DNA hydrogel biosensing composition for detecting pathogenic bacteria based on portable glucometer and application of DNA hydrogel biosensing composition

Abstract

The invention belongs to the field of food detection, and particularly relates to a DNA hydrogel biosensing composition based on a portable glucometer for detecting pathogenic bacteria and application of the DNA hydrogel biosensing composition. The DNA hydrogel biosensing composition comprises magnetic beads loaded with pathogenic bacterium aptamers and primer probes, a ring forming sequence 1, a ring forming sequence 2, glucose or sucrase and sucrose. According to the method, pathogenic bacteria are competitively combined with pathogenic bacteria aptamers, primer probes which are partially complementarily hybridized with the pathogenic bacteria aptamers are released, under the action of free primer probes, a cyclization sequence 1 and a cyclization sequence 2 are subjected to a pre-cyclization reaction and an RCA amplification reaction respectively, generated ssDNA is self-assembled into mutually-crosslinked DNA hydrogel, glucose or sucrase is wrapped in the DNA hydrogel, and the hydrogel is prepared. Further, the kit is combined with a portable glucometer to realize convenient, rapid, accurate and visual detection of the escherichia coli, and the kit is suitable for popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of food testing, specifically relating to a DNA hydrogel biosensor composition based on a portable blood glucose meter for the detection of pathogenic bacteria and its application. Background Technology

[0002] With the development of food processing technology, foodborne pathogens during food distribution have become a crucial risk control indicator for food safety in the food industry. In recent years, food safety incidents have occurred frequently, and enteropathogenic Escherichia coli (E. coli) is a representative pathogen among foodborne pathogens and one of the main transmission pathogens in the food industry. Hemorrhagic E. coli can cause severe acute gastroenteritis after infecting epithelial cells in the intestines. Among these, Shiga toxin-producing serotype E. coli O157:H7 (Escherichia coli, E. coli) has become an important food and waterborne pathogen. Currently, national standards stipulate that E. coli O157:H7 in food must not be detected. Therefore, rapid and sensitive detection of pathogenic E. coli is particularly important in the field of food safety.

[0003] Currently, traditional detection methods for pathogenic bacteria in food, such as microbial culture, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and magnetic fluorescent nanoparticle detection, suffer from low sensitivity, long processing times, and require trained personnel. Immunological analysis and molecular biological detection significantly shorten detection time, but require sample pretreatment, are prone to false positives, and are costly. With technological advancements, biosensor research has progressed rapidly, resulting in many novel biosensors. Among them, aptamer biosensors have attracted attention due to their sensitivity and speed. Nucleic acid aptamers, sequences screened from artificially synthesized oligonucleotide libraries, can recognize target molecules with high specificity, have a wide range of applications, high resolution, and a relatively simple and rapid screening process. Qi et al. combined split nucleic acid aptamers with chitosan and AuNPs to create nanocomposites with different morphologies, in order to develop label-free colorimetric aptamer sensors for rapid detection of kanamycin. Chu et al. proposed a functional DNA as a cross-linking agent and analyte-binding probe for label-free responsive detection of heavy metal environmental bacteria. Li et al. developed a mussel-responsive biosensor to detect Escherichia coli O157:H7. Although these methods have high sensitivity, they rely on sophisticated large instruments and have cumbersome operation procedures, which cannot meet the current demand for rapid on-site detection.

[0004] With the increasing development of the field of bioanalytical detection, the requirements for the accuracy and sensitivity of biosensors are becoming increasingly stringent. Sensor strategies based on magnetic beads (MBs) have emerged as a result, as MBs have broad application prospects due to their excellent surface modification capabilities. In 2012, Luo et al. prepared DNA hydrogel materials constructed from linear long-chain DNA (ssDNA) using various nucleic acid amplification methods. Rolling circle amplification (RCA) is an isothermal nucleic acid amplification technique that generates long single-stranded DNA with a periodic sequence by cyclically replicating a circular template. It has become one of the routine methods for rapid preparation of DNA hydrogels. DNA hydrogels are hydrophilic materials that exist between fluids and solids, formed by the self-assembly of DNA molecules; they are macroscopically visible hydrogel materials. Currently, there are numerous studies on biosensing strategies that combine nanomaterials with DNA hydrogels.

[0005] Point-of-care testing (POCT) is considered one of the technologies that meet the "point-of-care testing requirements" and is applied in home healthcare and environmental monitoring. Personal glucose meters (PGMs), as common POC devices, are widely used due to their ability to perform on-site testing, portability, and user-friendly operation. However, research on using them to construct biosensors for food detection is relatively limited. In recent years, DNA hydrogels assembled from nucleic acid aptamers and nanomaterials have exhibited excellent biological properties, and their combination with PGMs shows great promise for the point-of-care detection of foodborne pathogens. Summary of the Invention

[0006] This invention aims to provide a DNA hydrogel biosensor composition based on a portable blood glucose meter for the detection of pathogenic bacteria. It transforms the detection of pathogenic bacteria into glucose detection based on a portable blood glucose meter, offering simple operation, immediate results, portability, accuracy, and reliability, with promising application prospects.

[0007] This invention provides a DNA hydrogel biosensor composition based on a portable blood glucose meter for the detection of pathogenic bacteria, comprising magnetic beads loaded with pathogenic bacteria aptamers and primer probes, circular sequence 1, circular sequence 2, glucose or sucrase and sucrose, wherein the primer probes are hybridized and linked to the pathogenic bacteria aptamers, and the pathogenic bacteria aptamers are hybridized and loaded on the magnetic beads.

[0008] Circular sequence 1 is shown in SEQ ID NO:1, and circular sequence 2 is shown in SEQ ID NO:2.

[0009] A DNA hydrogel biosensor composition for detecting pathogenic bacteria based on a portable blood glucose meter, further comprising a pre-circularization system component and an RCA amplification system component. The pre-circularization system component includes T4 ligase, T4 ligase buffer, etc., and the RCA amplification system component includes phi 29 DNA polymerase, phi 29 DNA polymerase buffer, dNTPs, etc.

[0010] The pathogenic aptamer is loaded onto the magnetic beads by hybridization with the aldehyde group on the magnetic beads.

[0011] The primer probes are linked to the pathogenic aptamers via partial complementary hybridization.

[0012] The pathogenic bacterium is Escherichia coli, specifically Escherichia coli O157:H7. The aptamer for Escherichia coli O157:H7 is shown in SEQ ID NO:3, and the primer probe is shown in SEQ ID NO:4. This invention is validated by preparing a DNA hydrogel biosensor composition for detecting Escherichia coli O157:H7 based on a portable blood glucose meter. When E. coli O157:H7 is present, it competitively binds to the E. coli aptamer. Primers and probes that partially hybridize with the E. coli aptamer are released into a free state. Under the action of these free primers and probes, circular sequence 1 and circular sequence 2 undergo pre-circularization and RCA amplification reactions, respectively, generating ssDNA that self-assembles into a cross-linked DNA hydrogel. Added glucose or sucrase is gradually encapsulated within the DNA hydrogel. As glucose is gradually encapsulated, the amount of free glucose gradually decreases, resulting in no color change or insignificant color change on the blood glucose test strip, or a lower reading on the blood glucose meter. Similarly, as sucrase is gradually encapsulated, the amount of free sucrase gradually decreases, leading to a corresponding decrease in the glucose produced from sucrose, resulting in no color change or insignificant color change on the blood glucose test strip, and a lower reading on the blood glucose meter.

[0013] Therefore, the DNA hydrogel biosensor composition based on a portable blood glucose meter for pathogen detection provided by this invention can be used for the detection of pathogens, especially Escherichia coli (e.g., E. coli O157:H7); it can also be used to prepare biosensor reagents, kits, or biosensors for the qualitative and / or quantitative detection of pathogens, realizing the qualitative and / or quantitative detection of pathogens, especially Escherichia coli (e.g., E. coli O157:H7). It has the advantages of simple operation, rapid reaction, convenient visualization, and accurate and reliable results, and has broad application prospects.

[0014] A DNA hydrogel biosensor or biosensor based on a portable blood glucose meter for the detection of pathogenic bacteria, comprising the DNA hydrogel biosensor composition based on a portable blood glucose meter for the detection of pathogenic bacteria provided by the present invention.

[0015] The present invention provides a DNA hydrogel biosensor composition for detecting pathogenic bacteria based on a portable blood glucose meter. The prepared DNA hydrogel is in the form of nanoflowers and tightly connected into a network structure. The DNA hydrogel has a size of 500-1500 nm, preferably 800-1200 nm, and more preferably 1000 nm.

[0016] A method for detecting pathogenic bacteria includes the following steps: mixing magnetic beads loaded with pathogenic bacteria aptamers and primer probes with the analyte and incubating; taking the supernatant, adding circular sequence 1 and circular sequence 2, and sequentially performing a pre-circularization reaction and RCA amplification reaction; adjusting the pH to acidic and adding glucose for incubation; and taking the supernatant for qualitative or quantitative detection on a blood glucose test strip or blood glucose meter; or

[0017] Magnetic beads loaded with pathogenic bacterial aptamers and primer probes are mixed with the analyte and incubated. The supernatant is taken, and circular sequence 1 and circular sequence 2 are added. Pre-circulation reaction and RCA amplification reaction are performed sequentially. The pH is adjusted to acidic and sucrase is added for incubation. Sucrose is then added and the reaction is carried out at room temperature. The supernatant is taken and used on blood glucose test strips or blood glucose meters for qualitative or quantitative detection.

[0018] Further, magnetic beads loaded with pathogenic bacterial aptamers and primer probes were mixed with the analyte and incubated. The supernatant was collected and divided into two equal portions. Circular sequence 1 and circular sequence 2 were added to each portion, and each portion underwent a pre-circularization reaction and RCA amplification reaction sequentially. After adjusting the pH to acidic and adding glucose for incubation, the two portions were mixed for hybridization. The supernatant was then used for qualitative or quantitative detection on blood glucose test strips or a blood glucose meter; or

[0019] Magnetic beads loaded with pathogenic bacterial aptamers and primer probes were mixed with the analyte and incubated. The supernatant was taken and divided into two equal parts. Circular sequence 1 and circular sequence 2 were added to each part, and pre-circularization reaction and RCA amplification reaction were performed sequentially. The pH was adjusted to acidic and sucrase was added for incubation. The two parts were then mixed for hybridization. Sucrose was added and the reaction was carried out at room temperature. The supernatant was then used for qualitative or quantitative detection on blood glucose test strips or a blood glucose meter.

[0020] In quantitative detection, a standard curve is plotted using the concentration of pathogenic bacteria and the blood glucose meter reading as variables, and quantitative determination is performed.

[0021] The pH value is 3-7, preferably 3-5, and more preferably 3.

[0022] The pathogenic bacterium is *Escherichia coli*, specifically *E. coli* O157:H7, with a concentration range of 1×10⁻⁶. 3 ~1×10 7 CFU / mL, the standard curve is Y = 18.05 - 1.63X, R 2 =0.99.

[0023] Beneficial effects

[0024] This study selected *E. coli* O157:H7 as the research object and designed a DNA hydrogel instant detection aptamer sensor based on aldehyde-based biomagnetic bead activation and rolling circle amplification, which was then used in conjunction with a portable blood glucose meter. By simply collecting the supernatant of the generated DNA hydrogel and placing it onto the blood glucose meter test strip, on-site and rapid detection of food safety can be achieved. This strategy is simple and rapid, requiring no large instruments. Furthermore, by simply changing the aptamer sequence of the target, the visual quantification of other foodborne pathogens or food contaminants can be achieved, demonstrating good versatility. Attached Figure Description

[0025] Figure 1 A schematic diagram illustrating the construction principle of a portable blood glucose meter biosensor based on DNA hydrogel.

[0026] Figure 2 UV absorption spectra of magnetic bead hybrid DNA: (A) Absorption spectrum of magnetic bead-linked E. coli aptamers (MBs@EA); (B) UV absorption spectrum of magnetic bead-EA-CS complex (MBs@dsDNA).

[0027] Figure 3 (A) Agarose gel electrophoresis image for feasibility verification (lane 1: experimental group; lane 2: control group; M, molecular weight standard); (B) Agarose gel electrophoresis image of CS sequence rolling circle amplification products at gradient concentrations: lane 1, 25 μmol / L; lane 2, 5 μmol / L; lane 3, 1 μmol / L; lane 4, 0.2 μmol / L; lane 5, 0.04 μmol / L; M, molecular weight standard.

[0028] Figure 4 (A) Photograph of RCA products under white light (tubes 1 and 2 are partially complementary RCA products; tube 3 is the product after mixing the two tubes); (C) Fluorescence microscopy characterization of stained DNA hydrogel; (B) Morphology of DNA hydrogel in a culture dish; (D) Scanning electron microscopy image of DNA hydrogel (top) and magnified nanoflower structure (bottom).

[0029] Figure 5(A) Blood glucose meter readings after glucose was encapsulated in DNA hydrogels generated by CS sequence amplification at gradient concentrations; (B) Decreases in blood glucose meter readings after glucose was encapsulated in DNA hydrogels at different pH values; (C) Linear fitting curves.

[0030] Figure 6 (A) Blood glucose meter readings for detecting gradient concentrations of E. coli O157:H7; (B) Linear fitting curve.

[0031] Figure 7 Sensor specificity verification diagrams, including amplification products of different bacteria (top) and corresponding agarose gel electrophoresis diagrams (bottom) (2-5 are E. coli O6, S. typhimurium, S. aureus, and L. monocytogenes, respectively). Detailed Implementation

[0032] 1. Experimental Section

[0033] 1.1 Instruments and Reagents

[0034] UV-2540 UV-Vis spectrophotometer (Shimadzu Corporation, Japan); CKX-41 inverted fluorescence microscope (Olympus Corporation, Japan); ChemiDoc XRS+ gel imaging system (Bio-Rad Corporation, USA); 9600 gene amplification instrument (Zhuhai Heima Medical Instrument Co., Ltd.); TUS200P thermostatic oscillating metal bath (Shanghai Yiheng Technology Co., Ltd.); Mixer 4K vortex mixer (Sangon Biotech (Shanghai) Co., Ltd.); SU8600 field emission scanning electron microscope (Hitachi Corporation, Japan); Sinocare blood glucose meter (Dalian Kaimei Chemical Engineering Supporting Co., Ltd.).

[0035] Aminomagnetic particle kit (Bio-Mag Plus, Polysciences, USA); bovine serum albumin (BSA, Amresco, USA); phosphate buffer (1×PBS, pH 7.5), 50×TAE buffer (2 mol / L Tris-acetic acid, 100 mol / L EDTA, pH 8.4), high-strength agarose, D-glucose, 4S Red Plus nucleic acid staining agent (10000× aqueous solution), phi29 DNA polymerase (Thermo Fisher Scientific, USA); dNTPs, LB broth powder, nutrient agar, sucrose (20000 μg / ml in H2O), sucrase (100 u / mg), and other routine reagents were purchased from the CASMA online marketplace. E. coli O157∶H7 (ATCC 43889), Escherichia coli O6 (E. coli O6, ATCC 25922), Listeria monocytogenes (L. monocytogenes, ATCC 19115), Staphylococcus aureus (S. aureus, ATCC 25923), and Salmonella typhimurium (S. typhimurium, ATCC 19585) were all purchased from the American Type Culture Collection (ATCC). All reagents used were at least analytical grade. The water used in the experiments was ultrapure water prepared using the Milli-Q system (Barnstead, USA). DNA fragments were purified by HPLC. The E. coli aptamer sequence was provided by Sangon Biotech (Shanghai) Co., Ltd., and the specific DNA sequence is shown in Table 1.

[0036] 1.2 Experimental Methods

[0037] 1.2.1 Preparation of MBs@dsDNA

[0038] Activate 5 mL of magnetic beads (50 mg / mL, 1.5 μmol / L) according to the product instructions to convert the amino groups on the beads to aldehyde groups. Pipette 80 μL of the activated magnetic beads, wash three times with 1×PBS, magnetically separate, add 92 μL of 1×PBS buffer and 8 μL of 50 μmol / L *E. coli* aptamer (EA) denatured at 95°C, and incubate overnight at room temperature with shaking. After magnetic separation, wash twice with 200 μL of 1×PBS, add 100 μL of 2% BSA, and block at room temperature for 20 min. After magnetic separation, resuspend the magnetic beads in 100 μL of 1×PBS buffer and store at 4°C. 16 μL of complementary sequence primers and probes (CS) of 50 μmol / L E. coli aptamers were pipetted into a 100 μL MBs@EA centrifuge tube, incubated at 37 °C for 2 h, vortexed, magnetically separated, washed three times with 200 μL PBS buffer, and the precipitate was resuspended in 100 μL PBS solution. The obtained MBs@dsDNA complex was stored at 4 °C for later use.

[0039] 1.2.2 Bacterial Culture

[0040] E. coli O157∶H7 bacterial culture frozen at -20℃ was activated at room temperature, streaked, and inoculated into LB broth medium. The culture was then incubated overnight on a shaker (200 rpm, 37℃, 12 h). The overnight cultured bacteria were centrifuged at 8000 rpm for 5 min, washed three times with 1×PBS, and the supernatant was collected. The bacterial concentration was determined by plating for 24 h, and the culture was diluted to an appropriate concentration with TE buffer for further experiments.

[0041] Table 1. DNA sequences used in the experiment.

[0042]

[0043] *The italicized portion represents the aptamer sequence of E. coli O157∶H7. The primer-probe (CS) serves as the primer for the circular sequence. The bases in the single-underlined portion are complementary sequences. The double-underlined portion represents the bases complementary to the circular sequence and the primer.

[0044] 1.2.3 Determination of E. coli O157:H7

[0045] Take 16 μL of the previously prepared MBs@dsDNA complex and add 64 μL of E. coli O157:H7 diluted with TE buffer at gradient concentrations. Incubate at 37 °C for 10 min, shaking occasionally to ensure full contact between the magnetic bead complex and the bacterial culture, for specific capture of the bacteria releasing the CS sequence. After the reaction, magnetically separate the supernatant and divide it into two equal-volume tubes. Add circular sequence 1, circular sequence 2, and T4 ligase to the supernatant of each tube for pre-circularization for 16 h. Then add phi 29 DNA polymerase, dNTPs, etc., and incubate at 37 °C for 15 min for RCA. Then add 5 μL of sucrase solution and continue incubation for 10 min. After the reaction, hybridize the amplified products of both tubes at room temperature with shaking. Add 35 μL of sucrose solution and react at room temperature for 25 min. Take 3 μL of the supernatant and drop it onto a blood glucose meter test strip. Read the blood glucose meter reading after 20 s.

[0046] 2 Results and Discussion

[0047] 2.1 Design and Detection Principle of Aptamer Sensor

[0048] The biosensing strategy of portable blood glucose meters based on DNA hydrogels is as follows: Figure 1 As shown. In the first step, the activated magnetic bead surface is converted into aldehyde groups, which hybridize with the aptamer EA. Then, the added primer probe (CS) hybridizes with the partially complementary EA, forming a magnetic bead-dsDNA complex. When E. coli O157∶H7 is present in the detection system, the aptamer on the magnetic bead specifically recognizes and captures the bacteria, releasing CS as a primer for the circular sequence. After magnetic separation, the supernatant was collected and divided into two tubes. Two circular templates and phi29 DNA polymerase were added to each tube for RCA. During this process, deoxyribonucleotide triphosphates (dNTPs) were continuously added to the 3' sticky ends of the primers, resulting in two ssDNA amplification products. The two single strands were partially complementary, forming a cross-linked DNA hydrogel through DNA self-assembly. Sucrase solution was then added. A higher concentration of E. coli O157:H7 resulted in a larger amount of DNA hydrogel generated, leading to a greater amount of encapsulated sucrase. Encapsulated sucrase could not catalyze sucrose conversion, reducing the amount of free sucrase and thus decreasing glucose production from sucrose, resulting in a lower reading on the blood glucose meter. The two showed a negative correlation. In the control group without E. coli O157:H7, no DNA hydrogel was formed, and all the added sucrase was used to catalyze sucrose, resulting in a significantly higher blood glucose meter reading.

[0049] UV-Vis absorption spectrum of 2.2 MBs@dsDNA

[0050] The assembly of magnetic beads with aptamers EA and CS was characterized by ultraviolet absorption spectroscopy. For example... Figure 2As shown in Figure A, magnetic beads were connected to the EA chain. After magnetic separation, the supernatant was collected, and the absorbance at 260 nm was measured to be 0.8946. This is lower than the UV absorbance of the added EA chain at this point (0.8964), indicating that the amino-modified EA chain and aldehyde-based magnetic beads were successfully assembled into MBs@EA. Similarly, the UV absorption curves of the supernatant after magnetic separation and hybridization with CS were compared with those of the unincubated CS sequence. Figure 2 As shown in Figure B, the peak at 260 nm also decreased, indicating successful assembly of MBs@dsDNA.

[0051] 2.3 Feasibility Verification of Aptamer Sensors

[0052] Take 0 and 1×10 4 CFU / mL E. coli O157:H7 was added to the prepared magnetic bead-DNA complex for incubation. After magnetic separation, the supernatants of both were aspirated and added with circularization sequence and phi29 DNA polymerase for RCA. Due to the competitive binding of E. coli O157:H7 to the aptamer, CS was released, initiating RCA and forming a DNA hydrogel. No amplification products were generated without the target analyte. The amplification products after RCA were characterized by agarose gel electrophoresis. Figure 3 As shown in A, lane 1 has 1×10 4 The sample containing CFU / mL E. coli O157:H7 amplified linear ssDNA products, showing a bright band at the injection port. This phenomenon also indicates that the CS sequence was successfully released on the surface of the magnetic beads, triggering RCA and generating high molecular weight products. The control group without E. coli O157:H7 could not perform RCA (lane 2) and no band was displayed.

[0053] Before detecting gradient concentrations of E. coli O157∶H7, the amount of RCA product amplified was compared using primers and probes CS at gradient concentrations (25, 5, 1, 0.2, 0.04 μmol / L). Figure 3 As shown in Figure B, the amount of RCA product generated in lanes 1-5 decreased sequentially with decreasing CS chain concentration (CS chain concentrations in lanes 1-5 were 25, 5, 1, 0.2, and 0.04 μmol / L, respectively). When the CS concentration decreased to 0.04 μmol / L, the product band could no longer be observed (lane 5). These results indicate that when the concentration of the cyclic sequence remains constant, the amount of RCA product generated is positively correlated with the concentration of the CS sequence.

[0054] While the above-mentioned rolling circle amplification (RCA) validation confirmed that the *E. coli* aptamer successfully recognized *E. coli* O157:H7 and initiated RCA, the amplified ssDNA became difficult to observe visually as the concentration of the analyte decreased. Therefore, based on the existing results, an RCA biosensor prepared by adding two circular sequences (circular sequence 1 and circular sequence 2) to the above steps was used to detect *E. coli* O157:H7. The prepared MBs@dsDNA was mixed with 1×10 6 Incubate with CFU / mL E. coli O157:H7, then centrifuge with shaking after incubation. Figure 4 As shown in Figure A, flocculent RCA products are clearly visible in both tubes. After mixing and centrifugation, white clumps of DNA hydrogel form at the bottom of the tubes. Carefully aspirate the DNA hydrogel with a pipette and place it in a culture dish; it will exhibit a viscous, stringy consistency. Figure 4 (B, red arrow) This morphology exhibits a liquid-like state in water, but becomes transparent and solid-like after being absorbed. To observe the microstructure of the hydrogel, a red fluorescent dye was added to the generated DNA hydrogel, and a network structure was observed under a fluorescence microscope. Figure 4 C). Next, the microstructure of the DNA hydrogel was characterized. Figure 4 D is an image of the DNA hydrogel under a scanning electron microscope. The surface of the DNA hydrogel shows dense, uniformly sized nanoflower-like structures, which are clusters formed by RCA products in space. They are uniform in shape and size, with a diameter of about 1 μm.

[0055] 2.4 Detection performance of blood glucose meter biosensors based on rolling circle amplification technology for E. coli O157:H7

[0056] To demonstrate the speed and simplicity of this sensor, the encapsulation of glucose by the DNA hydrogel was first investigated. The encapsulation performance of the DNA hydrogel was verified using the five gradient concentrations of the aforementioned CS sequences. After pre-circulating with circularizing sequences 1 and 2 and initiating RCA with DNA polymerase, the two tubes were mixed, glucose was added beforehand, and incubation continued. The supernatant was then centrifuged and the reading was taken on a blood glucose meter. Figure 5 As shown in Figure A, the amount of glucose molecules encapsulated decreases with decreasing DNA hydrogel concentration, resulting in a lower reading on the blood glucose meter. Further investigation was conducted to explore the effect of pH on the encapsulation performance of the DNA hydrogel. A glucose solution was encapsulated using a 25 μmol / L DNA hydrogel with amplified CS sequences. Figure 5 As shown in Figure B, the blood glucose meter readings showed a significant change at pH 3. This may be because glucose molecules are positively charged and have a strong electrostatic adsorption effect on negatively charged DNA, resulting in strong encapsulation and a smaller number of glucose molecules remaining in the supernatant. For 1×10⁻⁶... 3 ~1×10 7The E. coli O157:H7 bacterial suspensions were tested using CFU / mL gradient concentrations. Figure 5 C) The bacterial concentration was linearly fitted to the PGM signal, yielding the equation Signal = 1.28lgC - 3.61, with a limit of detection (LOD) of 1 × 10⁻⁶. 4 CFU / mL. The above results show that although DNA hydrogels can double the encapsulation efficiency of glucose through electrostatic adsorption (CFU / mL). Figure 5 B), but due to the small molecular weight of glucose, the encapsulation efficiency was still not ideal. Therefore, we further tried to replace glucose with sucrase (261.940 KD) with a larger molecular weight and tested the encapsulation performance of the gel.

[0057] To evaluate the detection performance of the blood glucose meter's biosensor, a setting (1×10) was used. 3 ~1×10 7 The detection performance was evaluated using E. coli O157:H7 bacterial suspensions at gradient concentrations (CFU / mL). After RCA, sucrase and sucrose solution were added sequentially to tubes 1-5, followed by shaking and incubation. Since the isoelectric point of sucrase is 5, the pH of the system was adjusted to 3 to make it positively charged and adsorb onto the DNA hydrogel for encapsulation. Figure 6 As shown in Figure A, the blank control group, which does not contain E. coli O157:H7, showed no DNA hydrogel formation. With increasing E. coli O157:H7 concentration, the amount of DNA hydrogel formed in centrifuge tubes (1–5) increased sequentially. The supernatant was aspirated, and the reading was taken using a blood glucose meter. Figure 6 As can be seen, the signal (glucose concentration) measured by PGM gradually decreases with increasing E. coli O157:H7 concentration; for example... Figure 6 As shown in B, the PGM signal is negatively correlated with the concentration of E. coli O157:H7 at a concentration of 1×10⁻⁶. 3 ~1×10 7 The linear relationship was good within the CFU / mL range, and the linear equation was Signal = 18.05 - 1.63lgC(R). 2 =0.99), and its detection limit was calculated to be 1×10. 3 CFU / mL. We also compared this sensor with other detection methods (Table 2), showing that this method has the advantages of being rapid, simple, and providing visual quantification.

[0058] Table 2 Comparison of detection methods for E. coli O157:H7

[0059]

[0060] literature:

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[0065] To verify the selectivity of the biosensor of this method for Escherichia coli O157:H7, 1×10 5 E. coli O157:H7 at a concentration of CFU / mL and 1×10 7 The control group, consisting of four other pathogenic bacteria at CFU / mL (E. coli O6, S. typhimurium, S. aureus, and L. monocytogenes), was incubated and characterized by agarose gel electrophoresis. Results are as follows: Figure 7 The results showed that no obvious DNA hydrogel was generated in the control group and no bands were observed in lanes 2-5. When E. coli O157:H7 was detected, obvious DNA hydrogel appeared at the bottom of the tube, with only lane 1 showing a clear black band, consistent with naked-eye observation. This indicates that the sensor can only release the CS sequence to trigger RCA and generate DNA hydrogel when E. coli O157:H7 is present, which is then encapsulated with sucrase for blood glucose meter reading, demonstrating good recognition specificity.

[0066] 3. Conclusion

[0067] Currently, biosensing technology based on nanomaterials can effectively reduce the spread rate of foodborne pathogens during production and transportation. This study uses DNA hydrogel combined with a portable blood glucose meter for real-time detection, establishing a linear relationship between the target and the blood glucose meter signal, thus meeting the need for rapid, on-site detection. Compared with existing methods, this method has the following advantages: (1) It is highly modifiable and designable, requiring only changes to the nucleic acid aptamer sequence to detect other target substances; (2) It is easy to operate, requiring less skill from operators, and can detect pathogens without the need for large instruments; (3) The detection time is relatively fast, with a portable blood glucose meter providing readings in 20 seconds, meeting the requirements for rapid on-site detection. This sensor can be observed with the naked eye or used with a home portable blood glucose meter to obtain quantitative results, providing a reference method for future food safety risk assessment, environmental monitoring, and clinical diagnosis.

[0068] The above description of the embodiments is intended to enable those skilled in the art to understand and use them. The implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the technical essence and principle of the present invention are included within the protection scope of the present invention.

Claims

1. A DNA hydrogel biosensor composition based on a portable blood glucose meter for detecting pathogenic bacteria, characterized in that, The device includes magnetic beads loaded with pathogenic aptamers and primer probes, circular sequence 1, circular sequence 2, glucose or sucrase and sucrose, wherein the primer probes are hybridized and linked to the pathogenic aptamers, and the pathogenic aptamers are hybridized and loaded onto the magnetic beads.

2. The DNA hydrogel biosensing composition according to claim 1, characterized in that, The circulating sequence 1 is shown in SEQ ID NO:1, and the circulating sequence 2 is shown in SEQ ID NO:

2.

3. The DNA hydrogel biosensing composition according to claim 1 or 2, characterized in that, The pathogenic bacterium is Escherichia coli.

4. The DNA hydrogel biosensing composition according to any one of claims 3, characterized in that, The pathogenic bacterium is Escherichia coli O157:H7, the aptamer of Escherichia coli O157:H7 is shown in SEQ ID NO:3, and the primer probe is shown in SEQ ID NO:

4.

5. The use of the DNA hydrogel biosensing composition based on a portable blood glucose meter for the detection of pathogenic bacteria as described in any one of claims 1-4 in the preparation of biosensing reagents, kits, or biosensors for the qualitative and / or quantitative detection of pathogenic bacteria.

6. A DNA hydrogel biosensing reagent, kit, or biosensor based on a portable blood glucose meter for the detection of pathogenic bacteria, characterized in that, A DNA hydrogel biosensing composition based on a portable blood glucose meter for detecting pathogenic bacteria, as described in any one of claims 1-4.

7. The application of the DNA hydrogel biosensing composition based on a portable blood glucose meter for the detection of pathogenic bacteria according to any one of claims 1-4 in the qualitative and / or quantitative detection of pathogenic bacteria.

8. A method for detecting pathogenic bacteria, characterized in that the steps include... include: Magnetic beads loaded with pathogenic bacterial aptamers and primer probes are mixed with the analyte and incubated. The supernatant is taken, and circular sequence 1 and circular sequence 2 are added. The pre-circulation reaction and RCA amplification reaction are carried out in sequence. After adjusting the pH to acidic and adding glucose for incubation, the supernatant is taken and used on blood glucose test strips or blood glucose meters for qualitative or quantitative detection. or Magnetic beads loaded with pathogenic bacterial aptamers and primer probes are mixed with the analyte and incubated. The supernatant is taken, and circular sequence 1 and circular sequence 2 are added. Pre-circulation reaction and RCA amplification reaction are performed sequentially. The pH is adjusted to acidic and sucrase is added for incubation. Sucrose is then added and the reaction is carried out at room temperature. The supernatant is taken and used for qualitative or quantitative detection on blood glucose test strips or blood glucose meters.

9. The method for detecting pathogenic bacteria according to claim 8, characterized in that, The pH value is 3-7.

10. A method for detecting pathogenic bacteria according to claim 8 or 9, characterized in that, In quantitative detection, a standard curve is plotted using the concentration of pathogenic bacteria and the blood glucose meter reading as variables, and quantitative determination is performed.

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