CN112760221B - Vibrio parahaemolyticus detection device and method - Google Patents
Vibrio parahaemolyticus detection device and method Download PDFInfo
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Abstract
The invention discloses a vibrio parahemolyticus detection device and a method. The device comprises a computer, a data acquisition unit, an electrochemical workstation, a first detection mechanism and a second detection mechanism, wherein the first detection mechanism comprises a first base, a first detection container, a first detection module and a first lifter for driving the first detection module to lift are arranged on the first base, the second detection mechanism comprises a second base, and a second lifter for driving the second detection container, the second detection module and the second detection module to lift are arranged on the second base. The invention can accurately detect the concentration of the vibrio parahemolyticus and has simple operation.
Description
Technical Field
The invention relates to the technical field of bacteria detection, in particular to a vibrio parahemolyticus detection device and a method.
Background
Food-borne diseases refer to a general term for diseases which are caused by food contaminated by pathogenic bacteria entering human bodies by means of ingestion and causing human body infection or poisoning, and threaten human health and life safety. Although the development of modern science and technology reaches a certain level, food-borne diseases still seriously harm the health of people no matter in developed or developing countries, and the frequent food safety events in recent years also indicate that the food-borne diseases are not effectively controlled, so that the development of a novel detection technology for accurately detecting the food-borne pathogenic bacteria is very important, and the detection technology has important significance for preventing and controlling the food-borne diseases and guaranteeing the health of people.
Vibrio parahaemolyticus can cause diseases such as neonatal meningitis, septicemia and necrotizing small intestine conjunctivitis, and poses serious threats to human health and life safety, so that the research on the method for accurately detecting vibrio parahaemolyticus is of great significance. The existing vibrio parahaemolyticus detection method generally adopts a chemical detection method, and has the defects of complex operation and poor repeatability.
Disclosure of Invention
In order to solve the technical problems, the invention provides a vibrio parahemolyticus detection device and method, which can accurately detect the concentration of vibrio parahemolyticus and are simple to operate.
In order to solve the problems, the invention adopts the following technical scheme:
the invention relates to a vibrio parahemolyticus detection device, which comprises a computer, a data collector, an electrochemical workstation, a first detection mechanism and a second detection mechanism, wherein the first detection mechanism comprises a first base, a first detection container, a first detection module and a first lifter for driving the first detection module to lift are arranged on the first base, the first detection module comprises a first guide rail, a first slide block capable of sliding along the first guide rail and a first driving module for driving the first slide block to slide, the bottom of the first slide block is connected with a first bracket, the bottom of the first bracket is provided with m first connecting seats at equal intervals from left to right, the first connecting seat is detachably connected with a first working electrode, the bottom of the first guide rail is detachably connected with a first counter electrode and a first reference electrode, the first counter electrode and the first reference electrode are positioned right above the first detection container, still be equipped with first multichannel data selector on the first support, m input of first multichannel data selector are connected with m first connecting seat electricity respectively, first connecting seat is connected with the first working electrode electricity of being connected, second detection mechanism includes the second base, be equipped with the second on the second base and detect container, second detection module and drive the second riser that second detection module goes up and down, second detection module includes the second guide rail, can follow the gliding second slider of second guide rail and drive the gliding second drive module of second slider, second slider bottom is connected with the second support, second support bottom is equipped with m second connecting seats from a left side to the right equidistant, can dismantle on the second connecting seat and be connected with the second working electrode, second guide rail bottom can be dismantled and be connected with the gliding second counter electrode of second counter electrode, the second counter electrode and the second reference electrode are positioned right above the second detection container, the second support is further provided with a second multi-channel data selector, m input ends of the second multi-channel data selector are respectively and electrically connected with m second connecting seats, the second connecting seats are electrically connected with a second working electrode connected with the second connecting seats, the electrochemical workstation is respectively and electrically connected with an output end of the first multi-channel data selector, the first counter electrode, the first reference electrode, an output end of the second multi-channel data selector, the second counter electrode, the second reference electrode and an input end of the data collector, the computer is respectively and electrically connected with an output end of the data collector, the first lifter, the second lifter, the first driving module, the second driving module, a control end of the first multi-channel data selector and a control end of the second multi-channel data selector, vibrio parahemolyticus antigen is not cultured on the first working electrode, and vibrio parahemolyticus antigen is cultured on the second working electrode.
In the scheme, before detection, 2.5 mu l of vibrio parahaemolyticus suspension with the concentration is dripped on the m first working electrodes which do not participate in detection on the first detection module and stands for 25 minutes, and 2.5 mu l of vibrio parahaemolyticus suspension with the concentration is dripped on the m second working electrodes which do not participate in detection on the second detection module and stands for 25 minutes. Buffer solution is injected into the first detection container and the second detection container to bufferThe solution is washed by 0.5 mmol/1H 2 O 2 The solution and 1.0mol/1 Thi/HaC-NaAc solution are mixed uniformly according to the volume ratio of 1: 2.
The m first working electrodes on the first support are numbered 1, 2 and 3 … … m from left to right in sequence, and the m second working electrodes on the second support are numbered 1, 2 and 3 … … m from left to right in sequence.
During detection, firstly, a first counter electrode, a first reference electrode and a first working electrode with the number of 1 are inserted into a buffer solution in a first detection container, a second counter electrode, a second reference electrode and a second working electrode with the number of 1 are inserted into a buffer solution in a second detection container, the first working electrode with the number of 1 is communicated with an electrochemical workstation by a first multi-path data selector, the second working electrode with the number of 1 is communicated with the electrochemical workstation by a second multi-path data selector, the first counter electrode, the first reference electrode and the first working electrode which are inserted into the buffer solution form a first electrochemical sensor array, and the second counter electrode, the second reference electrode and the second working electrode which are inserted into the buffer solution form a second electrochemical sensor array;
the electrochemical workstation adopts cyclic voltammetry to switch n different scanning rates from small to large for detection, and n reduction peak current difference values delta Ip are obtained 1 、ΔIp 2 …ΔIp n ，ΔIp n ＝Ip1 n -Ip2 n ，ΔIp n Is the difference in reduction peak current at the nth scan rate, ip1 n Is the reduction peak current value, ip2, corresponding to the first electrochemical sensor array at the nth scanning rate n The data acquisition unit acquires data acquired by the electrochemical workstation and sends the data to the computer for the reduction peak current value corresponding to the second electrochemical sensor array at the nth scanning speed;
computer will delta Ip 1 、ΔIp 2 …ΔIp n Performing secondary spline interpolation to obtain curve x (t), and substituting x (t) into nonlinear directional saturated resonance model
In the step (1), the first step,
flat feedback cascade steady state potential function:
the detection signal loading component of ing (t) = cOS (kt + η) + x (t),
wherein t is an interpolation variable, k is a real parameter, η is a real parameter, nois (t) is colored noise with uneven power spectral density function, P is a real parameter, Q is a real parameter, a, b and c are real numbers, delta is a flat delay parameter,
adjusting the value of t, the nonlinear directional saturation resonance model is at t = t r And (3) when the point position reaches resonance, calculating a characteristic value F of a resonance state:
then, the first lifter drives the first guide rail to ascend to enable the first working electrode with the number of 1 to leave a first detection container, the second lifter drives the second guide rail to ascend to enable the second working electrode with the number of 1 to leave a second detection container, the first sliding block drives the first support to move leftwards to enable the first working electrode with the number of 2 to move to the upper side of the first detection container, the second sliding block drives the second support to move leftwards to enable the second working electrode with the number of 2 to move to the upper side of the second detection container, the first pair of electrodes, the first reference electrode and the first working electrode with the number of 2 are inserted into a buffer solution in the first detection container, the second pair of electrodes, the second reference electrode and the second working electrode with the number of 2 are inserted into a buffer solution in the second detection container, the first multi-path data selector connects the first working electrode with the number of 2 with an electrochemical workstation, the second multi-path data selector connects the second working electrode with the electrochemical workstation, the first working electrode and the reference electrode array form a second working sensor, and the second multi-path data selector is used for detecting characteristic value by adopting the electrochemical sensor;
and then, repeating the above operations, inserting the first counter electrode, the first reference electrode and the first working electrode with the number of 3 into the buffer solution in the first detection container, inserting the second counter electrode, the second reference electrode and the second working electrode with the number of 3 into the buffer solution in the second detection container, performing primary detection to obtain a corresponding characteristic value F, and repeating the steps until the first counter electrode, the first reference electrode and the first working electrode with the number of m are inserted into the buffer solution in the first detection container, and the second counter electrode, the second reference electrode and the second working electrode with the number of m are inserted into the buffer solution in the second detection container, performing primary detection to obtain a corresponding characteristic value F.
The whole detection process is carried out m times of detection in total to obtain m characteristic values F, the m characteristic values F are averaged to obtain an average value, the average value is a value of a characteristic value Y, and the value of the characteristic value Y is substituted into a formula: y =0.2+0.1 × LgX, and the concentration of the vibrio parahaemolyticus suspension to be detected is calculated, wherein X is the concentration of the vibrio parahaemolyticus.
Preferably, the first working electrode and the second working electrode are both copper film electrodes, the first counter electrode and the second counter electrode are both Pt electrodes, and the first reference electrode and the second reference electrode are both Ag/AgCl electrodes.
Preferably, the method for culturing the vibrio parahaemolyticus antigen on the second working electrode is as follows:
n1: mixing silver paste SL and sodium alginate SA according to the mass ratio of 1: 3 to prepare 10ml of sol water solution, then carrying out ultrasonic dispersion for 15min, and weighing 5mg of graphene oxide GO and 1mg of amino-functionalized organic metal framework material NH 2 dissolving-CuBTC in the sol water solution, and performing ultrasonic oscillation for 25min to obtain GO/NH 2 A mixture of-CuBTC/SL/SA, 1.5. Mu.L of GO/NH 2 Dropping the mixed solution of-CuBTC/SL/SA onto the surface of the second working electrode, drying for 5h at room temperature, and forming a layer of GO/NH on the second working electrode 2 -a film of CuBTC/SL/SA;
n2: diluting the vibrio parahaemolyticus polyclonal antibody solution by 300 times by using PBS buffer solution, then modifying 4 mu l of the diluted vibrio parahaemolyticus polyclonal antibody solution on the dried second working electrode, and standing and drying the modified second working electrode in a drying dish;
n3: dripping 3.5 μ l of vibrio parahemolyticus antigen solution on the second working electrode, culturing at 30 deg.C for 25min, washing off unbound vibrio parahemolyticus antigen on the second working electrode with distilled water, and air drying.
Selecting graphene oxide GO and amino functionalized organic metal framework material NH 2 -CuBTC is used as a second working electrode modification material, sodium alginate SA and silver paste SL are used as dispersing agents to enable graphene oxide GO and amino functionalized organic metal framework material NH 2 -CuBTC is dispersed to be stably fixed on the surface of the second working electrode, and NH is utilized 2 And (3) enriching the concentration of the detected substance by CuBTC, then fixing the vibrio parahaemolyticus antibody on the modified second working electrode, incubating vibrio parahaemolyticus antigen on the prepared second working electrode, and detecting the reduction peak current value by using a Cyclic Voltammetry (CV).
Preferably, the preparation method of the vibrio parahaemolyticus antigen solution is as follows: at a concentration of 2X 10 8 cfu/ml～2×10 9 Inactivating cfu/ml vibrio parahaemolyticus with 8% -12% formalin at 25-39 ℃, centrifuging to remove the formalin after inactivation, coating a flat plate for aseptic inspection, and resuspending a precipitate with equal volume of sterile physiological saline after determining the sterility, thereby obtaining the vibrio parahaemolyticus antigen solution.
Preferably, the preparation method of the vibrio parahaemolyticus polyclonal antibody solution is as follows:
after the rabbits are raised for 2 weeks, 15ml of blood is collected from the ear veins, and serum is taken out to be used as a negative serum sample; immunizing rabbit with vibrio parahaemolyticus antigen, performing second immunization at intervals of 3 days, performing booster immunization at intervals of 6 days, performing carotid artery one-time blood sampling at 4 days after booster immunization, standing at room temperature for 45min, transferring to 4 deg.C overnight, and centrifuging at 4 deg.C and 5000rpm for 45min the next day to obtain antiserum for storage;
fixing 1.5ml affinity chromatographic column on a protein purifier, washing out protective agent solution by deionized water, balancing the column by PBS buffer solution, loading 1.5ml antiserum sample on the column, eluting impurities by PBS buffer solution, and finally eluting vibrio parahaemolyticus polyclonal antibody by citric acid buffer solution to obtain vibrio parahaemolyticus polyclonal antibody solution.
The invention discloses a vibrio parahaemolyticus detection method, which is used for the vibrio parahaemolyticus detection device and comprises the following steps:
s1: preparing vibrio parahaemolyticus suspension liquid with different concentrations, detecting characteristic values Y corresponding to the vibrio parahaemolyticus suspension liquid with each concentration, and fitting the values to obtain a concentration calculation formula: y =0.2+ 0.1X LgX, X is the concentration of vibrio parahaemolyticus;
s2: taking the vibrio parahaemolyticus suspension to be detected, detecting the corresponding characteristic value Y, and calculating according to a concentration calculation formula: y =0.2+ 0.1X LgX, and the concentration of the vibrio parahaemolyticus suspension to be detected is calculated.
Preferably, the method for detecting the characteristic value Y corresponding to the vibrio parahaemolyticus suspension at a certain concentration comprises the following steps:
m1: dripping 2.5 mu l of vibrio parahaemolyticus suspension with the concentration on the m first working electrodes which do not participate in the detection on the first detection module and standing for 25 minutes, dripping 2.5 mu l of vibrio parahaemolyticus suspension with the concentration on the m second working electrodes which do not participate in the detection on the second detection module and standing for 25 minutes;
m2: the vibrio parahaemolyticus suspension with the concentration is detected for m times by the same method, and the detection steps of each detection are as follows:
inserting a first pair of electrodes, a first reference electrode and a first working electrode which does not participate in detection into a buffer solution in a first detection container, wherein the first pair of electrodes, the first reference electrode and the first working electrode which does not participate in detection form a first electrochemical sensor array;
inserting a second counter electrode, a second reference electrode and a second working electrode which does not participate in detection into a buffer solution in a second detection container, wherein the second counter electrode, the second reference electrode and the second working electrode which does not participate in detection form a second electrochemical sensor array;
sequentially switching n different types from small to large by adopting cyclic voltammetryThe scanning speed is detected to obtain n reduction peak current difference values delta Ip w1 、ΔIp w2 …ΔIp wn ，ΔIp wn ＝Ip1 wn -Ip2 wn ，ΔIp wn Is the reduction peak current difference at the nth scan rate in the w-th test, ip1 wn The reduction peak current value Ip2 corresponding to the first electrochemical sensor array at the nth scanning rate in the w-th detection wn The reduction peak current value corresponding to the second electrochemical sensor array under the nth scanning speed in the w detection is more than or equal to 1 and less than or equal to m;
m3: constructing a feature matrix D according to the detection data of m times of detection,
m4: performing secondary spline interpolation on each row of data of the feature matrix D to obtain m curves x (t) corresponding to each row of data, performing the same data processing on the m curves x (t) to obtain m feature values F, wherein the data processing on each curve x (t) comprises the following steps:
substituting x (t) into a nonlinear directional saturated resonance model
flat feedback cascaded steady state potential function:
the detection signal loading component of ing (t) = cOS (kt + η) + x (t),
wherein t is an interpolation variable, k is a real parameter, η is a real parameter, nois (t) is colored noise with uneven power spectral density function, P is a real parameter, Q is a real parameter, a, b and c are real numbers, delta is a flat delay parameter,
adjusting the value of t, and enabling the nonlinear directional saturated resonance model to be at t = t r The point location reaches resonance and is calculatedCharacteristic value F of vibration state:
m5: and averaging the m characteristic values F to obtain an average value which is the value of the characteristic value Y.
The vibrio parahemolyticus detection device is provided with m first working electrodes which do not participate in detection and m second working electrodes which do not participate in detection, one of the first working electrodes which do not participate in detection and one of the second working electrodes which do not participate in detection are adopted in each detection in the process of detecting the vibrio parahemolyticus suspension for m times, and the first working electrode and the second working electrode which participate in the detection for one time are not used for detection any more.
Preferably, the n different scan rates comprise 50mV/s, 100mV/s, 200mV/s, 300mV/s, 400mV/s, 500mV/s.
The invention has the beneficial effects that: can accurately detect the concentration of the vibrio parahaemolyticus, and is simple to operate.
Drawings
FIG. 1 is a circuit connection block diagram of an embodiment;
FIG. 2 is a schematic view of a first detection mechanism;
fig. 3 is a schematic structural view of the second detection mechanism.
In the figure: 1. the device comprises a computer, 2, a data collector, 3, an electrochemical workstation, 4, a first base, 5, a first detection container, 6, a first lifter, 7, a first guide rail, 8, a first sliding block, 9, a first driving module, 10, a first support, 11, a first working electrode, 12, a first pair of electrodes, 13, a first reference electrode, 14, a first multi-way data selector, 15, a first connecting seat, 16, a second base, 17, a second detection container, 18, a second lifter, 19, a second guide rail, 20, a second sliding block, 21, a second driving module, 22, a second support, 23, a second working electrode, 24, a second pair of electrodes, 25, a second reference electrode, 26, a second multi-way data selector, 27 and a second connecting seat.
Detailed Description
The technical scheme of the invention is further specifically described by the following embodiments and the accompanying drawings.
The embodiment is as follows: the vibrio parahemolyticus detecting device of the embodiment, as shown in fig. 1, 2, and 3, includes a computer 1, a data collector 2, an electrochemical workstation 3, a first detecting mechanism and a second detecting mechanism, the first detecting mechanism includes a first base 4, a first detecting container 5 is disposed on the first base 4, a first detecting module and a first lifter 6 for driving the first detecting module to ascend and descend, the first detecting module includes a first guide rail 7, a first slider 8 capable of sliding along the first guide rail 7 and a first driving module 9 for driving the first slider 8 to slide, the bottom of the first slider 8 is connected with a first support 10, the bottom of the first support 10 is provided with m first connecting seats 15 at equal intervals from left to right, the first connecting seat 15 is detachably connected with a first working electrode 11, the bottom of the first guide rail 7 is detachably connected with a first pair of electrodes 12, the first reference electrodes 13, the first pair of electrodes 12, the first reference electrodes 13 are disposed right above the first detecting container 5, the first support 10 is further provided with a first working electrode 14, the bottom of the first guide rail 7 is detachably connected with a second slider 16, the second slider 16 is electrically connected with a second detecting module 16, the second slider 16 and a second detecting module 16, the bottom of the second guide rail 19 is detachably connected with a second counter electrode 24 and a second reference electrode 25, the second counter electrode 24 and the second reference electrode 25 are positioned right above the second detection container 17, the second bracket 22 is further provided with a second multi-channel data selector 26, m input ends of the second multi-channel data selector 26 are respectively and electrically connected with m second connecting seats 27, the second connecting seats 27 are electrically connected with a connected second working electrode 23, the electrochemical workstation 3 is respectively and electrically connected with an output end of the first multi-channel data selector 14, the first counter electrode 12, the first reference electrode 13, an output end of the second multi-channel data selector 26, the second counter electrode 24, the second reference electrode 25 and an input end of the data collector 2, the computer 1 is respectively and electrically connected with an output end of the data collector 2, the first lifter 6, the second lifter 18, the first driving module 9, the second driving module 21, a control end of the first multi-channel data selector 14 and a control end of the second multi-channel data selector 26, and vibrio parahemolytic antigen is not cultured on the first working electrode 11 and vibrio parahemolytic antigen is cultured on the second working electrode 23.
In the scheme, before detection, 2.5 mu l of vibrio parahaemolyticus suspension with the concentration is dripped on the m first working electrodes which do not participate in detection on the first detection module and stands for 25 minutes, and 2.5 mu l of vibrio parahaemolyticus suspension with the concentration is dripped on the m second working electrodes which do not participate in detection on the second detection module and stands for 25 minutes. Buffer solution is injected into the first detection container and the second detection container, and the buffer solution is composed of 0.5 mmol/1H 2 O 2 The solution and 1.0mol/l Thi/HaC-NaAc solution are mixed uniformly according to the volume ratio of 1: 2.
The m first working electrodes on the first support are numbered 1, 2 and 3 … … m from left to right in sequence, and the m second working electrodes on the second support are numbered 1, 2 and 3 … … m from left to right in sequence.
During detection, firstly, a first counter electrode, a first reference electrode and a first working electrode with the number of 1 are inserted into a buffer solution in a first detection container, a second counter electrode, a second reference electrode and a second working electrode with the number of 1 are inserted into a buffer solution in a second detection container, the first working electrode with the number of 1 is communicated with an electrochemical workstation by a first multi-path data selector, the second working electrode with the number of 1 is communicated with the electrochemical workstation by a second multi-path data selector, the first counter electrode, the first reference electrode and the first working electrode which are inserted into the buffer solution form a first electrochemical sensor array, and the second counter electrode, the second reference electrode and the second working electrode which are inserted into the buffer solution form a second electrochemical sensor array;
the electrochemical workstation adopts cyclic voltammetry to switch n different scanning rates from small to large for detection, and n reduction peak current difference values delta Ip are obtained 1 、ΔIp 2 …ΔIp n ，ΔIp n ＝Ip1 n -Ip2 n ，ΔIp n Is the difference in reduction peak current at the nth scan rate, ip1 n Is the reduction peak current value, ip2, corresponding to the first electrochemical sensor array at the nth scanning rate n The data acquisition unit acquires data acquired by the electrochemical workstation and sends the data to the computer for the reduction peak current value corresponding to the second electrochemical sensor array at the nth scanning speed;
computer will delta Ip 1 、ΔIp 2 …ΔIp n Performing quadratic spline interpolation to obtain curve x (t), and substituting x (t) into nonlinear directional saturation resonance model
In (1),
flat feedback cascaded steady state potential function:
the detection signal loading component, ing (t) = cOS (kt + η) + x (t),
wherein t is an interpolation variable, k is a real parameter, η is a real parameter, nois (t) is colored noise with uneven power spectral density function, P is a real parameter, Q is a real parameter, a, b and c are real numbers, delta is a flat delay parameter,
adjusting the value of t, the nonlinear directional saturation resonance model is at t = t r And (3) when the point position reaches resonance, calculating a characteristic value F of a resonance state:
then, the first lifter drives the first guide rail to ascend to enable the first working electrode with the number of 1 to leave a first detection container, the second lifter drives the second guide rail to ascend to enable the second working electrode with the number of 1 to leave a second detection container, the first sliding block drives the first support to move leftwards to enable the first working electrode with the number of 2 to move to the upper side of the first detection container, the second sliding block drives the second support to move leftwards to enable the second working electrode with the number of 2 to move to the upper side of the second detection container, the first pair of electrodes, the first reference electrode and the first working electrode with the number of 2 are inserted into a buffer solution in the first detection container, the second pair of electrodes, the second reference electrode and the second working electrode with the number of 2 are inserted into a buffer solution in the second detection container, the first multi-path data selector connects the first working electrode with the number of 2 with an electrochemical workstation, the second multi-path data selector connects the second working electrode with the electrochemical workstation, the first working electrode and the reference electrode array form a second working sensor, and the second multi-path data selector is used for detecting characteristic value by adopting the electrochemical sensor;
and then, repeating the above operations, inserting the first counter electrode, the first reference electrode and the first working electrode with the number of 3 into the buffer solution in the first detection container, inserting the second counter electrode, the second reference electrode and the second working electrode with the number of 3 into the buffer solution in the second detection container, performing primary detection to obtain a corresponding characteristic value F, and repeating the steps until the first counter electrode, the first reference electrode and the first working electrode with the number of m are inserted into the buffer solution in the first detection container, and the second counter electrode, the second reference electrode and the second working electrode with the number of m are inserted into the buffer solution in the second detection container, performing primary detection to obtain a corresponding characteristic value F.
The whole detection process is carried out m times of detection in total to obtain m characteristic values F, the m characteristic values F are averaged to obtain an average value, the average value is a value of a characteristic value Y, and the value of the characteristic value Y is substituted into a formula: y =0.2+0.1 × LgX, and the concentration of the vibrio parahaemolyticus suspension to be detected is calculated, wherein X is the concentration of the vibrio parahaemolyticus.
The first working electrode and the second working electrode are both copper film electrodes, the first counter electrode and the second counter electrode are both Pt electrodes, and the first reference electrode and the second reference electrode are both Ag/AgCl electrodes.
The method for culturing the vibrio parahaemolyticus antigen on the second working electrode comprises the following steps:
n1: mixing silver paste SL and sodium alginate SA according to the mass ratio of 1: 3 to prepare 10ml of sol water solution, then carrying out ultrasonic dispersion for 15min, and weighing 5mg of graphene oxide GO and 1mg of amino-functionalized organic metal framework material NH 2 dissolving-CuBTC in sol water solution, and performing ultrasonic oscillation for 25min to obtain GO/NH 2 The mixture of-CuBTC/SL/SA was taken at 1.5. Mu.L of GO/NH 2 Dropping the mixed solution of-CuBTC/SL/SA on the surface of the second working electrode, drying for 5h at room temperature, and forming a layer of GO/NH on the second working electrode 2 -a film of CuBTC/SL/SA;
n2: diluting the vibrio parahaemolyticus polyclonal antibody solution by 300 times by using PBS buffer solution, then modifying 4 mu l of the diluted vibrio parahaemolyticus polyclonal antibody solution on the dried second working electrode, and standing and drying the modified vibrio parahaemolyticus polyclonal antibody solution in a drying dish;
n3: dripping 3.5 μ l of vibrio parahaemolyticus antigen solution on the second working electrode, culturing at 30 deg.C for 25min, washing off uncombined vibrio parahaemolyticus antigen on the second working electrode with distilled water, and air drying.
Selecting graphene oxide GO and amino functionalized organic metal framework material NH 2 -CuBTC is used as a second working electrode modification material, sodium alginate SA and silver paste SL are used as dispersing agents, and graphene oxide GO and amino functionalized organic metal framework material NH are used 2 -CuBTC is dispersed to be stably fixed on the surface of the second working electrode, and NH is utilized 2 And (3) enriching the concentration of the detected substance by CuBTC, then fixing the vibrio parahaemolyticus antibody on the modified second working electrode, incubating vibrio parahaemolyticus antigen on the prepared second working electrode, and detecting the reduction peak current value by using a Cyclic Voltammetry (CV).
The preparation method of the vibrio parahaemolyticus antigen solution comprises the following steps: the concentration is 2 x 10 8 cfu/ml～2×10 9 cfuInactivating/ml vibrio parahaemolyticus with 8% -12% formalin at 25-39 ℃, centrifuging to remove the formalin after inactivation, coating a flat plate for aseptic inspection, and resuspending a precipitate with equal volume of sterile physiological saline after determining the sterility, thereby obtaining a vibrio parahaemolyticus antigen solution.
The preparation method of the vibrio parahaemolyticus polyclonal antibody solution comprises the following steps:
after the rabbits are raised for 2 weeks, 15ml of blood is collected from the ear veins, and serum is taken out to be used as a negative serum sample; immunizing rabbit with vibrio parahaemolyticus antigen, performing second immunization at intervals of 3 days, performing booster immunization at intervals of 6 days, performing carotid artery one-time blood sampling at 4 days after booster immunization, standing at room temperature for 45min, transferring to 4 deg.C overnight, and centrifuging at 4 deg.C and 5000rpm for 45min the next day to obtain antiserum for storage;
fixing 1.5ml affinity chromatographic column on a protein purifier, washing out protective agent solution by deionized water, balancing the column by PBS buffer solution, loading 1.5ml antiserum sample on the column, eluting impurities by PBS buffer solution, and finally eluting vibrio parahaemolyticus polyclonal antibody by citric acid buffer solution to obtain vibrio parahaemolyticus polyclonal antibody solution.
The vibrio parahemolyticus detection method of the embodiment is used for the vibrio parahemolyticus detection device, and comprises the following steps:
s1: preparing vibrio parahaemolyticus suspensions with different concentrations, detecting characteristic values Y corresponding to the vibrio parahaemolyticus suspensions with each concentration, and fitting the values to obtain a concentration calculation formula: y =0.2+ 0.1X LgX, X is the concentration of vibrio parahaemolyticus;
s2: taking the vibrio parahaemolyticus suspension to be detected, detecting the corresponding characteristic value Y, and calculating according to a concentration calculation formula: y =0.2+0.1 × LgX, and the concentration of the vibrio parahaemolyticus suspension to be detected is calculated.
The method for detecting the characteristic value Y corresponding to the vibrio parahaemolyticus suspension with a certain concentration comprises the following steps of:
m1: dripping 2.5 mu l of vibrio parahaemolyticus suspension with the concentration on the m first working electrodes which do not participate in the detection on the first detection module and standing for 25 minutes, dripping 2.5 mu l of vibrio parahaemolyticus suspension with the concentration on the m second working electrodes which do not participate in the detection on the second detection module and standing for 25 minutes;
m2: the vibrio parahaemolyticus suspension with the concentration is detected for m times by adopting the same method, and the detection steps of each detection are as follows:
inserting a first pair of electrodes, a first reference electrode and a first working electrode which does not participate in detection into a buffer solution in a first detection container, wherein the first pair of electrodes, the first reference electrode and the first working electrode which does not participate in detection form a first electrochemical sensor array;
inserting a second counter electrode, a second reference electrode and a second working electrode which does not participate in detection into a buffer solution in a second detection container, wherein the second counter electrode, the second reference electrode and the second working electrode which does not participate in detection form a second electrochemical sensor array;
sequentially switching n different scanning rates from small to large by adopting cyclic voltammetry to detect to obtain n reduction peak current difference values delta Ip w1 、ΔIp w2 …ΔIp wn ，ΔIp wn ＝Ip1 wn -Ip2 wn ，ΔIp wn Is the difference of the reduction peak current at the nth scan rate in the w-th test, ip1 wn The reduction peak current value Ip2 corresponding to the first electrochemical sensor array at the nth scanning rate in the w-th detection wn The reduction peak current value corresponding to the second electrochemical sensor array under the nth scanning speed in the w detection is more than or equal to 1 and less than or equal to m;
m3: constructing a feature matrix D according to the detection data of m times of detection,
m4: performing quadratic spline interpolation on each line of data of the characteristic matrix D to obtain m curves x (t) corresponding to each line of data, performing the same data processing on the m curves x (t) to obtain m characteristic values F, wherein the data processing on each curve x (t) comprises the following steps:
substituting x (t) into a nonlinear directional saturated resonance model
flat feedback cascade steady state potential function:
the detection signal load component, ing (t) = cOs (kt + η) + x (t),
wherein t is an interpolation variable, k is a real parameter, η is a real parameter, nois (t) is colored noise with uneven power spectral density function, P is a real parameter, Q is a real parameter, a, b and c are real numbers, delta is a flat delay parameter,
adjusting the value of t, and enabling the nonlinear directional saturated resonance model to be at t = t r And (3) when the point position reaches resonance, calculating a characteristic value F of a resonance state:
m5: and averaging the m characteristic values F to obtain an average value which is the value of the characteristic value Y.
The vibrio parahaemolyticus detection device is provided with m first working electrodes which do not participate in detection and m second working electrodes which do not participate in detection, in the process of detecting the vibrio parahaemolyticus suspension for m times, one first working electrode which does not participate in detection and one second working electrode which does not participate in detection are adopted in each detection, and the first working electrode and the second working electrode which participate in the detection for one time are not used for detection any more.
The n different scan rates include 50mV/s, 100mV/s, 200mV/s, 300mV/s, 400mV/s, and 500mV/s.
Claims (4)
1. The detection device comprises a computer (1), a data acquisition unit (2), an electrochemical workstation (3), a first detection mechanism and a second detection mechanism, wherein the first detection mechanism comprises a first base (4), a first detection container (5), a first detection module and a first lifter (6) for driving the first detection module to lift are arranged on the first base (4), the first detection module comprises a first guide rail (7), a first sliding block (8) capable of sliding along the first guide rail (7) and a first driving module (9) for driving the first sliding block (8) to slide, the bottom of the first sliding block (8) is connected with a first support (10), m first connecting seats (15) are arranged at equal intervals from left to right at the bottom of the first support (10), a first working electrode (11) is detachably connected to the first connecting seat (15), a first counter electrode (12) and a first reference electrode (13) are detachably connected to the bottom of the first guide rail (7), the first counter electrode (12) and the first reference electrode (13) are arranged above the first connecting seats, the first counter electrode (12) and the first counter electrode (13), the first counter electrode (14) is electrically connected to a plurality of first data selector (14) respectively, and the first counter electrode (14) is electrically connected to a plurality of first counter electrodes (14), the first connecting seat (15) is electrically connected with a first working electrode (11) which is connected with the first connecting seat, the second detection mechanism comprises a second base (16), a second detection container (17), a second detection module and a second lifter (18) which drives the second detection module to lift are arranged on the second base (16), the second detection module comprises a second guide rail (19), a second sliding block (20) which can slide along the second guide rail (19) and a second driving module (21) which drives the second sliding block (20) to slide, the bottom of the second sliding block (20) is connected with a second support (22), m second connecting seats (27) are arranged at equal intervals from the bottom of the second support (22) to the right, a second working electrode (23) is detachably connected on the second connecting seat (27), a second counter electrode (24) and a second reference electrode (25) are detachably connected to the bottom of the second guide rail (19), the second counter electrode (24) and the second reference electrode (25) are positioned right above the second detection container (17), a second multiplexer (26) is arranged on the second support (22), and a plurality of second working electrode (26) and a plurality of data input ends (26) are respectively electrically connected with the second lifter (27), the electrochemical workstation (3) is respectively and electrically connected with the output end of a first multi-channel data selector (14), a first pair of electrodes (12), a first reference electrode (13), the output end of a second multi-channel data selector (26), a second pair of electrodes (24), a second reference electrode (25) and the input end of a data acquisition unit (2), the computer (1) is respectively and electrically connected with the output end of the data acquisition unit (2), a first lifter (6), a second lifter (18), a first driving module (9), a second driving module (21), the control end of the first multi-channel data selector (14) and the control end of the second multi-channel data selector (26), vibrio parahemolyticus antigens are not cultured on the first working electrode (11), vibrio parahemolyticus antigens are cultured on the second working electrode (23), and the vibrio parahemolyticus antigens are characterized by comprising the following steps of:
s1: preparing vibrio parahaemolyticus suspension liquid with different concentrations, detecting characteristic values Y corresponding to the vibrio parahaemolyticus suspension liquid with each concentration, and fitting the values to obtain a concentration calculation formula: y =0.2+ 0.1X LgX, X is the concentration of vibrio parahaemolyticus;
s2: taking the vibrio parahaemolyticus suspension to be detected, detecting the corresponding characteristic value Y, and calculating according to a concentration calculation formula: y =0.2+0.1 × LgX, and calculating the concentration of the vibrio parahaemolyticus suspension to be detected;
the method for detecting the characteristic value Y corresponding to the vibrio parahaemolyticus suspension with a certain concentration comprises the following steps of:
m1: dripping 2.5 mu l of vibrio parahaemolyticus suspension with the concentration on the m first working electrodes which do not participate in the detection on the first detection module and standing for 25 minutes, dripping 2.5 mu l of vibrio parahaemolyticus suspension with the concentration on the m second working electrodes which do not participate in the detection on the second detection module and standing for 25 minutes;
m2: the vibrio parahaemolyticus suspension with the concentration is detected for m times by the same method, and the detection steps of each detection are as follows:
inserting a first pair of electrodes, a first reference electrode and a first working electrode which does not participate in detection into a buffer solution in a first detection container, wherein the first pair of electrodes, the first reference electrode and the first working electrode which does not participate in detection form a first electrochemical sensor array;
inserting a second counter electrode, a second reference electrode and a second working electrode which does not participate in detection into a buffer solution in a second detection container, wherein the second counter electrode, the second reference electrode and the second working electrode which does not participate in detection form a second electrochemical sensor array;
sequentially switching n different scanning rates from small to large by adopting cyclic voltammetry to detect to obtain n reduction peak current difference values delta Ip w1 、ΔIp w2 …ΔIp wn ，ΔIp wn ＝Ip1 wn -Ip2 wn ，ΔIp wn Is the reduction peak current difference at the nth scan rate in the w-th test, ip1 wn Is the reduction peak current value corresponding to the first electrochemical sensor array under the nth scanning speed in the w detection, ip2 wn The reduction peak current value corresponding to the second electrochemical sensor array under the nth scanning speed in the w detection is more than or equal to 1 and less than or equal to m;
m3: constructing a feature matrix D according to the detection data of m times of detection,
m4: performing quadratic spline interpolation on each line of data of the characteristic matrix D to obtain m curves x (t) corresponding to each line of data, performing the same data processing on the m curves x (t) to obtain m characteristic values F, wherein the data processing on each curve x (t) comprises the following steps: substituting x (t) into a nonlinear directional saturated resonance model
the detection signal loading component, ing (t) = cos (kt + η) + x (t),
wherein t is an interpolation variable, k is a real parameter, η is a real parameter, nois (t) is colored noise with uneven power spectral density function, P is a real parameter, Q is a real parameter, a, b and c are real numbers, delta is a flat delay parameter,
adjusting the value of t, the nonlinear directional saturation resonance model is at t = t r And (3) when the point position reaches resonance, calculating a characteristic value F of a resonance state:
m5: averaging the m characteristic values F to obtain an average value which is the value of the characteristic value Y; the method for culturing the vibrio parahaemolyticus antigen on the second working electrode comprises the following steps:
n1: mixing silver paste SL and sodium alginate SA according to the mass ratio of 1: 3 to prepare 10ml of sol water solution, then carrying out ultrasonic dispersion for 15min, and weighing 5mg of graphene oxide GO and 1mg of amino-functionalized organic metal framework material NH 2 dissolving-CuBTC in sol water solution, and performing ultrasonic oscillation for 25min to obtain GO/NH 2 The mixture of-CuBTC/SL/SA was taken at 1.5. Mu.L of GO/NH 2 Dropping the mixed solution of-CuBTC/SL/SA on the surface of the second working electrode, drying for 5h at room temperature, and forming a layer of GO/NH on the second working electrode 2 -a film of CuBTC/SL/SA;
n2: diluting the vibrio parahaemolyticus polyclonal antibody solution by 300 times by using PBS buffer solution, then modifying 4 mu l of the diluted vibrio parahaemolyticus polyclonal antibody solution on the dried second working electrode, and standing and drying the modified vibrio parahaemolyticus polyclonal antibody solution in a drying dish;
n3: dripping 3.5 μ l of vibrio parahaemolyticus antigen solution on the second working electrode, culturing at 30 deg.C for 25min, washing off uncombined vibrio parahaemolyticus antigen on the second working electrode with distilled water, and air drying;
the preparation method of the vibrio parahaemolyticus antigen solution comprises the following steps: the concentration is 2 x 10 8 cfu/ml～2×10 9 Inactivating cfu/ml vibrio parahaemolyticus with 8% -12% formalin at 25-39 deg.C, centrifuging to remove formalin after inactivation, coating flat plate for aseptic test, precipitating with equal volume of aseptic physiologicalAnd (4) resuspending the saline water so as to obtain the vibrio parahaemolyticus antigen solution.
2. The detection method of the Vibrio parahemolyticus detection device for non-disease diagnosis purpose according to claim 1, wherein the first working electrode (11) and the second working electrode (23) are both copper film electrodes, the first counter electrode (12) and the second counter electrode (24) are both Pt electrodes, and the first reference electrode (13) and the second reference electrode (25) are both Ag/AgCl electrodes.
3. The method of claim 1, wherein the Vibrio parahaemolyticus polyclonal antibody solution is prepared by the following steps:
after the rabbits are raised for 2 weeks, 15ml of blood is collected from the ear veins, and serum is taken out to be used as a negative serum sample; immunizing rabbit with vibrio parahaemolyticus antigen, performing second immunization at intervals of 3 days, performing booster immunization at intervals of 6 days, performing carotid artery one-time blood sampling at 4 days after booster immunization, standing at room temperature for 45min, transferring to 4 deg.C overnight, and centrifuging at 4 deg.C and 5000rpm for 45min the next day to obtain antiserum for storage;
fixing 1.5ml affinity chromatographic column on a protein purifier, washing out protective agent solution by deionized water, balancing the column by PBS buffer solution, loading 1.5ml antiserum sample on the column, eluting impurities by PBS buffer solution, and finally eluting vibrio parahaemolyticus polyclonal antibody by citric acid buffer solution to obtain vibrio parahaemolyticus polyclonal antibody solution.
4. The method for detecting the Vibrio parahaemolyticus detection device for non-disease diagnostic purposes as claimed in claim 1, wherein the n different scan rates include 50mV/s, 100mV/s, 200mV/s, 300mV/s, 400mV/s, and 500mV/s.
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