A microfluidic electrochemical immunosensor for periodontitis detection and its preparation method

Abstract

The present invention relates to a microfluidic electrochemical immunosensor for periodontitis detection and a preparation method thereof, belonging to the technical field of medical devices. The microfluidic electrochemical immunosensor for periodontitis detection comprises: a microfluidic chip; a planar immunosensor, mounted on the chip, the planar immunosensor comprising: an MMP-8 immunosensor; an IL-1β immunosensor, used to cooperate with the MMP-8 immunosensor for early diagnosis and monitoring of periodontitis through saliva. The sensor of the present invention is of great significance for the early diagnosis and monitoring of periodontitis, and solves the problems of high cost, need for professionals, complicated operation, and not conducive to chairside operation in traditional detection methods.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, in particular to a microfluidic electrochemical immunosensor for periodontitis detection and a preparation method thereof. Background Art

[0002] Periodontitis is the sixth largest chronic inflammatory disease in the world characterized by damage to teeth and their supporting tissues. It not only affects oral health but is also closely related to some systemic diseases. Since there are no obvious clinical symptoms in the early stages of the disease, patients may miss the best time for treatment and suffer permanent damage. Therefore, early diagnosis and monitoring of periodontitis are very important for the prevention and control of the disease. However, traditional clinical diagnostic methods (such as visual examination, probing, and imaging examinations) are difficult to operate, expensive, and can only reflect the cumulative damage of periodontitis rather than the current status of the disease, let alone monitor the progression of periodontitis. Therefore, there is an urgent need to develop a new, simple, and low-cost method for diagnosing periodontitis.

[0003] In recent years, periodontitis-related biomarkers have been widely used to reflect the status of the disease, predict the progression of the disease, and the prognosis. Matrix metalloproteinase-8 (MMP-8) and the proinflammatory cytokine interleukin-1β (IL-1β) are the most typical periodontitis biomarkers present in oral fluids such as saliva and gingival crevicular fluid. Importantly, their levels are significantly increased compared with healthy people and change with the progression and treatment of periodontal damage. Among them, the levels of MMP-8 and IL-1β are significantly increased in the early stage of periodontitis, and IL-1β is also associated with the late stage of periodontitis in a dose-dependent manner. In fact, a large number of studies have shown that the simultaneous detection of multiple biomarkers is more feasible for the diagnosis of the disease. In addition, compared with gingival crevicular fluid, the collection of saliva is a more convenient, non-invasive, blood-free, and professional-free method.

[0004] Therefore, simultaneous detection of the concentrations of MMP-8 and IL-1β in saliva is necessary for accurately diagnosing the severity of periodontitis and monitoring the progression of periodontitis. However, the levels of biomarkers in saliva are low and contain some interfering substances. Therefore, high-sensitivity and specificity of salivary multi-marker combined detection still faces great challenges.

[0005] Currently, in clinical research, the detection of salivary biomarkers is usually performed by enzyme-linked immunosorbent assay and immunofluorescence assay. These methods have disadvantages such as expensive equipment, high reagent consumption, low efficiency, and inconvenience for chairside operation. In recent years, microfluidics technology has been considered as one of the most promising clinical diagnostic platforms due to its high volumetric rate, low sample consumption, and the ability to integrate other functional components. On the other hand, among the various strategies for converting biochemical information into measurable signals, electrochemical methods stand out due to their high sensitivity and low detection limit, and electrodes of different sizes and models are also easy to integrate into microfluidic chips. Therefore, the combination of microfluidics and electrochemical sensing technology is an attractive diagnostic analysis platform, especially for disease diagnosis and monitoring in a POCT manner.

[0006] With the development of materials science and sensor technology, there are some biosensors used for diagnosing periodontitis at the laboratory level. However, these biosensors can only detect a single biomarker, which is not reliable enough for diagnosing the disease; or use paper-based sensors to judge the disease status by color changes, which has limited detection sensitivity; or can only be used in the laboratory, which is not conducive to operation and chairside testing. To the best of our knowledge, there is currently no microfluidic electrochemical immunosensor that can simultaneously detect multiple biomarkers in saliva with high sensitivity and specificity in a POCT manner for the diagnosis and monitoring of periodontitis. Summary of the invention

[0007] The object of the present invention is to provide a microfluidic electrochemical immunosensor for periodontitis detection and a preparation method thereof, so as to solve the problems raised in the above background technology.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A microfluidic electrochemical immunosensor for periodontitis detection, comprising:

[0010] Microfluidic chips;

[0011] A planar immune sensor component is mounted on the chip component, and the planar immune sensor component includes:

[0012] MMP-8 immunosensor;

[0013] IL-1β immunosensor is used in conjunction with MMP-8 immunosensor for early diagnosis and monitoring of periodontitis through saliva.

[0014] As a further technical solution of the present invention, the MMP-8 immunosensor is prepared by the following method:

[0015] Synthesis of iridium oxide and titanium carbide composite nanomaterials;

[0016] The screen-printed electrodes were washed with deionized water and dried at room temperature;

[0017] The synthesized iridium oxide titanium carbide composite nanomaterials and chitosan are ultrasonically mixed evenly, and then the mixed solution is dropped onto the screen-printed electrode and dried under an infrared lamp;

[0018] The infrared-dried electrodes were immersed in glutaraldehyde aqueous solution at room temperature and then rinsed with deionized water;

[0019] The rinsed electrode was immersed in the MMP-8 antibody solution, and after incubation, the electrode surface was rinsed with a biological buffer solution;

[0020] The electrode rinsed with biological buffer was incubated with bovine serum albumin solution, rinsed with biological buffer solution after completion, and then stored in biological buffer for future use.

[0021] As a further technical solution of the present invention, the IL-1β immunosensor is prepared by the following method:

[0022] Synthesis of iridium oxide and titanium carbide composite nanomaterials;

[0023] The screen-printed electrodes were washed with deionized water and dried at room temperature;

[0024] The synthesized iridium oxide titanium carbide composite nanomaterials and chitosan are ultrasonically mixed evenly, and then the mixed solution is dropped onto the screen-printed electrode and dried under an infrared lamp;

[0025] The infrared-dried electrodes were immersed in glutaraldehyde aqueous solution at room temperature and then rinsed with deionized water;

[0026] The rinsed electrode was immersed in the IL-1β antibody solution, and after incubation, the electrode surface was rinsed with a biological buffer;

[0027] The electrode rinsed with biological buffer was incubated with bovine serum albumin solution, rinsed with biological buffer solution after completion, and then stored in biological buffer for future use.

[0028] As a further technical solution of the present invention, the synthesis of iridium oxide titanium carbide composite nanomaterials is: synthesizing titanium carbide nanosheets by top-down selective etching, and synthesizing iridium oxide nanotubes by electrostatic spinning process.

[0029] As a further technical solution of the present invention, the mass concentration of the chitosan is 1%; the mass concentration of the glutaraldehyde aqueous solution is 1%, and the immersion time is 30 minutes.

[0030] As a further technical solution of the present invention, the mass concentration of the bovine serum albumin solution is 1%.

[0031] As a further technical solution of the present invention, the microfluidic chip comprises:

[0032] Chip parts;

[0033] A sample feeding part, connected to the chip part, for providing a test sample;

[0034] The sample outlet tube has one end connected to the detection pool and is used to send out the sample in the detection pool.

[0035] As a further technical solution of the present invention, the chip component includes:

[0036] The chip substrate has two electrode fixing grooves;

[0037] The top layer of the chip is provided with a detection pool and a liquid microchannel, wherein the detection pool is connected to the sample injection part through the liquid microchannel, and is used to receive the sample provided by the sample injection part and make the sample react with the planar immune sensor part;

[0038] The chip fixing screws are used to connect and fix the chip top layer and the chip base layer to the planar immune sensor element.

[0039] As a further technical solution of the present invention, the sample injection part comprises:

[0040] Sample injection tube;

[0041] PBS buffer injection tube;

[0042] Redox couple injection tube.

[0043] A method for preparing the microfluidic electrochemical immunosensor for periodontitis detection as described above comprises the following steps:

[0044] Preparation of MMP-8 immunosensor;

[0045] Preparation of IL-1β immunosensor;

[0046] preparing a microfluidic chip;

[0047] The MMP-8 immunosensor and the IL-1β immunosensor are assembled on a microfluidic chip.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] 1. It is of great significance for the early diagnosis and monitoring of periodontitis, solving the problems of traditional detection methods, such as high cost, need for professionals, complicated operation, and not conducive to chairside operation;

[0050] 2. Use a microfluidic device to transport the saliva sample to the detection pool. After the sample reacts with the MMP-8 immunosensor and IL-1β immunosensor installed in the detection pool, the patient's periodontal health status is obtained based on the current signal;

[0051] 3. Simple operation, low reagent consumption, convenient and fast, a non-invasive method for diagnosing early periodontitis;

[0052] 4. It paves the way for the future POCT approach to predict the progression of periodontitis and guide periodontitis treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Exploded diagram of the immunosensor;

[0054] Figure 2 Iridium oxide nanotubes and titanium carbide nanosheets (IrO x / Ti3C2T x ) Composite material morphology;

[0055] Figure 3 For the preparation of IrO x / Ti3C2T x Composite material surface morphology analysis diagram;

[0056] Figure 4 Optimization diagram of electrochemical performance and experimental conditions of immunosensor;

[0057] Figure 5 The schematic diagram of the fabrication process of the planar immunosensor and the use of microfluidic immunosensor for early diagnosis and monitoring of periodontitis;

[0058] Figure 6 Figure 2 shows the performance of the immunosensor.

[0059] In the figure: 1-injection piece, 11-sample injection tube, 12-PBS buffer injection tube, 13-redox pair injection tube, 2-chip fixing screws, 3-chip top layer, 4-liquid microchannel, 5-planar immunosensor, 51-MMP-8 immunosensor, 52-IL-1β immunosensor, 6-chip base layer, 7-electrode fixing groove, 8-working electrode, 9-detection cell, 10-sample outlet tube. DETAILED DESCRIPTION

[0060] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0061] The embodiment of the present invention is implemented as follows: Figure 1 The microfluidic electrochemical immunosensor for periodontitis detection shown comprises:

[0062] Microfluidic chips;

[0063] A planar immune sensor 5 is mounted on the chip component, and the planar immune sensor 5 includes:

[0064] MMP-8 immunosensor;

[0065] IL-1β immunosensor is used in conjunction with MMP-8 immunosensor for early diagnosis and monitoring of periodontitis through saliva.

[0066] In actual application, the present invention detects saliva through the MMP-8 immunosensor 51 and the IL-1β immunosensor 52 in conjunction with the working electrode 8, thereby realizing early diagnosis and early monitoring of periodontitis. The two markers are different, and the two monitorings are more comprehensive and accurate; the patient's periodontal health status results are obtained based on the current signal; the operation is simple, the reagent consumption is small, it is convenient and fast, and it is a non-invasive early periodontitis diagnosis method, paving the way for future POCT methods to predict the progression of periodontitis and guide periodontitis treatment.

[0067] Specifically, the MMP-8 immunosensor 51 and the IL-1β immunosensor 52 are prepared by the following method:

[0068] Synthesis of iridium oxide titanium carbide composite nanomaterials: The surface of the screen-printed electrode (SPEs) working electrode 8 is made of iridium oxide nanotubes and titanium carbide nanosheets (IrO x / Ti3C2T x ) composite materials to amplify the sensing signal; two-dimensional Ti3C2T x NSs were synthesized by a top-down selective etching process, and iridium oxide nanotubes (IrO x NTs) are obtained by electrospinning; because they are negatively charged and positively charged, stable iridium oxide titanium carbide composite nanomaterials (IrO x / Ti3C2T x );

[0069] The screen-printed electrodes (SPEs) were washed with deionized water and dried at room temperature;

[0070] Will IrO x / Ti3C2T x The composite nanomaterials were ultrasonically mixed with 1 wt% chitosan (CS) and then 5 μL of the mixed solution was dripped onto the working electrode 8 of the SPEs and dried under an infrared lamp.

[0071] The infrared-dried electrode was immersed in a 1% by mass glutaraldehyde (GA) aqueous solution at room temperature for 30 min to activate the amino groups on the surface of CS to couple the antibody (Ab); then the modified SPE electrode was rinsed with deionized water to remove excess GA molecules;

[0072] The electrodes without GA were immersed in 3.12 μg / mL MMP-8 antibody solution and 6.25 μg / mL IL-1β antibody solution, respectively, and after incubation at 4 °C for 12 h, the electrode surface was rinsed with biological buffer (PBS) to remove unbound antibodies and physical adsorption;

[0073] The electrode rinsed with biological buffer was incubated with 1% bovine serum albumin (BSA) solution at 37°C for 1 h to further block the nonspecific active sites on the electrode surface. After the reaction was completed, it was rinsed with biological buffer solution to remove excess BSA on the electrode surface. The corresponding MMP-8 immunosensor 51 and IL-1β immunosensor 52 were stored in PBS solution at 4°C when not in use.

[0074] Furthermore, titanium carbide nanosheets (Ti3C2T x NSs) were prepared as follows (see Figure 2 , where: A is multilayer Ti3C2T x NSs scanning electron microscope image, B is a few-layer Ti3C2T x Transmission electron microscopy image of NSs, C is IrO x NTs scanning electron microscopy image, D is IrO x Transmission electron microscopy image of NTs, E is IrO x / Ti3C2T x SEM images of composite materials, F is IrO x / Ti3C2T x Transmission electron microscopy images of the composite materials (white and gray arrows are IrO x NTs and Ti3C2T x NSs), G is IrO x / Ti3C2T x Scanning transmission electron microscope image of the composite material, Ha-Hd is the corresponding energy dispersion X-ray spectrum of Ir, O, Ti and C elements):

[0075] 2D Ti3C2T x NSs were synthesized via a top-down selective etching procedure.

[0076] 1) Multilayer Ti3C2T xPreparation: First, 2 g of lithium fluoride (LiF) was added to 20 mL of 9 M dilute hydrochloric acid (diluted with concentrated hydrochloric acid) and stirred with a magnetic stirrer; then 1 g of Ti3AlC2 powder was slowly dispersed in the above solution and stirred at 35 °C for 24 h to form a uniform suspension; the particles were then washed 4 times with deionized water and ethanol until the pH value of the supernatant was about 6, at which time multilayer Ti3C2T x The morphology of the material is shown in Figure 2 As shown in A.

[0077] 2) Few-layer Ti3C2T x Preparation: The mixture was diluted to 200 mL and ultrasonically treated for 1 h, and then the supernatant was collected by centrifugation at a speed of 3500 rpm / min; the colloidal solution was vacuum filtered on a porous membrane with a pore size of 20 nm, and the obtained solid was dried at 40 ° C for 8 hours to obtain a few-layer Ti3C2T x NSs powder, the morphology of the material is as follows Figure 2 As shown in B.

[0078] Iridium oxide nanotubes (IrO x Preparation of NTs: IrO x NTs were obtained by electrospinning process.

[0079] 1) Preparation of precursor solution: First, weigh 0.25 g of hydrated iridium trichloride (IrCl3·xH2O) and add it to 2 mL of N,N-dimethylformamide (DMF) solution, and stir it magnetically in a water bath at 80°C for 10 min. Then, add 0.4 g of polyvinyl pyrrolidone (PVP) (Mw=1300000) to the mixed solution, and continue to stir magnetically until a transparent electrospinning precursor solution is obtained.

[0080] 2) Electrospinning parameter setting: The prepared precursor solution was loaded into a 5 mL plastic syringe for electrospinning, a stainless steel needle with an inner diameter of 0.8 mm was used as the spinneret for electrospinning, and aluminum foil was covered on the roller as a receiving plate; the distance between the spinneret and the receiving plate was 17 cm, the injection speed was 0.05 mm / min, and the positive and negative voltages were 18 kV and -1 kV, respectively.

[0081] 3) IrO x Preparation of tubular structure: After the electrospun membrane was dried in a drying tower for 12 h, it was heated in a muffle furnace from room temperature to 500 °C at a heating rate of 1 °C / min for 3 h to remove the PVP nanofiber template and to allow IrO x Crystallization, then the furnace is naturally cooled to room temperature, and the product obtained by calcination is IrO x NTs, the morphology of the material is shown in Figure 2 C, shown in 2D.

[0082] (3) Iridium oxide titanium carbide composite material (IrO x / Ti3C2T x ) preparation: IrO x NTs and Ti3C2T x NSs were mixed in water and magnetically stirred for 2 h. Since they were positively and negatively charged, the Ti3C2T x Mass fraction of 5mg / mL IrO x The stable composite solution, the morphology of the material is as follows Figure 2 E, 2F, the scanning transmission electron microscope image and energy dispersive X-ray spectrometer are shown in Figure 2 G, shown in 2H.

[0083] To further prove that IrO x / Ti3C2T x The successful preparation of composite materials is followed by surface morphology analysis, such as Figure 3 As shown (where A is IrO x 、Ti3C2T x and IrO x / Ti3C2T x X-ray diffraction diagram of the composite material, B is Raman spectrum, C is Fourier transform infrared spectrum), X-ray diffraction analysis, Raman spectrum analysis and Fourier transform infrared spectrum analysis were performed on it, and characteristic signals of the material can be obtained; all of the above results can prove that IrO was successfully synthesized. x / Ti3C2T x Composite materials.

[0084] Optimization of experimental conditions

[0085] To obtain the best electrochemical performance, the experimental conditions such as material ratio, pH value, Ab immobilization concentration and antigen incubation time were optimized by differential pulse voltammetry (DPV) (see Figure 4 , where A and B are the CV and EIS graphs of the MMP-8 immunosensor 51, respectively; C and D are the CV and EIS graphs of the IL-1β immunosensor 52, respectively (a is SPE, b is CS / IrO x / Ti3C2T x / SPE,c is Ab / CS / IrO x / Ti3C2T x / SPE,d is BSA / Ab / CS / IrO x / Ti3C2T x / SPE,e is Ag / BSA / Ab / CS / IrO x / Ti3C2Tx / SPE;);E is IrO x / Ti3C2T x Four different mass fractions of Ti3C2T in the composites x DPV peak; F is the pH value of the buffered salt solution of MMP-8 immunosensor 51 and IL-1β immunosensor 52, G is the Ab concentration of MMP-8, H is the Ab concentration of IL-1β, and I is the optimization of incubation time (except for Figure E, all experiments were incubated with 150 ng / mL MMP-8 antigen or 100 ng / mL IL-1β antigen solution, and all experiments were carried out at 3.0 mM [Fe(CN)6] 3- / 4- and 0.1 M KCl buffer solution).

[0086] (1) Ti3C2T x Optimization of mass fraction: Since the electrochemical performance is affected by the different ratios of the two substances in the composite material, the mass fraction of Ti3C2T x The mass fraction of is optimized to be 1%, 2%, 5% and 10%; Figure 4 As shown in E, the peak value of the current reaches the highest value when the mass fraction is 2%, so the content of Ti3C2T x 5mg / mL IrO x / Ti3C2T x The composite material was selected as the final material for modified electrode.

[0087] (2) Optimization of pH value of buffered saline solution: Since pH value can affect the activity of antigens and Abs immobilized on the immunosensor, the immunosensor incubated with 150 ng / mL MMP-8 and 100 ng / mL IL-1β antigen solutions at 37°C was placed in a buffer solution with a pH value of 5.0-9.0 for testing. Figure 4 As can be seen from Figure F, the current intensity reaches a maximum at a pH of 7.4; a similar variation trend can also be found in the IL-1β immunosensor 52; therefore, a pH value of 7.4 was selected as the buffer electrolyte in subsequent measurements.

[0088] (3) Optimization of antibody concentration: covalently linked to CS / IrO x / Ti3C2T x / The amount of Ab on the SPE determines its binding efficiency with the target analyte; therefore, five different concentrations of MMP-8 and IL-1β antibodies (0.78-12.50 μg / mL) were incubated with 150 ng / mL MMP-8 and 100 ng / mL IL-1β antigen solutions at 37 °C to optimize the sensing performance; Figure 4 G and 4H, MMP-8 and IL-1β antigen / Ab / CS / IrOx / Ti3C2T x The / SPEs had the smallest current intensity when incubated with 3.12 and 6.25 μg / mL Ab concentrations, respectively, indicating that the specific binding between the antigen and Ab was the greatest at this time.

[0089] (4) Optimization of antigen incubation time: Using the above optimized optimal conditions, the MMP-8 immunosensor 51 and the IL-1β immunosensor 52 were incubated with 150 ng / mL MMP-8 and 100 ng / mL IL-1β antigen solutions for 10-50 min, respectively; Figure 4 As shown in I, the current peak gradually decreased from 10 min to 40 min, and then reached a stable state, indicating that the antigen-antibody had reached the maximum binding efficiency when the incubation time was 40 min; therefore, all subsequent tests were carried out under the conditions of pH 7.4, MMP-8 and IL-1βAb concentrations of 3.12 μg / mL and 6.25 μg / mL, respectively, and antigen incubation time of 40 min.

[0090] like Figure 1 As shown, as a preferred embodiment of the present invention, the microfluidic chip includes:

[0091] A chip component, the chip component comprising:

[0092] The chip substrate 6 is provided with two electrode fixing grooves 7;

[0093] The top layer 3 of the chip is provided with a detection pool 9 and a liquid microchannel 4. The detection pool 9 is connected to the sample injection part through the liquid microchannel 4, and is used to receive the sample provided by the sample injection part and make the sample react with the planar immunosensor.

[0094] Chip fixing screws 2, used to connect and fix the chip top layer 3 and the chip base layer 6 to the planar immune sensor;

[0095] The sample injection part 1 is connected to the chip part and is used to provide a test sample. The sample injection part 1 includes:

[0096] Sample injection tube 11;

[0097] PBS buffer injection tube 12;

[0098] Redox pair injection tube 13;

[0099] One end of the sample outlet tube 10 is connected to the detection pool 9 and is used to send out the sample in the detection pool 9 .

[0100] In one case of the present embodiment, the working electrode 8 is a part of a planar immune sensor component, and the chip component is composed of a chip base layer 6, a chip top layer 3 and a chip fixing screw 2; during assembly, the planar immune sensor component 5 is first installed in the electrode fixing groove 7 of the chip base layer 6, and then the chip top layer 3, the chip fixing screw 2, the sample injection component 1 and the sample outlet tube 10 are assembled in sequence; when in use, the sample is pumped in by the sample injection tube 11, flows into the sample detection pool 9 through the liquid microchannel 4, reacts with the planar immune sensor component assembled therein, causes the current signal to change, and thus obtains the patient's periodontal health status result based on the current signal.

[0101] This embodiment provides a method for preparing a microfluidic electrochemical immunosensor for periodontitis detection, comprising the following steps:

[0102] Preparation of MMP-8 immunosensor 51;

[0103] Preparation of IL-1β immunosensor 52;

[0104] preparing a microfluidic chip;

[0105] The MMP-8 immunosensor 51 and the IL-1β immunosensor 52 are assembled on a microfluidic chip.

[0106] In one case of the present embodiment, the microfluidic chip is carefully designed using 3ds Max 2020 software (Autodesk, USA) and is produced using a HP Multi Jet Fusion 3D printer (Hewlett-Packard, Inc., Palo Alto, CA) through stereolithography (SLA) technology; the microfluidic chip consists of three input ports (for sample, buffer and redox couple inflow, respectively), two detection chambers (diameter: 7 mm; depth: 0.8 mm), an outlet for waste liquid outflow and some microchannels (width: 1 mm; depth: 0.5 mm); the prepared planar MMP-8 immunosensor 51 and IL-1β immunosensor 52 are assembled with the prepared microfluidic chip, as shown in FIG. Figure 5 As shown; when the detection liquids are introduced through the liquid microchannel 4, they will be transferred to different detection pools 9 and react with the planar MMP-8 immunosensor 51 and IL-1β immunosensor 52 assembled in the detection pools 9, causing the current signal to change; the electrochemical performance is characterized and tested by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV).

[0107] Experimental example

[0108] Detection of MMP-8 and IL-1β (see Figure 6, where A and B are the DPV and IC diagrams of the MMP-8 immunosensor 51, respectively; C and D are the DPV and IC diagrams of the IL-1β immunosensor 52, respectively; E, F, and G are the repeatability, long-term stability, anti-interference and selectivity diagrams of the prepared planar immunosensor, respectively; H is the result diagram of the microfluidic device detecting clinical samples)

[0109] (1) Preparation of standard solutions: Prepare standard solutions of MMP-8 and IL-1β antigens with different concentrations (including a set of blank solutions with no concentration).

[0110] (2) Modifying the working electrode 8: The MMP-8 and IL-1β standard solutions prepared in (1) are respectively applied to the surfaces of the working electrodes 8 of the prepared MMP-8 immunosensor 51 and IL-1β immunosensor 52, and incubated at 37°C for 40 min for antigen-antibody specific binding, followed by rinsing with a biological buffer to remove unbound antigens, thereby obtaining the modified electrode to be tested.

[0111] (3) Drawing a working curve: The electrode prepared in (2) was placed in a phosphate-buffered saline (PBS) solution containing 3 mM potassium ferrocyanide / potassium ferrocyanide (K3[Fe(CN)6] / K4[Fe(CN)6]) (1:1) and 0.1 M potassium chloride, pH = 7.4, and the DPV curves of different concentrations of MMP-8 and IL-1β antigen standard solutions were tested. Figure 6 A, 6C, and detect the linear relationship between the concentration C of the MMP-8 and IL-1β antigen standard solution and the current I, and draw the IC curve. The test results of the present invention are as follows Figure 6 B, as shown in 6D.

[0112] (4) Performance investigation: The prepared MMP-8 immunosensor 51 and IL-1β immunosensor 52 were incubated with a solution containing 50 ng / mL of MMP-8 and 100 ng / mL of IL-1β, and five different immunosensors were examined under the same conditions to evaluate the repeatability. Figure 6 As shown in Figure E, the RSDs of MMP-8 immunosensor 51 and IL-1β immunosensor 52 were 1.1% and 1.3%, respectively. Subsequently, the long-term stability of the immunosensor was studied by continuously testing the current response of the prepared immunosensor within 4 weeks. Figure 6As shown in Figure 1, MMP-8 immunosensor 51 and IL-1β immunosensor 52 retained 96.1% and 94.9% of the original response values ​​for 50 ng / mL MMP-8 and 100 ng / mL IL-1β, respectively, showing good long-term stability. In addition, in practical applications, the lack of cross-interference between analytes and between analytes and other similar substances is an important and necessary factor, so the cross-interference and selectivity of MMP-8 immunosensor 51 and IL-1β immunosensor 52 with similar substances were also investigated. Figure 6 As shown in columns 1-4 of G, the current of the prepared MMP-8 immunosensor 51 and IL-1β immunosensor 52 decreased significantly only in PBS containing relevant target biomarkers (150 ng / mL MMP-8 and 100 ng / mL IL-1β antigen solution), indicating that an immunosensor without cross-interference that can simultaneously detect MMP-8 and IL-1β was successfully fabricated. In addition, the selective specificity of the MMP-8 immunosensor 51 and IL-1β immunosensor 52 was further studied by introducing 10-fold concentrations of other potential interfering substances in PBS containing MMP-8 and IL-1β antigens, such as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), glucose (GLU), and ascorbic acid (AA) in saliva. Figure 6 Other columns of G show that even in the face of high concentrations of interferents, the changes in electrochemical reactions are slight, indicating that the immunosensor has good selectivity. The cross-reactions between the prepared immunosensors and with other high concentrations of interferents in saliva are negligible, which enables the prepared immunosensor to accurately detect MMP-8 and IL-1β in saliva, thereby diagnosing periodontitis.

[0113] (5) Detection in actual environment: Different concentrations of MMP-8 and IL-1β antigen standard solutions were added to the prepared artificial saliva, and the artificial saliva without the standard solution was the blank sample. Then, 20 μL of the artificial saliva with the addition of MMP-8 and IL-1β antigens was transported from the sample channel to the detection pool 9 of the microfluidic device by a peristaltic pump. After the device was incubated at 37°C for 40 minutes, the biological buffer was pumped into the detection pool 9 by a peristaltic pump. After cleaning, the electrolyte containing the redox pair K3[Fe(CN)6] / K4[Fe(CN)6] was pumped into the detection pool 9, and the test of step (3) was performed on it.

[0114] (6) Clinical saliva sample testing: All clinical saliva samples were obtained from the Jilin University Stomatological Hospital. A total of 30 subjects’ whole oral saliva was collected, including 10 healthy people, 10 patients with gingivitis and 10 patients with periodontitis. All subjects were required not to eat, drink, brush their teeth or rinse their mouths within 1 hour before sampling, and saliva was collected between 9:00 and 11:00 in the morning. The subjects spit out unstimulated whole saliva into a plastic tube and frozen it at -80°C. Before testing, the frozen saliva samples were thawed and centrifuged to remove insoluble particles and possible interference from highly viscous mucins in the saliva, and then the supernatant was taken and 0.01M PBS was added to dilute and preserve the antigen components in the saliva. Thereafter, 20 μL of the above sample was pumped into the microfluidic device for testing. The test results are as follows. Figure 6 As shown in Figure 3, the MMP-8 and IL-1β response currents of 10 healthy subjects were the highest, while the corresponding current intensities of patients with gingivitis and periodontitis were significantly reduced. As expected, the periodontitis group was significantly lower than the gingivitis group, indicating that the sensor can not only diagnose periodontitis in the early stages, but also has the ability to distinguish the severity of the disease, thus providing valuable evidence for guiding clinical treatment. In addition, the current response critical values ​​of MMP-8 or IL-1β biomarkers are relatively close in some patients with different disease states, which may be due to individual differences or other systemic factors. The simultaneous analysis of the current response values ​​of the two biomarkers can more accurately confirm different disease states. In summary, this microfluidic device with good immunosensing performance can simultaneously detect two key biomarkers by POCT to achieve early diagnosis and monitoring of periodontitis, and is expected to be applied to the clinical diagnosis of periodontitis and guide clinical treatment, especially for vulnerable groups living with insufficient medical conditions.

[0115] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0116] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A microfluidic electrochemical immunosensor for periodontitis detection, characterized in that: include: A microfluidic chip, the microfluidic chip comprising: a chip component, the chip component comprising: a chip base layer, provided with two electrode fixing grooves; a chip top layer, provided with a detection pool and a liquid microchannel, the detection pool being connected to a sample injection component through the liquid microchannel, for receiving a sample provided by the sample injection component and allowing the sample to react with a planar immunosensor component; a chip fixing screw, for connecting and fixing the chip top layer and the chip base layer to the planar immunosensor component; a sample injection component, connected to the chip component, for providing a detection sample; a sample outlet tube, one end of which is connected to the detection pool, for sending out the sample in the detection pool; A planar immune sensor component is mounted on the chip component, and the planar immune sensor component includes: MMP-8 immunosensor; IL-1β immunosensor, used in conjunction with MMP-8 immunosensor for early diagnosis and monitoring of periodontitis through saliva; The MMP-8 immunosensor is prepared by the following method: Synthesis of iridium oxide and titanium carbide composite nanomaterials; The screen-printed electrodes were washed with deionized water and dried at room temperature; The synthesized iridium oxide titanium carbide composite nanomaterials and chitosan are ultrasonically mixed evenly, and then the mixed solution is dropped onto the screen-printed electrode and dried under an infrared lamp; The infrared-dried electrode was immersed in a glutaraldehyde aqueous solution at room temperature and then rinsed with deionized water; The rinsed electrode was immersed in the MMP-8 antibody solution, and after incubation, the electrode surface was rinsed with a biological buffer solution; The electrode rinsed with biological buffer is incubated with bovine serum albumin solution, rinsed with biological buffer solution after completion, and then stored in biological buffer for future use; The IL-1β immunosensor is prepared by the following method: Synthesis of iridium oxide and titanium carbide composite nanomaterials; The screen-printed electrodes were washed with deionized water and dried at room temperature; The synthesized iridium oxide titanium carbide composite nanomaterials and chitosan are ultrasonically mixed evenly, and then the mixed solution is dropped onto the screen-printed electrode and dried under an infrared lamp; The infrared-dried electrodes were immersed in glutaraldehyde aqueous solution at room temperature and then rinsed with deionized water; The rinsed electrode was immersed in the IL-1β antibody solution, and after incubation, the electrode surface was rinsed with a biological buffer; The electrode rinsed with biological buffer is incubated with bovine serum albumin solution, rinsed with biological buffer solution after completion, and then stored in biological buffer for future use; The synthesis of the iridium oxide titanium carbide composite nanomaterial comprises: synthesizing titanium carbide nanosheets by top-down selective etching, and synthesizing iridium oxide nanotubes by electrostatic spinning; The mass concentration of the chitosan is 1%; the mass concentration of the glutaraldehyde aqueous solution is 1%, and the soaking time is 30 minutes; The mass concentration of the bovine serum albumin solution is 1%.

2. The microfluidic electrochemical immunosensor for periodontitis detection according to claim 1, characterized in that: The sample injection part comprises: Sample injection tube; PBS buffer injection tube; Redox couple injection tube.

3. The method for preparing a microfluidic electrochemical immunosensor for periodontitis detection according to any one of claims 1-2, characterized in that: The following steps are involved: Preparation of MMP-8 immunosensor; Preparation of IL-1β immunosensor; preparing a microfluidic chip; The MMP-8 immunosensor and the IL-1β immunosensor are assembled on a microfluidic chip.

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