An electrochemical array sensor for rapid identification of bacteria and its detection method

Abstract

The present invention discloses an electrochemical array sensor for rapid bacterial identification and a detection method thereof, and relates to the technical field of bacterial identification. The electrochemical array sensor is composed of 6 different modified silver nanoparticles, and utilizes the different binding forces of different bacterial species to the 6 silver nanoparticles to generate electrochemical signals of different sizes, and uses linear discriminant analysis of electrochemical data to create different response patterns to achieve differentiation and identification of different bacteria. The present invention discloses an electrochemical array sensor for rapid bacterial identification and a detection method thereof, and the preparation method thereof is simple, and the detection thereof has the characteristics of high accuracy, short cycle, strong controllability, simple instrumentation, and intuitive result determination.

Description

Technical Field

[0001] The present invention relates to the technical field of bacteria identification, and in particular to an electrochemical array sensor for rapid bacteria identification and a detection method thereof. Background Art

[0002] Bacterial infection has always been a clinical focus. At present, the most commonly used bacterial detection method in clinical practice is culture medium. Although this method is the gold standard for bacterial detection, the detection process is cumbersome and time-consuming. For example, for Campylobacter, it takes 4-9 days to obtain a negative result and 14-16 days to confirm a positive result.

[0003] Polymerase chain reaction (PCR) has also been widely used in bacterial detection. This method only takes 5 to 24 hours to obtain test results, but the sample processing process is cumbersome and requires personnel with professional backgrounds and expensive instruments.

[0004] In addition, the immunology-based enzyme-linked immunosorbent assay (ELISA) is also widely used in bacterial detection. The ELISA detection method has the advantages of economical equipment and high specificity. However, its ability to detect unknown pathogens is poor, and its specificity is easily interfered by antibodies, which affects its detection sensitivity.

[0005] Therefore, developing a platform that can quickly and easily identify multiple bacteria simultaneously becomes a current challenge.

[0006] Array-based sensing methods mainly achieve the purpose of detection by distinguishing the overall characteristics of the analyte, and can replace the traditional "lock-key" detection method that relies on specific recognition for use in the field of bioanalysis. The interaction between a series of recognition units in the sensor array and each analyte will produce a characteristic signal fingerprint pattern for the analyte, thereby realizing the identification and classification of the analyte. This special detection method is similar to the sense of smell of mammals, so it is also called the "chemical nose or tongue" detection method. It is an effective method for analyzing subtle differences between different samples, complex sample mixtures, and even samples to be analyzed in biological matrices. Therefore, even without any prior knowledge of specific chemical or biological targets, the array-based "chemical nose" method can be used to detect and identify bacteria. Summary of the invention

[0007] To solve the above problems, the present invention provides an electrochemical array sensor for rapid bacterial identification and a detection method thereof. The sensor array preparation method adopted is simple, low-cost, and requires a small amount of sample; the sensor array can achieve 100% bacterial identification accuracy; the detection cycle is short and the controllability is strong; the instrument used is simple and the result judgment is intuitive.

[0008] To achieve the above-mentioned purpose, the present invention provides an electrochemical array sensor for rapid identification of bacteria, comprising cystamine-modified silver nanoparticles, glutaraldehyde-modified silver nanoparticles, vancomycin-modified silver nanoparticles, galactose-modified silver nanoparticles, immunoglobulin-modified silver nanoparticles, and lysozyme-modified silver nanoparticles having different binding forces with different bacterial species.

[0009] A detection method of an electrochemical array sensor for rapid identification of bacteria, based on the above electrochemical array sensor, utilizes the characteristics that different bacterial species have different binding forces with different silver nanoparticles in the electrochemical array sensor, thereby generating different electrical signals to distinguish and identify bacterial species;

[0010] The method for acquiring the electrical signal comprises the following steps:

[0011] S1, incubate the bacterial solution to be identified with the electrode, adsorb the bacteria on the conductive side of the electrode, and obtain an electrode adsorbing bacteria;

[0012] S2, co-incubating the electrode adsorbing bacteria with the electrochemical array sensor to allow the bacteria to bind to the electrochemical array sensor to obtain a working electrode;

[0013] S3. The working electrode is immersed in the electrolyte solution, and a linear sweep voltammetric measurement is performed on an electrochemical workstation using a three-electrode test system to obtain the oxidation current values ​​of different silver nanoparticles in the electrochemical array sensor.

[0014] Preferably, the electrode is amino- and aldehyde-modified indium tin oxide conductive glass.

[0015] Preferably, the specific steps of co-incubating the bacterial solution to be identified with the electrode in step S1 include: fixing an electrode area of ​​1 cm×0.5 cm on the surface of the modified indium tin oxide conductive glass with a high temperature resistant tape, placing 100 μL OD 600 =0.1 bacterial suspension was dropped on the conductive side surface of the modified indium tin oxide conductive glass. After adsorption for 1 hour, the surface of the modified indium tin oxide conductive glass was cleaned with PBS solution to obtain modified indium tin oxide conductive glass with adsorbed bacteria.

[0016] Preferably, in step S2, the modified indium tin oxide conductive glass adsorbing bacteria is incubated with 100 μL of six types of silver nanoprobes for 1 hour respectively, and washed with PBS solution after the incubation.

[0017] Preferably, in step S3, in the three-electrode test system, the counter electrode is a platinum wire, the reference electrode is a saturated calomel electrode, the electrolyte solution is an aqueous solution containing 0.1M NaCl and 0.1M NaNO3, the scanning range is 0-0.6V, and the scanning speed is 100mV / s.

[0018] Preferably, the bacterial species are differentiated and identified according to the oxidation current values ​​obtained when different bacteria are combined with different silver nanoparticles in the electrochemical array sensor;

[0019] If n types of bacteria are distinguished and measured five times in parallel, a 6×n×5 data array will be obtained for all bacteria. Linear discriminant analysis (LDA) will be performed on the obtained data array, and each type of bacteria will be displayed as a non-overlapping cluster on the two-dimensional LDA graph.

[0020] The present invention provides an electrochemical array sensor for rapid identification of bacteria and a detection method thereof, which has the following beneficial effects:

[0021] (1) The sensor array preparation method used in the present invention is simple, low-cost, and requires a small amount of sample;

[0022] (2) The sensor array of the present invention can achieve 100% accuracy in bacterial identification;

[0023] (3) The present invention can detect and distinguish multiple bacteria simultaneously;

[0024] (4) The detection cycle of the present invention is short and the controllability is strong;

[0025] (5) The instrument used in the present invention is simple and the result determination is intuitive.

[0026] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the scanning electron microscopy image of Cit-AgNPs;

[0028] Figure 2 is the transmission electron microscopy image of Cit-AgNPs;

[0029] Figure 3 is the ZETA potential of Cit-AgNPs and their functionalized forms;

[0030] Figure 4 Fluorescence microscopy images of E. coli after adsorption to the adhesion interface, where (A) is a bright field image, (B) is a fluorescence image, and (C) is a merged image;

[0031] Figure 5 This is the electrochemical impedance spectroscopy of ITO electrode after modification and adsorption of bacteria;

[0032] Figure 6 The electrochemical response patterns of the array sensor to eight types of bacteria;

[0033] Figure 7 Schematic diagram of linear discrimination of eight bacteria based on six probes. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages disclosed in the embodiments of the present invention clearer, the embodiments of the present invention are further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the embodiments of the present invention and are not intended to limit the embodiments of the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application. Examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions.

[0035] It should be noted that the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or server that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units that are not explicitly listed or inherent to these processes, methods, products or devices.

[0036] Like reference numerals and letters denote similar items in the following drawings, and thus, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0037] Embodiment 1

[0038] An electrochemical array sensor for rapid bacterial identification is composed of six differently modified silver nanoparticles: cystamine-modified silver nanoparticles (Cyst-AgNPs), glutaraldehyde-modified silver nanoparticles (GA-AgNPs), vancomycin-modified silver nanoparticles (Van-AgNPs), galactose-modified silver nanoparticles (Gly-AgNPs), immunoglobulin-modified silver nanoparticles (IgG-AgNPs), and lysozyme-modified silver nanoparticles (LZM-AgNPs).

[0039] The preparation methods of 6 different modified silver nanoparticles are as follows:

[0040] (1) Preparation of citrate-stabilized silver nanoparticles

[0041] 18 mg of AgNO3 was placed in 100 mL of deionized water, heated to boiling, and stirred continuously. After the AgNO3 was completely dissolved, 2 mL of 1% sodium citrate aqueous solution was added and heated to boiling again under continuous stirring. After reacting for 1 h, the resulting solution was cooled to room temperature to obtain citrate-stabilized silver nanoparticles Cit-AgNPs.

[0042] The scanning electron microscopy images of Cit-AgNPs are shown in Figure 1As shown in the transmission electron microscopy image Figure 2 Scanning electron microscopy showed that Cit-AgNPs were well dispersed, and transmission electron microscopy showed that their average particle size was about 29.3nm.

[0043] (2) Preparation of cystamine-modified silver nanoparticles

[0044] 54 mg of cystamine was added to 10 mL of deionized water and stirred to dissolve, and 80 mL of Cit-AgNPs prepared in Example 1 was added. After ultrasonic reaction at 50°C for 3 h, the product was collected by centrifugation at 6000 rpm. The product was washed three times by centrifugation with deionized water and dispersed in 20 mL of deionized water to obtain cystamine-modified silver nanoparticles Cyst-AgNPs.

[0045] (3) Preparation of glutaraldehyde-modified silver nanoparticles

[0046] 50 mL of Cyst-AgNPs prepared in Example 2 was dropped into a mixed solution of 5 mL of 50% glutaraldehyde and 5 mL of PBS buffer (100 mM, pH = 7.4), and dispersed by ultrasound and continuous stirring. After 12 h, the product was collected by centrifugation at 6000 rpm for 5 min. After washing three times with deionized water, it was dispersed in 50 mL of deionized water to obtain glutaraldehyde-modified silver nanoparticles GA-AgNPs.

[0047] (4) Preparation of vancomycin-modified silver nanoparticles

[0048] 500 μL of 100 mM vancomycin was added to 10 mL of GA-AgNPs prepared in Example 3, shaken overnight at 4°C, and the product was collected by centrifugation at 6000 rpm. The product was washed three times by centrifugation with deionized water and then dispersed in 10 mL of deionized water to obtain vancomycin-modified silver nanoparticles Van-AgNPs.

[0049] (5) Preparation of galactose-modified silver nanoparticles

[0050] 500 μL of 500 mM galactose was added to 10 mL of GA-AgNPs prepared in Example 3, shaken overnight at 4°C, and the product was collected by centrifugation at 6000 rpm. After centrifugation washing three times with deionized water, it was dispersed in 10 mL of deionized water to obtain galactose-modified silver nanoparticles Gly-AgNPs.

[0051] (6) Preparation of immunoglobulin (IgG) modified silver nanoparticles

[0052] 500 μL of 2 mg / mL IgG was added to 10 mL of GA-AgNPs prepared in Example 3, shaken overnight at 4°C, and the product was collected by centrifugation at 6000 rpm. After centrifugation washing three times with deionized water, it was dispersed in 10 mL of deionized water to obtain immunoglobulin-modified silver nanoparticles IgG-AgNPs.

[0053] (7) Preparation of lysozyme-modified silver nanoparticles

[0054] 500 μL of 2 mg / mL lysozyme was added to 10 mL of GA-AgNPs prepared in Example 3, shaken overnight at 4°C, and the product was collected by centrifugation at 6000 rpm. After centrifugation washing three times with deionized water, it was dispersed in 10 mL of deionized water to obtain lysozyme-modified silver nanoparticles LZM-AgNPs.

[0055] like Figure 3 As shown, the zeta potential of silver nanoparticles changed significantly after being modified with different materials, confirming the successful modification of the six types of silver nanoparticles.

[0056] Embodiment 2

[0057] The method for bacterial identification using the electrochemical array sensor in Example 1 utilizes the different binding forces of different bacterial species to the six types of silver nanoparticles to generate electrochemical signals of different sizes, and uses linear discriminant analysis of electrochemical data to create different response patterns, thereby achieving differentiation and identification of different bacteria.

[0058] This embodiment involves the identification of 8 bacterial species, including: Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Alcaligenes faecalis (A. faecalis), Staphylococcus epidermidis (S. epidermidis), Yeast, kanamycin-resistant Escherichia coli (KREC) and methicillin-resistant Staphylococcus aureus (MRSA).

[0059] Bacterial culture: Inoculate the bacterial substrate on an agar plate by streaking and incubate overnight in an incubator at 37°C. Select a single colony with good growth and place it in LB medium, and culture it at 37°C with shaking for 8-12 hours. Collect the bacteria by centrifugation at 5000 rpm for 5 minutes, wash three times with PBS (pH=7.0), measure the optical density (OD) at λ=600nm, and dilute to the required concentration.

[0060] The identification method specifically includes the following steps:

[0061] (1) Aminated and aldehyde-modified indium tin oxide conductive glass (ITO) was used as an electrode. A 1 cm × 0.5 cm electrode area was fixed on the electrode surface with a high temperature resistant tape. 100 μL of OD600 = 0.1 bacterial suspension was dropped on the conductive side of the electrode. After adsorption for 1 h, the electrode surface was cleaned with PBS solution to obtain modified ITO with adsorbed bacteria.

[0062] The preparation method of the modified ITO by amination and formaldehyde includes:

[0063] ITO was cut into 2cm×0.5cm strips, ultrasonicated for 5min in acetone, isopropanol and deionized water respectively, and then dried in vacuum. The dried ITO was placed in a mixed solution of 30mL1M HCl and 30% H2O2 (1:1, v:v) and soaked for 30min, and then washed with water. Then it was transferred to a 1MNaOH ethanol solution (ethanol: water = 1:1, v:v), ultrasonicated for 10min, and then washed with water. Then 5% (3-aminopropyl) trimethoxysilane aqueous solution was added, soaked for 1h for amination (ITO-NH2), washed with water 3 times, and dried at 80℃ overnight. Finally, it was added to 20mL 5% glutaraldehyde, reacted for 12h, washed with water, and dried.

[0064] One aldehyde group of glutaraldehyde reacts with the amino group on ITO-NH2 to undergo an amidation reaction, and the other aldehyde group is exposed on the ITO surface to achieve aldehyde modification (ITO-GA). Bacteria can bind to the aldehyde group and adsorb on the modified ITO surface.

[0065] After the modification, Escherichia coli (ITO-E.Coli) was attached to the ITO surface to test the bacterial adsorption performance of the surface.

[0066] like Figure 4 As shown, the constructed bacterial adhesion interface can adsorb Escherichia coli well.

[0067] like Figure 5 As shown, the electrochemical impedance spectrum of ITO changed after both amination and aldehyde modification, which confirmed that the ITO modification was successful, and the impedance value further increased after the adsorption of Escherichia coli.

[0068] (2) The modified ITO adsorbed with bacteria was co-incubated with 100 μL of the six silver nanoprobes in Example 1 for 1 h, respectively, to allow the bacteria to bind to different silver nanoparticles. After the incubation, the electrodes were washed with PBS solution to obtain working electrodes.

[0069] (3) The working electrode was immersed in the electrolyte solution and linear scanning voltammetry was performed on an electrochemical workstation using a conventional three-electrode test system. The counter electrode was a platinum wire and the reference electrode was a saturated calomel electrode (SCE). The electrolyte used was an aqueous solution containing 0.1 M NaCl and 0.1 M NaNO3. The scanning range was 0-0.6 V and the scanning rate was 100 mV / s.

[0070] The electrochemical response data were obtained through the oxidation current of six types of silver nanoparticles adsorbed on the bacterial surface. The current values ​​were recorded to obtain the oxidation current values ​​of different silver nanoprobes.

[0071] The electrochemical response data of the six functional nanoprobes are shown in Figure 2. Figure 6 As shown, multiple bacteria are distinguished according to the electrochemical response data of each bacterium. The method for obtaining the electrochemical response data of each bacterium is the same as the above-mentioned bacterial species identification method. When preparing the working electrode, only the bacterial species solution is added dropwise to make it adsorbed on the modified ITO, and then it is co-incubated with 6 types of silver nanoparticles, and finally the linear scanning voltammogram of the bacterial species combined with different silver nanoprobes is measured. Since different bacterial species have different binding forces with different silver nanoparticles, the electrochemical response data obtained are also different.

[0072] In this example, eight bacterial samples were measured five times in parallel, so a 6×8×5 data array was obtained for all bacteria. Linear discriminant analysis (LDA) was performed on the obtained data using SYSTAT (version 13.1). Figure 7 As shown, according to the results of linear discriminant analysis, these eight bacterial species were well distinguished without any overlap.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A detection method of an electrochemical array sensor for rapid identification of bacteria, characterized in that: The electrochemical array sensor includes cystamine-modified silver nanoparticles, glutaraldehyde-modified silver nanoparticles, vancomycin-modified silver nanoparticles, galactose-modified silver nanoparticles, immunoglobulin-modified silver nanoparticles, and lysozyme-modified silver nanoparticles, which have different binding forces with different bacterial species. The bacterial species are distinguished and identified by utilizing the different binding forces between different bacterial species and different silver nanoparticles in the electrochemical array sensor, thereby generating different electrical signals. The method for acquiring the electrical signal comprises the following steps: S1, incubate the bacterial solution to be identified with the electrode, adsorb the bacteria on the conductive side of the electrode, and obtain an electrode adsorbing bacteria; S2, co-incubating the electrode adsorbing bacteria with the electrochemical array sensor to allow the bacteria to bind to the electrochemical array sensor to obtain a working electrode; S3, the working electrode is immersed in the electrolyte solution, and a linear sweep voltammetric measurement is performed on an electrochemical workstation using a three-electrode test system to obtain the oxidation current values ​​of different silver nanoparticles in the electrochemical array sensor; The bacterial species are distinguished and identified based on the oxidation current values ​​obtained by combining different bacteria with different silver nanoparticles in the electrochemical array sensor; If n types of bacteria are distinguished and measured five times in parallel, a 6×n×5 data array will be obtained for all bacteria. Linear discriminant analysis will be performed on the obtained data array, and each type of bacteria will be displayed as a non-overlapping cluster on the two-dimensional linear discriminant analysis graph.

2. The detection method of an electrochemical array sensor for rapid bacterial identification according to claim 1, characterized in that: The electrode is amino- and aldehyde-modified indium tin oxide conductive glass.

3. The detection method of an electrochemical array sensor for rapid bacterial identification according to claim 2, characterized in that: The specific steps of co-incubating the bacterial solution to be identified and the electrode in step S1 include: fixing an electrode area of ​​1 cm × 0.5 cm on the surface of the modified indium tin oxide conductive glass with a high temperature resistant tape, and placing 100 μL OD 600 =0.1 bacterial suspension was dropped on the conductive side surface of the modified indium tin oxide conductive glass. After adsorption for 1 h, the surface of the modified indium tin oxide conductive glass was cleaned with PBS solution to obtain the modified indium tin oxide conductive glass with adsorbed bacteria.

4. The detection method of an electrochemical array sensor for rapid bacterial identification according to claim 2, characterized in that: In step S2, the modified indium tin oxide conductive glass adsorbed with bacteria was incubated with 100 μL of the six silver nanoprobes for 1 h respectively, and then washed with PBS solution after the incubation.

5. The detection method of an electrochemical array sensor for rapid bacterial identification according to claim 1, characterized in that: In step S3, in the three-electrode test system, the counter electrode is a platinum wire, the reference electrode is a saturated calomel electrode, the electrolyte solution is an aqueous solution containing 0.1 M NaCl and 0.1 M NaNO3, the scanning range is 0-0.6 V, and the scanning speed is 100 mV / s.

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