water samples to obtain bacterial suspensions for each water sample. Then, the bacterial concentration in the bacterial suspensions is determined by plate counting. Finally, the electrochemical sensor is used to detect the bacterial concentration in the bacterial suspensions. The specific experimental method is as follows: Three different water samples (20 mL each) were collected: surface water (from Nanhu Park), tap water (from a laboratory tap), and drinking water (from a conventional water purifier). Each sample was added to 200 mL of LB liquid medium and incubated overnight at 37°C and 220 rpm with shaking. The bacterial cells were collected by centrifugation, the supernatant was discarded, and the suspension was resuspended in 0.01 M Tris-HCl buffer (pH 7.4) to obtain a bacterial suspension. The bacterial count was determined by serial dilution and incubation at 37°C for 18 h in solid LB medium. The 16S rRNA gene was then extracted using a boiling extraction method, and the supernatant was detected using the electrochemical sensor provided in Example 1.

[0057] The test results of three different water samples from actual bodies are as follows: Figure 7 As shown, in the three water samples, the peak current of the SWV curve gradually increased with the increase of bacterial concentration. Figure 7 (a, c, e in the equation). A good linear relationship was observed between the peak current and the logarithm of the bacterial concentration. Specifically, the fitted regression equation for surface water was ΔI = 0.746 lgC. 地表水中的细菌 +2.845, correlation coefficient R 2 =0.994 ( Figure 7 (b) The fitted regression equation for tap water is ΔI = 0.784lgC. 自来水中的细菌 +2.579, correlation coefficient R 2 =0.998 ( Figure 7 (d) The fitted regression equation for drinking water is ΔI = 0.812 lgC 饮用水中的细菌 +2.537, correlation coefficient R 2 =0.996 ( Figure 7 (f in the figure). The above results demonstrate that the electrochemical sensor provided by this invention is suitable for detecting the total number of bacteria in complex real-world water bodies.

[0058] Furthermore, the correlation between the total bacterial count detected using the electrochemical sensor provided by this invention and the total bacterial count detected using the traditional plate-mount technique was verified. The specific evaluation method is as follows: Different concentrations of *E. coli* were added to three actual water samples that had undergone autoclaving, and then the total bacterial count in the obtained water samples was detected using the two methods described above. Figure 8 As shown, the correlation coefficient R between the two methods is [value missing] in the three water samples. 2 All values ​​were greater than 97%, showing a significant correlation. Statistical analysis showed that the slopes of all linear regression equations were close to 1, and the intercepts were close to zero, demonstrating that there was no significant difference between the detection method using the electrochemical sensor provided by this invention and the standard culture method. This indicates that the electrochemical sensor provided by this invention possesses accuracy comparable to the "gold standard" plate counting method.

[0059] When using the electrochemical sensor provided by this invention to perform actual detection on water samples, a pretreatment step is performed to avoid interference from environmental DNA (eDNA) and ensure that the detection results are within the detection limits of the method. Specifically, the pretreatment method involves collecting a large volume of water sample of known volume (sufficient to meet subsequent detection requirements), filtering it through a 0.22 μm filter membrane to remove eDNA from the water, and collecting the bacteria enriched on the filter membrane for subsequent 16S rRNA gene extraction and electrochemical detection, thereby achieving accurate determination of the total bacterial count in the sample.

[0060] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. An electrochemical sensor for detecting total bacterial count in water, characterized in that, The electrochemical sensor includes target DNA for detecting total bacterial count in water, CMPs formed by coupling an amino-modified hairpin-structured capture probe with carboxyl-modified polystyrene magnetic particles, a hairpin probe, potassium ions, methylene blue, and an artificial nuclease 1MIA-Zr; the nucleotide sequence of the target DNA is shown in SEQ ID NO.21, the nucleotide sequence of the capture probe is shown in SEQ ID NO.22, and the hairpin probe is composed of H1 and H2, the nucleotide sequences of H1 and H2 are shown in SEQ ID NO.23 and SEQ ID NO.24, respectively; When bacteria are present in the water, CMPs can specifically recognize the conserved region of the 16S rRNA gene of aquatic bacteria, open the hairpin structure of the capture probe, and then undergo a hybridization chain reaction with the designed hairpin probes H1 and H2 to achieve nucleic acid chain amplification, resulting in the first amplification of the signal. Subsequently, under the action of potassium ions, a G-quadruplex spatial structure is formed to embed more electrochemical signal molecules, methylene blue, resulting in the second amplification of the signal. After magnetic separation and washing, the artificial nucleic acid hydrolase 1MIA-Zr is used to hydrolyze the phosphodiester bond, cut the nucleic acid chain, and release the electrochemical signal molecule MB, achieving the third amplification of the signal. By detecting the electrochemical signal molecule MB, the total number of bacterial colonies in the water can be quantified.

2. The method for preparing the electrochemical sensor according to claim 1, characterized in that, Includes the following steps: 1) Prepare a Tris-HCl buffer solution containing H1, H2 and KCl; 2) Prepare a Tris-HCl buffer solution containing methylene blue; 3) Prepare Tris-HCl buffer containing artificial nuclease 1MIA-Zr; 4) Preparation of CMP suspension: After magnetic separation and washing, the carboxyl-modified polystyrene magnetic particle solution was placed in imidazole-hydrochloric acid buffer containing NHS and EDC and activated at room temperature in the dark for 30 min; an amino-modified hairpin-structured capture probe was added and the reaction was carried out overnight with gentle shaking at 37°C; after the reaction, after magnetic separation and washing, the solution was placed in PBS buffer containing BSA and Tween-20 and blocked at 37°C for 1 h; after blocking, the solution was washed with PBS buffer, and finally the CMP product was resuspended in Tris-HCl buffer.

3. The preparation method according to claim 2, characterized in that, In step 1), the concentrations of H1, H2, and KCl in the Tris-HCl buffer are 1 μM, 1 μM, and 100 mM, respectively. The concentration of Tris-HCl in the Tris-HCl buffer is 0.01 M, and the pH of the Tris-HCl buffer is 7.

4.

4. The preparation method according to claim 2, characterized in that, In step 2), the concentration of methylene blue in the Tris-HCl buffer is 10-25 mM, the concentration of Tris-HCl in the Tris-HCl buffer is 0.1 M, and the pH of the Tris-HCl buffer is 7.

4.

5. The preparation method according to claim 2, characterized in that, In step 3), the concentration of the artificial nuclease 1MIA-Zr in the Tris-HCl buffer is 2 mg / mL, the concentration of Tris-HCl in the Tris-HCl buffer is 0.1M, and the pH of the Tris-HCl buffer is 7.

4.

6. The preparation method according to claim 2, characterized in that, The specific method for step 4) is as follows: Take 50 μL of a carboxyl-modified polystyrene magnetic particle solution with a concentration of 5 mg / mL, perform magnetic separation, wash three times with 200 μL of imidazole-hydrochloric acid buffer containing 0.02% Tween-20, and place it in 50 μL of imidazole-hydrochloric acid buffer containing 10 mg / mL NHS and 20 mg / mL EDC. Activate at room temperature in the dark for 30 min. After activation, add 50 μL of 1 μM amino-modified capture probe and react gently at 37°C overnight. After the reaction, perform magnetic separation, wash three times with imidazole-hydrochloric acid buffer, and then place it in 100 μL of PBS buffer containing 3% BSA and 0.02% Tween-20. Block at 37°C for 1 h. After blocking, wash three times with PBS buffer. Finally, resuspend the product CMPs in 100 μL of Tris-HCl buffer to prepare a suspension of 2.5 mg / mL.

7. The preparation method according to claim 6, characterized in that, The concentration of the imidazole-hydrochloric acid buffer was 0.1 M and the pH was 7.4; the concentration of the PBS buffer was 0.1 M and the pH was 7.

4. The Tris-HCl buffer solution has a concentration of 0.01 M and a pH of 7.

4.

8. A method for detecting the total number of bacterial colonies in water using the electrochemical sensor described in claim 1, characterized in that, The procedure includes the following steps: Collect water samples of known volume that meet the requirements for subsequent testing; filter the water through a 0.22 μm filter membrane to remove environmental DNA; collect bacteria enriched on the filter membrane; extract the 16S rRNA gene to obtain the test sample; mix the test sample with a 2.5 mg / mL CMP suspension and incubate for 1 h; after magnetic separation, wash three times with PBS buffer, then add Tris-HCl buffer containing 1 μM H1, 1 μM H2, and 100 mM KCl, and perform a hybridization chain reaction at 37°C for 0.5–4 h; after magnetic separation, add Tris-HCl buffer containing 10–25 mM methylene blue, and after magnetic separation, wash multiple times until no residual MB is detected in the supernatant; add Tris-HCl buffer containing 2 mg / mL 1MIA-Zr, and perform a hydrolysis reaction at 37°C for 0.5–6 h. h, obtain the hydrolysate; using a screen-printed carbon electrode as a three-electrode system, take the supernatant of the hydrolysate after vortex mixing and magnetic separation, and drop it onto the three-electrode region of the screen-printed carbon electrode (or immerse the three-electrode region of the screen-printed carbon electrode in the solution), and detect it using square wave voltammetry.

9. The method according to claim 8, characterized in that, In the three-electrode system, both the working electrode and the counter electrode are carbon electrodes, and the reference electrode is an Ag / AgCl electrode.

10. The method according to claim 8, characterized in that, The square wave voltammetry scanning parameters are set as follows: potential range -0.5 to 0 V; step potential 0.005 V; amplitude 20 mV; frequency 25 Hz; resting time 2 s.

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