Porphyromonas gingivalis detection method based on portable glucometer

Abstract

The invention relates to the field of pathogen detection, in particular to a porphyromonas gingivalis detection method based on a portable glucometer. The method provided by the invention comprises the following steps: S101, adding a sample into a first reaction system and incubating to obtain a first mixture; s102, a modified matrix electrode is in contact with the first mixture and then washed; s103, inserting the washed modified substrate electrode into a blood glucose meter; s104, obtaining a glucometer reading, and further obtaining the concentration of the porphyromonas gingivalis in the sample; wherein the modified matrix electrode is modified by an Au (at) V4C3MXene material and is combined with an antibody for specifically recognizing porphyromonas gingivalis and a sealing agent. The detection method provided by the invention is sensitive, rapid, stable, accurate and simple, and accurate detection of the single-digit porphyromonas gingivalis in an actual sample can be realized in a relatively short time (such as 20 minutes).

Description

Technical Field

[0001] The present invention relates to the field of pathogen detection, and in particular to a method for detecting Porphyromonas gingivalis based on a portable blood glucose meter. Background Art

[0002] Periodontal disease is one of the most common oral diseases caused by subgingival plaque. Periodontitis is known as the "number one killer" of tooth loss in adults and is closely related to the occurrence and development of systemic diseases such as diabetes, autoimmune diseases, and cancer. The clinical diagnosis of periodontal disease mainly relies on clinical examinations by dentists, which is subjective and lacks quantitative standards. Periodontal pathogens stimulate the body's immune response to cause overexpression of local inflammatory mediators, which is considered to be the fundamental mechanism for the occurrence of clinical symptoms of periodontal disease. Porphyromonas gingivalis (Pg), Treponema denticola (Td), and Bacteroides forsythiae (Bf) are considered to be closely related to periodontal disease. Therefore, quantitative detection of periodontal pathogens is of great significance for early and timely intervention, rational treatment, and improved prognosis of periodontal disease.

[0003] Currently, many analytical methods have been used for the quantitative detection of periodontal pathogens, such as (I) bacterial culture-based detection, (II) DNA detection using polymerase chain reaction (PCR) and loop-mediated isothermal amplification, and (III) immunology. However, these methods usually take a long time to process, lack sufficient sensitivity and specificity, or require complex primer design and expensive equipment and reagents. However, most of the existing quantitative detection methods for periodontal pathogens are not suitable for rapid and accurate detection in complex or special occasions (e.g., bedside, chairside, home), and have narrow linear ranges and low detection limits (e.g., >10 3 CFU / mL) and other issues. Summary of the invention

[0004] In a first aspect, the present invention provides a method for detecting Porphyromonas gingivalis for non-diagnostic purposes, characterized in that the method comprises:

[0005] S101: adding a sample to a first reaction system and incubating the mixture to obtain a first mixture, wherein the first reaction system includes a first nucleic acid molecule, a second nucleic acid molecule and an aptamer molecule; the aptamer molecule is used to capture the Porphyromonas gingivalis; after the aptamer molecule is combined with the Porphyromonas gingivalis, the first nucleic acid molecule and the second nucleic acid molecule are hybridized on the surface of the Porphyromonas gingivalis through a hybridization chain reaction to form a bionic nano-wall;

[0006] S102: contacting the modified substrate electrode with the first mixture and then washing;

[0007] S103 inserting the washed modified substrate electrode into the blood glucose meter;

[0008] S104: obtaining a blood glucose meter reading, and then obtaining the concentration of Porphyromonas gingivalis in the sample;

[0009] The modified substrate electrode is modified with Au@V4C3 MXene material and combined with antibodies and blocking agents that specifically recognize Porphyromonas gingivalis.

[0010] In some embodiments, the preparation method of the Au@V4C3 MXene material is as follows:

[0011] a) dispersing V4C3 MXene powder in an organic alkaline compound, stirring and centrifuging to obtain a first precipitate;

[0012] In some embodiments, the V4C3 MXene powder is a multilayer V4C3 MXene.

[0013] In some embodiments, in the step a), the stirring time comprises 24 hours.

[0014] In some embodiments, in the step a), the centrifugation method includes centrifugation at 8500 rpm for 5 min.

[0015] In some embodiments, the organic basic compound includes tetrapropylammonium hydroxide.

[0016] In some embodiments, the V4C3 MXene powder can be obtained by etching V4AlC3 powder, which is not limited in the present invention.

[0017] b) adding a first solvent to the first precipitate and centrifuging to obtain a single-layer V4C3 MXene suspension; It should be noted that the present invention adopts an organic alkaline compound to prepare a single-layer V4C3 MXene, thereby avoiding the problem of residual aluminum inside the multi-layer V4C3 MXene and enabling gold nanoparticles to better adhere to the single-layer V4C3 MXene, thereby overcoming the problem that gold nanoparticles can only adhere to the upper and lower surfaces of the multi-layer V4C3 MXene.

[0018] In some embodiments, the first solvent includes one or more of ddH2O, ultrapure water, deionized water, water for injection, and pure water.

[0019] In some embodiments, in the step b), the centrifugation method includes centrifugation at 3500 rpm for 30 min.

[0020] c) mixing the KAuCl4 solution and the single-layer V4C3 MXene suspension and stirring until the solution turns red, and then adding a reducing agent to obtain a second precipitate, i.e., the electrode modification material;

[0021] In some embodiments, the concentration of the monolayer V4C3 MXene suspension comprises 1-10 mg / mL. In some embodiments, the concentration of the monolayer V4C3 MXene suspension comprises 5 mg / mL.

[0022] In some embodiments, the concentration ratio of the KAuCl4 solution to the single-layer V4C3 MXene suspension is 0.30-0.45: 1. It has been verified that by controlling the above concentration ratio, the conductivity of the electrode modification material prepared by the present invention is significantly improved.

[0023] In some embodiments, the reducing agent is used to further reduce KAuCl4 into gold nanoparticles. In some embodiments, the reducing agent comprises sodium borohydride. In some embodiments, the concentration of the reducing agent comprises 1 M. In some embodiments, the volume ratio of the reducing agent to the KAuCl4 solution comprises 1:1.

[0024] In some embodiments, the reducing agent is added dropwise.

[0025] In some embodiments, a concentration ratio of the KAuCl4 solution to the single-layer V4C3 MXene suspension comprises 0.38:1.

[0026] In some embodiments, the method further comprises washing the second precipitate and subjecting it to redistribution in the first solvent.

[0027] In the present invention, the Au@V4C3 MXene material can not only improve the electron transfer efficiency of the electrode, but also increase the active specific surface area of ​​the electrode, showing a strong advantage in improving the sensitivity of the electrochemical sensor. Compared with V4C3MXene, the conductivity of the Au@V4C3 MXene material provided by the present invention is increased by 4-6 times. In addition, the Au@V4C3 MXene material provided by the present invention has excellent stability, and the gold nanoparticles deposited in situ on the V4C3 MXene will not further dissolve or fall off, and is suitable for electrode modification. In addition, the Au@V4C3 MXene material also increases the bindability of the substrate electrode, so that macromolecular substances such as antibodies can be bound to the substrate electrode through the gold nanoparticles of the electrode modification material.

[0028] In some embodiments, the substrate electrode comprises a glassy carbon electrode or a bare gold electrode.

[0029] In some embodiments, the substrate electrode comprises a blood glucose chip.

[0030] In some embodiments, the blocking agent includes bovine serum albumin. The addition of the blocking agent in the present invention allows the inactive sites of the Au@V4C3 MXene material to be blocked.

[0031] In some embodiments, the method for preparing the modified substrate electrode comprises: mixing the Au@V4C3 MXene solution with the substrate electrode and then drying. In some embodiments, the concentration of the Au@V4C3 MXene solution comprises 1–10 mg / mL. In some embodiments, the concentration of the Au@V4C3 MXene solution comprises 5 mg / mL. In some embodiments, in order to enable the Au@V4C3 MXene material to better adhere to the substrate electrode, the Au@V4C3MXene solution may further comprise chitosan. In some embodiments, the concentration of the chitosan comprises 1–10 mg / mL.

[0032] In some embodiments, the antibody that specifically recognizes Porphyromonas gingivalis and the blocking agent are combined with the modified substrate electrode by incubating with the modified substrate electrode one after another.

[0033] In some embodiments, the concentration of the antibody that specifically recognizes Porphyromonas gingivalis includes 10-100 μg / mL. In some embodiments, the concentration of the antibody that specifically recognizes Porphyromonas gingivalis includes 50 μg / mL. In some embodiments, the volume ratio of the antibody that specifically recognizes Porphyromonas gingivalis to the modified substrate electrode includes 5:1.

[0034] In some embodiments, the sample comprises a saliva sample and / or a gingival crevicular fluid sample.

[0035] In some embodiments, the incubation time in step S101 is 20–60 min.

[0036] In some embodiments, the second nucleic acid molecule is conjugated to alginate.

[0037] In some embodiments, the nucleotide sequence of the first nucleic acid molecule is shown in SEQ ID NO: 1, and the nucleotide sequence of the second nucleic acid molecule is shown in SEQ ID NO: 2. In some embodiments, the first nucleic acid molecule and the second nucleic acid molecule are hairpin structures. It should be understood that other suitable combinations of the first nucleic acid molecule and the second nucleic acid molecule can also be selected, as long as they can perform a hybridization chain reaction on the surface of Porphyromonas gingivalis.

[0038] In some embodiments, the nucleotide sequence of the aptamer molecule is shown in SEQ ID NO: 3. It should be understood that other suitable aptamer molecules can also be selected as long as they can capture Porphyromonas gingivalis. The setting of the antibody and aptamer molecules that specifically recognize Porphyromonas gingivalis in the present invention realizes dual recognition of Porphyromonas gingivalis and improves the accuracy of the method of the present invention.

[0039] In some embodiments, the blood glucose meter comprises a portable blood glucose meter.

[0040] In some embodiments, the volume of the sample comprises 10-200 μL.

[0041] In some embodiments, the concentration ratio of the first nucleic acid molecule, the second nucleic acid molecule and the aptamer molecule in the first mixture includes 1:1:1 to 3:3:1. The above concentration ratio ensures that sufficient aptamer molecules can capture potential Porphyromonas gingivalis in the sample, and ensures that the first nucleic acid molecule and the second nucleic acid molecule can undergo a hybrid chain reaction on the surface of potential Porphyromonas gingivalis and form a bionic nanowall, thereby ensuring that the method of the present invention can detect trace amounts of Porphyromonas gingivalis.

[0042] In some embodiments, the concentrations of the first nucleic acid molecule, the second nucleic acid molecule, and the aptamer molecule are 30 μM, 30 μM, and 10 μM, respectively.

[0043] In some embodiments, the step S104 further includes drawing a standard curve of the blood glucose meter reading and the concentration of Porphyromonas gingivalis and obtaining a linear equation of the blood glucose meter reading and the concentration of Porphyromonas gingivalis, and obtaining the concentration of Porphyromonas gingivalis in the sample based on the linear equation.

[0044] In the present invention, the first reaction system designed by the present invention can form a negatively charged DNA biomimetic nanowall on the surface of Porphyromonas gingivalis, thereby improving the steric hindrance effect of monomeric bacteria and the efficiency of hindering electron transfer, thereby improving the detection rate of trace bacteria by the method of the present invention. 0.38 The substrate electrode modified with @V4C3MXene) has excellent conductivity, binding and stability, and can significantly improve the electrochemical performance of the substrate electrode. Even when combined with antibodies and blocking agents that specifically recognize Porphyromonas gingivalis, it still shows excellent electrochemical response capabilities. The setting of antibodies and aptamer molecules that specifically recognize Porphyromonas gingivalis in the present invention realizes dual recognition of Porphyromonas gingivalis, further ensuring the recognition and detection of Porphyromonas gingivalis by the method of the present invention.

[0045] In summary, under the synergistic effect of electrode modification materials and DNA bionic nanowalls, the detection method provided by the present invention is sensitive, rapid, stable, accurate, and simple. Only a blood glucose meter is required to accurately detect single-digit Porphyromonas gingivalis in actual samples (especially in clinical saliva samples) in a relatively short period of time (e.g., 20 minutes). The detection method provided by the present invention can meet the detection needs of various special environments (such as chairside testing or self-monitoring at home), and has demonstrated extremely high applicability in actual sample testing, overcoming the shortcomings of clinical detection methods such as difficulty in quantification, time-consuming, and complex operation. The detection method provided by the present invention can effectively detect 10~5×10 6 The detection limit of Porphyromonas gingivalis in the CFU / mL range is 9 CFU / mL, and it shows great advantages in terms of time consumption and portability, making it suitable for accurate detection of periodontal pathogens in chairside or home environments.

[0046] In a second aspect, the present invention provides a kit for detecting Porphyromonas gingivalis, characterized in that the kit comprises:

[0047] a) a modified blood glucose chip, wherein the modified blood glucose chip is modified with Au@V4C3 MXene material and combined with an antibody and a blocking agent that specifically recognizes Porphyromonas gingivalis;

[0048] b) a collecting tube, wherein the collecting tube is pre-installed with a first reaction system, wherein the first reaction system comprises a first nucleic acid molecule, a second nucleic acid molecule and an aptamer molecule, wherein the aptamer molecule is used to capture Porphyromonas gingivalis, and after the aptamer molecule binds to the Porphyromonas gingivalis, the first nucleic acid molecule and the second nucleic acid molecule hybridize on the surface of the Porphyromonas gingivalis through a hybridization chain reaction and form a bionic nanowall.

[0049] In some embodiments, the nucleotide sequence of the first nucleic acid molecule is shown in SEQ ID NO: 1, and the nucleotide sequence of the second nucleic acid molecule is shown in SEQ ID NO: 2. In some embodiments, the second nucleic acid molecule is coupled to alginate.

[0050] In some embodiments, the nucleotide sequence of the aptamer molecule is shown in SEQ ID NO:3.

[0051] In some embodiments, the kit further comprises a K4(FeCN)6 solution.

[0052] In some embodiments, the kit further comprises a portable blood glucose meter.

[0053] In some embodiments, the blocking agent comprises bovine serum albumin.

[0054] In some embodiments, the kit is for saliva and / or gingival crevicular fluid testing.

[0055] In some embodiments, the Au@V4C3 MXene material is Au 0.38 @V4C3 MXene material. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings without paying creative labor.

[0057] Figure 1 For Au 0.38 @V4C3 / MXene synthesis and characterization results;

[0058] Figure 2 For Au 0.38 @V4C3 / MXene performance verification results diagram;

[0059] Figure 3 This is a result diagram of the characterization of the DNA bionic nanowall;

[0060] Figure 4 This is the result diagram of electrochemical sensor performance analysis;

[0061] Figure 5 This is a result diagram of the performance analysis of the handheld electrochemical sensor;

[0062] Fig. 6A The results of the effect of different KAuCl4 and V4C3 MXene concentration ratios on the conductivity of bare gold electrode are shown in the figure;

[0063] Figure 6B The result diagram of ultraviolet absorption spectrum;

[0064] Figure 6C Cyclic voltammetry response spectra of gold electrodes modified with different V4C3 michelenes;

[0065] Fig.6D The cyclic voltammetry response spectrum of Ag@V4C3 michelene-modified gold electrode;

[0066] Fig. 6E Cyclic voltammetry response spectra of gold nanoparticles and Au@V4C3 michelene-modified gold electrodes;

[0067] Figure 7 The DPV results of Pg before and after modification with DNA bionic nanowall;

[0068] Figure 8 The results of the effects of temperature, pH value and incubation time on the reaction system are shown in the figure;

[0069] Fig. 9 This is the result of Hebs' actual saliva sample test;

[0070] Fig. 10A The results of the specific analysis of bacterial detection in artificial saliva samples are shown in FIG.

[0071] Fig. 10B It is the result diagram of the linear relationship between the current signal and the Pg-IgG concentration tested by the enzyme-linked immunosorbent assay kit;

[0072] Fig.11 Schematic diagram of the Hebs platform. DETAILED DESCRIPTION

[0073] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0074] Herein, suffixes such as "module", "component" or "unit" used to represent elements are only used to facilitate the description of the present invention, and have no specific meanings by themselves. Therefore, "module", "component" or "unit" can be used mixedly.

[0075] In this document, the directions or positional relationships indicated by the terms "upper", "lower", "inner", "outer", "front", "rear", "one end", "the other end", etc. are based on the directions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific direction, be constructed and operate in a specific direction, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0076] In this document, unless otherwise clearly specified and limited, the terms "installed", "provided with", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, a direct connection, or an indirect connection through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in this invention can be understood according to specific circumstances.

[0077] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0078] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0079] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0080] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0081] In this specification, certain embodiments may be disclosed in a format of being in a certain range. It should be understood that such description of "being in a certain range" is merely for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0082] Embodiment 1

[0083] 1.1 Materials and reagents

[0084] Potassium chloroaurate (KAuCl4) was purchased from Sigma-Aldrich (St. Louis, MO, USA). BSA, ascorbic acid (AA), and chitosan (CS) were purchased from Sangon Biotech Co. (Shanghai, China). MAbs were ordered from Sigma-Aldrich (St. Louis, MO, USA). Monolayer graphene was purchased from Dachang Co., Ltd., Hong Kong, China (Shenzhen, China). Sodium alginate (medium viscosity, 80–120 kDa) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). O-(2-aminoethyl)-O′-(2-aminoethyl) pentaethylene glycol (NH2PEG6-N3) was purchased from Sigma-Aldrich (St. Louis, MO, USA). N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were obtained from Sigma-Aldrich (St. Louis, MO, USA). DNAMarker and agarose were purchased from Takara (Dalian, China). All nucleic acid chains were synthesized by Shanghai Sangong Biotechnology Co., Ltd. (Shanghai, China). Water was purified using a Millipore-Q water purification system (resistivity 18.2 MΩ). Artificial saliva was purchased from Phygene Life Sciences Co., Ltd (Fujian, China). Saliva samples were obtained from the Affiliated Stomatological Hospital of Chongqing Medical University.

[0085] 1.2 Instruments

[0086] Electrochemical experiments were performed using an electrochemical workstation (CHI760D, Shanghai Chenhua Instrument Co., Ltd.). The three-electrode system consisted of a gold electrode, a platinum wire electrode, and a saturated calomel electrode. DPV measurements were performed in the potential range of -0.2 to 0.6 V with an amplitude of 50 mV. CV measurements were performed at 100 mV.s -1 The DPV and CV measurements were performed in phosphate buffered saline (10 mM PBS, pH = 7.4) containing 0.1 M KCl and 5.0 mM [Fe(CN)6] 3– / 4– .

[0087] 1.3 Statistical analysis

[0088] All data are expressed as mean ± standard deviation (SD). The variance analysis was performed and the t test was used for comparison among the groups. Statistical significance was set at *p<0.05.

[0089] Embodiment 2

[0090] 2.1 Synthesis of V4C3 MXene

[0091] V4C3 MXene (vanadium carbide MXene) was prepared by selectively etching the atomic layer of V4AlC3 Max phase powder. 0.3 g of V4AlC3 (vanadium aluminum carbide MXene) powder was slowly added to 15 mL of hydrofluoric acid (HF) solution and stirred at 55 °C for 96 h. After that, the sample was centrifuged at 8000 rpm for 5 min and washed with deionized water until the pH of the supernatant reached ~6.0. After drying in a vacuum oven at 60 °C for 12 h, V4C3 MXene powder was obtained. The obtained V4C3 MXene powder was dispersed in 15 mL of tetrapropylammonium hydroxide (TPAOH) and stirred at room temperature (RT) for 1 day. The mixture was centrifuged at 8500 rpm for 5 min and washed three times with deionized water to remove residual TPAOH. After removing the supernatant, 5 mL of deionized water was added to the precipitate. Then, the mixture was shaken for 2 minutes and centrifuged at 3500 rpm for 30 minutes to obtain a stable single-layer V4C3 MXene suspension.

[0092] 2.2 Synthesis of Au@V4C3 / MXene (gold@vanadium carbide MXene)

[0093] The KAuCl4 aqueous solution and the prepared V4C3 MXene suspension (5 mg / mL, 5 mL) were mixed in a 20 mL beaker at a concentration ratio of 0.08: 1, 0.15: 1, 0.23: 1, 0.30: 1, 0.38: 1, and 0.45: 1, respectively. The mixture was stirred at RT for 30 min, and the solution color turned brick red. Then, sodium borohydride (1 M, 250 μL) was added dropwise to the solution. After stirring for 30 minutes and leaving at room temperature overnight, Au@V4C3 / MXene was successfully formed. Subsequently, the product was collected by centrifugation (3000 rpm, 5 min) and washed three times with water and ethanol. The final precipitate was redistributed in deionized water for subsequent use.

[0094] 2.3 Preparation of gold electrodes

[0095] Polishing and cleaning of electrodes: First, the gold electrode (3 mm in diameter) was carefully polished to a mirror surface with 0.3 mm and 0.05 mm alumina powders, and then continuously ultrasonicated in ultrapure water, ethanol and ultrapure water for 10 min, activated with freshly prepared piranha solution (98% H2SO4:30% H2O2, volume ratio of 3:1) for 5 min, rinsed thoroughly with ultrapure water and dried.

[0096] Modification of the working electrode: 10 mL of Au@V4C3 / MXene (5 mg / mL, chitosan as solvent) was added to the prepared gold electrode (blood glucose chip, 2 μL) and allowed to dry naturally at room temperature until Au@V4C3 / MXene was fully adhered.

[0097] In this example, the electrochemical properties of Au@V4C3 / MXene were preliminarily studied using cyclic voltammetry. Fig. 6A The results show that when the concentration ratio (mg / mL) of KAuCl4 aqueous solution and V4C3 MXene suspension is 0.30:1-0.45:1, the electrochemical performance of Au@V4C3MXene is significantly improved. After comprehensively considering the electrochemical performance and preparation cost, the present invention selects the concentration ratio (mg / mL) of KAuCl4 aqueous solution and V4C3 MXene suspension to be 0.38:1, and the prepared Au@V4C3 MXene material (i.e., Au 0.38 @V4C3 / MXene) for subsequent testing.

[0098] 2.4Au 0.38 Synthesis and characterization of @V4C3 / MXene

[0099] Figure 1 A shows Au 0.38 Schematic diagram of the synthesis process of @V4C3 / MXene. Next, this example 0.38 @V4C3 / MXene was morphologically analyzed. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to analyze the morphology of multilayer V4AlC3MXene, multilayer V4C3 MXene, single-layer V4C3 MXene and Au 0.38 The morphology of @V4C3 / MXene was characterized. Figure 1 B shows the SEM image of V4AlC3 MXene, which is a multilayer, flaky, micrometer-scale structure with a rough surface. In contrast, the surface of multilayer V4C3 MXene is smoother, which may be due to the etching of aluminum. Figure 1 C shows the TEM image of V4C3 MXene, which is also micrometer-sized but has a single-layer structure. However, under TEM, Au 0.38 A large number of gold nanoparticles can be seen on the surface of @V4C3 / MXene, indicating that gold nanoparticles were successfully deposited in situ on V4C3 MXene ( Figure 1 D) High-angle annular dark field scanning transmission and element mapping results show that Au 0.38 @V4C3 / MXene is composed of C, V and Au ( Figure 1 E).

[0100] Figure 6B Display, Au 0.38 In the absorption spectrum of @V4C3 / MXene, there is an absorption peak at 619nm, which belongs to Au. Compared with the absorption peak of Au nanoparticles at 520nm, Au 0.38The absorption peak of @V4C3 / MXene has an obvious red shift of the plasma resonance peak, which may be the result of the in-situ deposition of Au nanoparticles on the surface of V4C3 MXene. 0.38 The preparation of @V4C3 / MXene was successful.

[0101] The Au 0.38 @V4C3 / MXene element valence and surface properties ( Figure 1 F. Figure 1 G. Figure 1 H) In Au 0.38 In the V2P XPS spectrum of @V4C3 / MXene, two double peaks were formed by spectral deconvolution. 3 / 2 ) and 520.73eV(V2P 1 / 2 ) can be classified as V 2+ , at 516.49(V2p 3 / 2 ) and 523.36eV(V2P 1 / 2 The other two shoulders at ) can be classified as V 4+ The XPS spectrum of C1 consists of four peaks located at 284.8, 286.29, 288.54 and 282.51 eV, corresponding to CC, CO, C=O and CV bonds, respectively. The XPS spectrum of Au 4f shows that Au4f 7 / 2 and Au 4f 5 / 2 There are two strong peaks at 83.8 and 87.46 eV respectively.

[0102] To further characterize the Au 0.38 The crystal plane and lattice spacing of @V4C3 / MXene were also analyzed by XRD patterns, such as Figure 1 I. Compared with V4AlC3, V4C3 MXene has a new (002) crystal plane on the left side of the spectrum, proving that Al is etched out in V4AlC3 MXene. However, the reflection of the (002) crystal plane is not reflected in Au. 0.38 @V4C3 / MXene, which can be attributed to the shielding effect of Au nanoparticles grown on the MXene surface. 0.38 In the XRD spectrum of @V4C3 / MXene, the characteristic crystal planes of Au crystals are also at 2θ values ​​of 38.14°, 44.4° and 64.4°, corresponding to (111), (200) and (220) crystal planes, respectively. In addition, high-resolution transmission electron microscopy (HRTEM) further verified the in-situ deposition of gold nanoparticles on the surface of V4C3 MXene. Figure 1J clearly depicts the Au nanoparticles attached to the V4C3MXene surface, with a lattice spacing of 0.24 nm in the selected area, corresponding to the Au(111) crystal plane. Meanwhile, the selected area electron diffraction (SAED) rings characterize the crystal plane and lattice spacing of the gold nanoparticles in more detail ( Figure 1 The d=K values ​​are 111 (d=0.243 nm), 200 (d=0.208 nm), 222 (d=0.121 nm) and 420 (d=0.092 nm) respectively.

[0103] Embodiment 3

[0104] Au 0.38 @V4C3 / MXene performance verification

[0105] First, this example compares Au 0.38 @Conductivity and resistivity of V4C3 / MXene, V4C3 MXene, and single-layer graphene (a well-known excellent electrode modification material) ( Figure 2 A and Figure 2 B) The results show that under the same pressure, Au 0.38 The conductivity of @V4C3 / MXene is 4.26 times higher than that of V4C3 MXene, and the resistivity is 4.26 times lower than that of V4C3 MXene. 0.38 The electrical conductivity of @V4C3 / MXene is very close to that of single-layer graphene. The above results show that the Au prepared by the present invention 0.38 @V4C3 / MXene is an excellent conductive material.

[0106] In addition to cyclic voltammetry, electrochemical impedance spectroscopy (EIS) was also used to study the Au 0.38 @V4C3 / MXene electrochemical properties (cyclic voltammetry and electrochemical impedance spectroscopy are considered to be effective methods for studying the interface characteristics and electron transfer impedance of the electrode surface). This example further compares the electrochemical properties of V4C3 MXene, Au 0.38 The electron transfer efficiency of bare gold electrodes modified with @V4C3 / MXene and single-layer graphene ( Figure 2 C) Figure 2 D. Figure 2 E and Figure 2 As shown in F, V4C3 MXene increases the conductivity of the bare gold electrode by approximately 1.8 times; however, Au 0.38 The electrode modification effect of @V4C3 / MXene is more obvious, which can increase the conductivity of bare gold electrode by about 6.15 times, which is very close to that of single-layer graphene and significantly better than other V4C3 / MXene materials (such as Cu@V4C3 / MXene and Pt@V4C3 / MXene, such as Figure 6CAs shown in (the preparation methods of Cu@V4C3 / MXene and Pt@V4C3 / MXene are similar to those of Au@V4C3 / MXene, Figure 6C The tested V4C3 / MXene material is prepared by using potassium chloroaurate or copper sulfate or potassium chloroplatinate and V4C3 / MXene at a concentration ratio of 0.38:1). In addition, after multiple tests and practical application verification, the Au@V4C3 / MXene (for example, Au 0.38 @V4C3 / MXene) has strong stability and does not dissolve or fall off gold nanoparticles. However, in the cyclic voltammetry test of Ag@V4C3 / MXene prepared by a similar method, silver will be stripped to silver (I) ions, which leads to the dissolution and fall off of silver nanoparticles, which has a serious impact on the stability of the current signal ( Fig.6D ). The above experimental results prove that the Au@V4C3 / MXene prepared by the present invention is an excellent electrode modification material.

[0107] This example also compares the conductivity of an electrode made by directly depositing gold nanoparticles on a bare gold electrode (i.e., "bare gold electrode + gold nanoparticles") with the conductivity of the Au0.38@V4C3 / MXene of the present invention. Fig. 6E As shown in Figure 2, although the direct deposition of gold nanoparticles can improve the conductivity of the electrode to a certain extent, it is still not as good as Au@V4C3 chemicene (Au 0.38 @V4C3 / MXene), there is a big gap.

[0108] Embodiment 4

[0109] Working electrode pre-assembly: First, 10 ml Au 0.38 @V4C3 / MXene (5 mg / mL, chitosan as solvent) was added to the prepared gold electrode (blood glucose chip, 2 μL) and dried naturally at room temperature until Au@V4C3 / MXene was completely adhered. After complete adhesion, anti-Pg mAb (10 μL, 50 μg / mL) was added and incubated at 37 °C for 1 h. After thorough washing with 1× PBS, 10 μL of 1% BSA was dropped onto the electrode surface and incubated for 1 h to seal excess binding sites. After repeated washing with 1× PBS, the prepared gold electrode was stored at 4 °C for further use.

[0110] Characterization of working electrode:

[0111] The Au 0.38 Electrochemical responses of gold electrodes modified with @V4C3 / MXene, mAb, and BSA ( Figure 2 C) Figure 2 As shown in G, compared with the bare gold electrode, Au0.38 The current of bare gold electrode after @V4C3 / MXene modification increased significantly, which can be attributed to the Au 0.38 @V4C3 / MXene has high specific surface area and ultra-high electrical conductivity. Figure 2 As shown in H, Au 0.38 After @V4C3 / MXene modified the bare gold electrode, the impedance value dropped sharply, indicating that Au 0.38 @V4C3 / MXene effectively improves the electron transfer efficiency of bare gold electrodes.

[0112] This example attempts to 0.38 @V4C3 / MXene, mAb and BSA are assembled on the electrode surface. Figure 2 As shown in G, when the monoclonal antibody is immobilized on the electrode surface, it will hinder the electrode from interacting with the redox probe ([Fe(CN)6] 3- / 4- ) caused a significant decrease in the current. With the addition of BSA, the nonspecific sites on the electrode were blocked and the current response was further reduced. EIS was used to confirm the cyclic voltammetry (CV) results and further characterize the self-assembly of the electrode. 0.38 The impedance of bare gold electrode modified with @V4C3 / MXene gradually increases with the addition of non-conductive monoclonal antibody and bovine serum albumin. The CV and EIS results strongly indicate that Au 0.38 @V4C3 / MXene, mAb and BSA were successfully assembled on the electrode surface. 0.38 @V4C3 / MXene modification, even if mAb and BSA are assembled on the electrode surface, resulting in a decrease in current response, 0.38 @V4C3 / MXene modified bare gold electrode still has excellent electrochemical response ability.

[0113] Embodiment 5

[0114] 5.1 Preparation of azide-modified sodium alginate (Alg-N3)

[0115] Alg (50mg) was dissolved in MES buffer (5mL, 50mM, pH=5). NH2-PEG6-N3 (28mg, 0.08mM), NHS (14mg, 0.12mM) and EDC (116mg, 0.60mM) were added to the solution and stirred for 30min at RT. Subsequently, 55mL 6M NaOH was added to the mixture to adjust its pH to 7.5. The mixture was placed at RT and reacted overnight. The product was purified by dialysis for 3 days in water (10000MWCO membrane). In order to further remove residual reagents, the Alg-N3 solution was precipitated in cold acetone, filtered and dried. Alg-N3 was dissolved in DDH2O and filtered with a 0.22μm filter membrane. Finally, Alg-N3 was lyophilized for use.

[0116] 5.2 Preparation of Alg-H2 macromonomer

[0117] Alg-H2 was prepared by copper-free hit reaction. 50 mL of 1% w / v Alg-N3 solution was incubated with 15 mL of 1 mM H2-DBCO at 37 ° C for 4 h. After the reaction, the prepared Alg-H2 was purified using a 100 kDa Amicon ultracentrifugal filter. In order to modify carboxy doxorubicin, Alg-H2 solution (50 mL, 1% w / v) was incubated with 15 mL MES buffer containing 1 mM DOX-COOH, 0.12 mM NHS and 0.60 mM EDC in a dark room at 37 ° C for 2 h.

[0118] 5.3 Agarose gel electrophoresis experiment

[0119] The buffer was 1×TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA), the agarose concentration was 3%, and the fluorescent dye was Goldview. Electrophoresis was performed at a constant voltage of 110 V for 30 min. The gel was observed under UV light and visualized and analyzed using a gel imaging analysis system (Bio-Rad, USA).

[0120] 5.4 Assembly of DNA biomimetic nanowalls on the living Pg surface

[0121] The collected Pg was centrifuged and washed twice, and then suspended in saline. Pg was incubated in 400 μL of aptamer solution (1×SSC buffer) for 30 min, and spontaneously bound to Pg by the aptamer. The aptamer-bound Pg was mixed with H1-FAM (1 μM) and Alg-H2 (1 μM) in saline for 3 h to form negatively charged DNA biomimetic nanowalls. To study the formation of DNA biomimetic nanowalls, Pg was collected and its fluorescence signals (DOX signal from Alg-H2 sodium and FAM signal from H1) were analyzed by flow cytometry.

[0122] Among them, the sequences of aptamer, H1 and H2 are shown below.

[0123] 5.5 Characterization of DNA biomimetic nanowall

[0124] In this embodiment, a DNA biomimetic nanowall rich in negative charge is assembled on the bacterial surface by hybrid chain reaction (HCR). The assembly steps are as follows: Figure 3 A. First, alginate (Alg) and hairpin DNA2 (H2) were coupled by copper-free click chemistry to synthesize Alg-H2 macromer ( Figure 3 B).

[0125] Since both Alg and H2 are negatively charged, this example further investigated whether the macromolecular monomer formed by coupling Alg and H2 would hinder the H1 / / H2 hybridization during the HCR process due to steric hindrance, that is, the continuous hybridization efficiency of H1 and H2 after connecting Alg was evaluated. Figure 3 As shown in Figure C, the agarose gel electrophoresis image shows that Alg-H2 can combine to form a hybrid macromer. At the same time, the gel image also shows that H1 and Alg-H2 hybridize continuously with the assistance of the aptamer, indicating that the steric hindrance and charge interaction caused by the macromer have no significant effect on HCR.

[0126] Flow cytometry was used to detect the fluorescence signals of H1-FAM and H2-Alg-DOX and analyze the formation of DNA bionic nanowall on the bacterial surface. Figure 3 D. Figure 3 E and Figure 3 F shows that the FAM and DOX fluorescence signals of the assembled bacteria increased dramatically compared with those of the control (unassembled bacteria). In addition, the changes in bacterial size and surface potential were detected by a dynamic light scattering (DLS) analyzer to further study the assembly efficiency of the DNA bionic nanowall on the bacterial surface. Figure 3 G shows that the bacterial radius increased by about 245 nm. Assuming that the length of a pair of H1 / / H2 is about 16 nm, the increase in size indicates that the hybridization between H1 and Alg-H2 leads to the effective formation of supramolecular DNA hybrid chains. At the same time, due to the increase in negative charge density on the bacterial surface, its surface potential also decreased significantly ( Figure 3 H) Use DPV to detect the efficiency of bacteria blocking electron transfer before and after DNA biomimetic nanowall wrapping. Figure 7 As shown, the current signal of bacteria wrapped in the DNA bionic nanowall is significantly reduced, which helps to improve the detection rate of trace bacteria. The above results show that the DNA bionic nanowall designed by the present invention increases the negative charge density on the bacterial surface, improves the steric hindrance effect of monomeric bacteria and the ability to hinder electron transfer, and further improves the detection rate of bacteria.

[0127] This example further compares the effects of temperature and pH value of the reaction system on HCR efficiency to improve the binding efficiency of bacteria to mAb and aptamers. Figure 8 A and Figure 8 As shown in B, the DPV signal was the lowest at 37.5°C and pH = 7.5, indicating that under this condition, the efficiency of mAb capturing bacteria and assembling DNA bionic nanowalls on the bacterial surface was the highest. In addition, the effect of incubation time on the reaction system was also investigated. Figure 8 As shown in Figure C, as the incubation time increases, the DPV signal gradually decreases, the decrease is most obvious within 20 minutes, and reaches a plateau at 60 minutes. In order to balance the time consumption and performance of Hebs, the present invention selects 20 minutes as the incubation time for subsequent detection, so as to be suitable for scenarios such as chairside detection and home monitoring.

[0128] Embodiment 6

[0129] Hebs assembly and performance analysis

[0130] Firstly, the detection performance of the developed electrochemical sensor was verified on an electrochemical workstation ( Figure 4 A). Figure 4 As shown in Figure B, the current signal decreases with the increase of Pg concentration, which confirms the high feasibility of the electrochemical sensor. Figure 4 C shows that in 5~5×10 6 The linear relationship between the current signal and the logarithm of the Pg concentration was within the range of 1:1. The linear regression equation was Y=40.365lgc-15.896, and the detection limit (LOD) was 3 CFU / mL (LOD=3α / S, α was 11 times the standard deviation of the blank signal, and S was the slope of the linear equation).

[0131] Specificity is an important indicator of the sensor. In order to evaluate the specificity of the electrochemical sensor, four periodontal nonspecific pathogens: Fusobacterium nucleatum (Fn), Streptococcus mutans (Sm), Escherichia coli (Ec) and Staphylococcus aureus (Sa) were selected as interfering substances and detected under the same experimental conditions. Figure 4 D shows that Pg and mixed bacterial suspension containing Pg triggered obvious DPV reactions, while the DPV reactions of Fn, Sm, Ec and Sa were not significantly different from those of blank buffer. The results show that the electrochemical sensor has good specificity and anti-interference performance.

[0132] In order to adapt to many special testing environments (such as chairside and home), electrochemical sensors are organically combined with personal blood glucose meters (PGMs), mainly based on the transformation of blood glucose chips ( Figure 5 A) Replace the glucose oxidase and ferrocene immobilized on the surface of the blood glucose chip with Au 0.38@V4C3 / MXene, mAb and BSA are prefixed. Figure 5 As shown in B, the current response of PGM also changed with the gradual modification, thus confirming the successful conversion of the blood glucose chip.

[0133] In order to verify the detection performance of Hebs, a series of concentrations of Pg standard suspension and the converted blood glucose chip were added to the prepared DNA bionic nanowall reaction system. After incubation, the blood glucose chip was washed and detected with PGM. The results showed that the PGM signal decreased with the increase of Pg concentration, indicating that Hebs is highly feasible for detecting bacteria. Figure 5 C shows that the logarithm of the current signal and Pg concentration is between 10 and 5×10 6 The linear relationship is within the range. The linear regression equation is Y=1.136LgC-0.069, and the detection limit is 9CFU / mL. The Hebs provided by the present invention have significant advantages in sensitivity and detection range, which is mainly attributed to Au 0.38 @V4C3 / MXene (improves the conductivity of the electrode) and DNA bionic nanowall (increases the steric hindrance and negative charge of bacteria).

[0134] In order to explore the specificity of Hebs, four nonspecific pathogenic bacteria, Fn, Sm, Ec and Sa, were selected as interfering bacteria and tested under the same experimental conditions. Figure 5 D shows that only when detecting Pg bacteria and mixed bacterial suspensions containing Pg, a low signal response is shown. This indicates that Hebs has good specificity and anti-interference performance. In addition, the reproducibility of Hebs inter-batch and intra-batch determination was explored by detecting 1000, 10000 and 100000 CFU / mL of Pg ( Figure 5 E). The results showed that the coefficient of variation was between 0.84% ​​and 2.96%, and the Hebs-based Pg detection method had high sensitivity, specificity and acceptable repeatability.

[0135] Hebs has comparable specificity, sensitivity, and LOD to the electrochemical workstation. However, Hebs has absolute advantages in operability, portability, and point-of-care testing capabilities, which are very beneficial for bedside testing, chairside testing, or home pathogen monitoring.

[0136] Actual saliva sample testing

[0137] In order to evaluate the detection effect of Hebs on saliva samples, a series of spiking experiments were performed ( Fig. 9 A). Add different concentrations of Pg standard to artificial saliva (similar to real human saliva greater than 99%), the Pg recovery rate is 95.4-103.5, CV% is 3.08-4.25 ( Fig. 9 B. Fig. 9C) These spiking experiments demonstrate that Hebs can be accurately detected in human saliva samples.

[0138] In addition, 100 CFU / mL of Pg, Fn, Sm, Ec, Sa and their mixed bacterial suspensions were added to artificial saliva for Hebs detection to evaluate the anti-interference effect and applicability of Hebs. Fig. 10A It was shown that only the current signal of saliva samples containing Pg was significantly reduced compared with other nonspecific bacteria.

[0139] To further verify the performance of Hebs in actual saliva samples, saliva samples from 10 healthy subjects and 30 patients with periodontitis were tested. Fig. 10B ) were used to detect the Pg antibody levels in the above saliva samples, and the Pg content in these saliva samples was detected by Hebs. Fig. 9 D shows that the Pg antibody level in saliva detected by ELISA kit is basically consistent with the Pg content detected by Hebs, which proves that the Hebs test result is reliable. Through the analysis of Pg levels in 40 saliva samples, it was found that the Pg level in the saliva of healthy people was generally lower than that of periodontitis patients (p = 0.0012, Fig. 9 E). In addition, 9 patients with periodontitis were selected according to the different levels of Pg in saliva, and their teeth were scanned by X-ray computed tomography ( Fig. 9 F). The red dotted line represents the actual alveolar bone height, and the green dotted line represents the enamel-cementum connection of the tooth, which is the ideal height of the alveolar bone in a healthy state. The distance between the red line and the green line represents the amount of alveolar bone loss caused by periodontitis. The larger the distance, the more alveolar bone loss caused by periodontitis, and the more severe the periodontitis. CT results show that the Pg level is highly correlated with the severity of periodontitis. Therefore, the Hebs provided by the present invention has the ability to efficiently detect and distinguish Pg in saliva, and shows broad application prospects in the evaluation of periodontal disease.

[0140] Fig.11 The working principle of the Hebs platform is summarized, which mainly includes: module ① saliva collection tube (pre-installed with DNA bionic nanowall reaction system), module ② modified blood glucose chip, module ③ blood glucose meter, module ④ reaction electrode of blood glucose chip, module ⑤ pathogenic bacteria, module ⑥ DNA bionic nanowall, module ⑦ monoclonal antibody, module ⑧ calf serum albumin, module ⑨ electrode modification material. Compared with the single-component V4C3 MXene, the electrode modification material synthesized by the present invention (such as optimized Au 0.38@V4C3 MXene) has excellent conductivity and stability, which can improve the electrochemical performance of bare gold electrodes by about 6 times and the electron transfer efficiency by about 3 times. In addition, the negatively charged DNA bionic nanowall designed by the present invention improves the steric hindrance effect of monomeric bacteria and the efficiency of hindering electron transfer, thereby improving the detection rate of trace bacteria. The saliva sample was added to the prepared DNA bionic nanowall reaction system and inserted into the converted blood glucose chip. The DNA bionic nanowall reaction system (bionic nanowall self-assembly system) gradually forms a negatively charged nanolayer on the surface of the target bacteria. The monoclonal antibodies fixed on the blood glucose chip capture the target bacteria. The blood glucose chip is then washed and inserted into the PGM. At a constant voltage of 400mV, the count was performed for 5 seconds, and then the current signal generated by the oxidation reaction of potassium ferrocyanide (working solution, 5 mg / ml) was detected. Since the target bacteria wrapped by the DNA bionic nanowall have a strong blocking effect on electron transfer, the current intensity is negatively correlated with the concentration of the target bacteria.

[0141] The dynamic changes of periodontal pathogens in saliva are closely related to the progression of periodontal disease. 0.38 @ Under the synergistic effect of V4C3MXene and DNA bionic nanowall, the present invention successfully developed a non-invasive and sensitive handheld electrochemical bacterial sensor (Hebs) and method for detecting periodontal pathogens. The Hebs platform has superior performance and good stability, and achieves accurate detection of single-digit bacteria, which can meet the detection needs of various special environments, such as chairside detection or self-monitoring at home. Especially in the detection of clinical saliva samples, Hebs has high accuracy and portability, and has high applicability in domestic clinical chairside periodontal disease assessment and self-monitoring, overcoming the shortcomings of clinical detection methods such as difficulty in quantification, time-consuming and complicated operation. Hebs can effectively detect 10~5×10 6 The detection limit of Pg in the CFU / mL range is 9 CFU / mL. Hebs is able to quantify Pg in saliva samples and shows great advantages in terms of time consumption and portability, making it suitable for accurate detection of periodontal pathogens in chairside or home environments.

[0142] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A method for detecting Porphyromonas gingivalis for non-diagnostic purposes, characterized in that: The method comprises: S101: adding a sample to a first reaction system and incubating the mixture to obtain a first mixture, wherein the first reaction system includes a first nucleic acid molecule, a second nucleic acid molecule and an aptamer molecule; the aptamer molecule is used to capture the Porphyromonas gingivalis; after the aptamer molecule is combined with the Porphyromonas gingivalis, the first nucleic acid molecule and the second nucleic acid molecule are hybridized on the surface of the Porphyromonas gingivalis through a hybridization chain reaction to form a bionic nano-wall; S102: contacting the modified substrate electrode with the first mixture and then washing; S103 inserting the washed modified substrate electrode into the blood glucose meter; S104 obtains the blood glucose meter reading, and then obtains the concentration of the Porphyromonas gingivalis in the sample; wherein the modified substrate electrode is modified with Au@V4C3 MXene material and combined with antibodies and blocking agents that specifically recognize Porphyromonas gingivalis.

2. The method according to claim 1, characterized in that The preparation method of the Au@V4C3 MXene material is as follows: a) dispersing V4C3 MXene powder in an organic alkaline compound, stirring and centrifuging to obtain a first precipitate; b) adding a first solvent to the first precipitate and centrifuging to obtain a single-layer V4C3 MXene suspension; c) mixing the KAuCl4 solution and the single-layer V4C3 MXene suspension and stirring until the solution turns red, adding a reducing agent to obtain a second precipitate, i.e., the electrode modification material; wherein the concentration ratio of the KAuCl4 solution to the single-layer V4C3 MXene suspension is 0.30-0.45:1; and the reducing agent comprises sodium borohydride.

3. The method according to claim 1, characterized in that The sample includes a saliva sample and / or a gingival crevicular fluid sample.

4. The method according to claim 1, characterized in that The nucleotide sequence of the first nucleic acid molecule is shown as SEQ ID NO:1, the nucleotide sequence of the second nucleic acid molecule is shown as SEQ ID NO:2, and the nucleotide sequence of the aptamer molecule is shown as SEQ ID NO:

3.

5. The method according to claim 1, characterized in that The incubation time in step S101 is 20–60 min.

6. The method according to claim 1, characterized in that The second nucleic acid molecule is coupled to alginate.

7. The method according to claim 1, characterized in that The sample volumes ranged from 10–200 μL.

8. The method according to claim 7, characterized in that The concentrations of the first nucleic acid molecule, the second nucleic acid molecule, and the aptamer molecule in the first mixture include 1:1:1 to 3:3:

1.

9. The method according to claim 1, characterized in that The substrate electrode includes a blood sugar chip.

10. The method according to claim 1, characterized in that The step S104 also includes drawing a standard curve of the blood glucose meter reading and the concentration of Porphyromonas gingivalis and obtaining a linear equation of the blood glucose meter reading and the concentration of Porphyromonas gingivalis, and obtaining the concentration of Porphyromonas gingivalis in the sample based on the linear equation.

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