阅读说明：本技术 一种基于绝缘微球浓度变化导致微通道电阻改变的生物传感检测方法 (Biosensing detection method based on microchannel resistance change caused by concentration change of insulating microspheres ) 是由 陈翊平 何慧禹 聂荣彬 王知龙 于 2020-11-09 设计创作，主要内容包括：本发明公开了一种基于绝缘微球浓度变化导致微通道电阻改变的生物传感检测方法,将生物识别分子分别修饰在磁珠和绝缘微球的表面,然后加入待测目标物进行生物反应,磁分离后,在微通道两端施以恒定的电压或电流,反应液中的绝缘微球在电渗流的作用下通过微通道并产生明显的阻塞效应,从而导致微通道电阻的改变,进而使电流或电压发生改变,而电流或电压的改变量又与绝缘微球的浓度相关,据此原理可检测绝缘微球浓度并间接得到待测目标物含量。本发明实现了对一系列目标物的检测,展现出良好的分析性能,而且设备成本低、简单高效,在体外诊断、食品安全、环境监测等领域具有很好的应用前景。(The invention discloses a biosensing detection method based on the change of the concentration of insulating microspheres to cause the change of the resistance of a micro-channel, which is characterized in that biological recognition molecules are respectively modified on the surfaces of magnetic beads and the insulating microspheres, then a target object to be detected is added for biological reaction, after magnetic separation, constant voltage or current is applied to two ends of the micro-channel, the insulating microspheres in reaction liquid pass through the micro-channel under the action of electroosmotic flow and generate obvious blocking effect, thereby causing the change of the resistance of the micro-channel, further causing the current or voltage to change, and the change of the current or voltage is related to the concentration of the insulating microspheres, thus the concentration of the insulating microspheres can be detected according to the principle and the content of the target object to be detected can be indirectly obtained. The invention realizes the detection of a series of target objects, shows good analysis performance, has low equipment cost, is simple and efficient, and has good application prospect in the fields of in-vitro diagnosis, food safety, environmental monitoring and the like.)

1. A biosensing detection method based on the change of the resistance of a micro-channel caused by the change of the concentration of insulating microspheres is characterized by comprising the following steps:

(1) respectively modifying the surfaces of the magnetic beads and the insulating microspheres with biological recognition molecules, and then adding a target object to be detected to perform biological reaction;

(2) carrying out magnetic separation on the reacted solution, taking the separated mixed solution, wherein the concentration of the unreacted insulating microspheres in the mixed solution is related to the concentration of the target object to be detected;

(3) placing an anode electrode and a cathode electrode at two ends of a micro-channel and applying constant voltage or current to form a closed-loop circuit, enabling mixed liquid to flow through the micro-channel arranged between the anode electrode and the cathode electrode under the driving of electroosmotic flow, enabling insulating microspheres to generate a blocking effect when the insulating microspheres flow through the micro-channel to cause the resistance of the closed-loop circuit to rise, enabling the rising amplitude to be positively correlated with the concentration of the insulating microspheres, and calculating the concentration of the insulating microspheres and indirectly obtaining the content of a target object to be detected by detecting the change value of the current or the voltage of the closed-loop circuit;

wherein the micro-channel has an inner diameter of 10-500 μm and a length of 0.1-5 mm.

2. The biosensor detection method of claim 1, wherein: the biological recognition molecule is an antibody and an antigen thereof which can generate competitive immune reaction with a target object to be detected; or the biological recognition molecule is a coated antibody and a labeled antibody thereof which can generate double-antibody sandwich immunoreaction with a target object to be detected; or the biological recognition molecule is a pair of probes capable of generating DNA molecular hybridization reaction with the target object to be detected.

3. The biosensor detection method of claim 1, wherein: the microchannel has an inner diameter of 75 μm and a length of 2 mm.

4. The biosensor detection method of claim 1, wherein: the insulating microspheres are polystyrene microspheres or polybutadiene microspheres or polyisoprene microspheres.

5. The biosensor detection method of claim 1, wherein: the particle size of the insulating microspheres is 0.1-50 microns, wherein when the particle size of the insulating microspheres is 1 micron, the mass ratio of the insulating microspheres to the magnetic beads is 1: 1; when the particle size of the insulating microspheres is 3 micrometers, the mass ratio of the insulating microspheres to the magnetic beads is 1: 3.

6. The microchannel-based electrochemical biosensor assay of claim 5, wherein: the voltage applied to the closed loop circuit is 1-1000 v, preferably 200 v.

7. A biosensing detection device based on microchannel resistance change caused by concentration change of insulating microspheres is characterized by comprising a reaction device and a conductive pool which are communicated through a pipeline, wherein a magnetic separator is arranged outside the reaction device, a valve is arranged on the pipeline, the conductive pool is composed of a first conductive pool and a second conductive pool, a microchannel is arranged between the first conductive pool and the second conductive pool, the inner diameter of the microchannel is 10-500 mu m, the length of the microchannel is 0.1-5 mm, a positive electrode and a negative electrode are respectively arranged inside the first conductive pool and the second conductive pool, the positive electrode and the negative electrode are respectively connected with a positive electrode and a negative electrode of a direct-current power supply through wires to form a closed-loop circuit, and a detection instrument and a power output controller are arranged on the closed-loop circuit.

8. The biosensing detection device of claim 7, wherein: and a piston is arranged on the reaction device.

9. A micro-fluidic chip for biosensing detection, includes the chip body, its characterized in that: be equipped with reaction channel and microchannel on the chip body, microchannel's internal diameter is 10 ~ 500 mu m, and length is 0.1 ~ 5mm, be equipped with the separation tank between reaction channel and the microchannel, the separation tank is equipped with magnetic separator outward, microchannel's both ends are equipped with first conductive cell and the conductive cell of second, separation tank and first conductive cell intercommunication, be equipped with positive electrode and negative electrode in first conductive cell and the second conductive cell respectively, positive electrode and negative electrode pass through the wire and establish and be connected at chip body outside DC power supply's positive, negative pole, form closed loop circuit, still be equipped with detection instrument and power output controller on the closed loop circuit, reaction channel's end is equipped with two introduction ports, two introduction ports and the peristaltic pump intercommunication of establishing at chip body outside.

10. Use of the biosensing detection device of claim 7 or 8, or the microfluidic chip of claim 9 in biosensing detection.

Technical Field

The invention belongs to the field of biosensing, relates to a biosensing detection method, in particular to a biosensing detection method based on the change of the resistance of a microchannel caused by the change of the concentration of insulating microspheres, and also relates to a biosensing detection device and a microfluidic chip for biosensing detection.

Background

The development of the on-site rapid detection technology with excellent performance, low cost and simple and convenient operation plays an important role in the aspects of food safety, disease diagnosis, environmental monitoring and the like. In recent years, biosensors have attracted much attention in the field of rapid on-site detection due to their advantages such as good sensitivity and specificity, and easy integration. Biosensors often use biomolecules (e.g., antigens, antibodies, nucleic acids) as recognition elements, which are converted into detectable signals by a transducer. Heretofore, signal readout methods such as optical, electrochemical, and magnetic are still widely used. However, for rapid detection in the field, these methods are still limited by such factors as complicated instrumentation, high cost, susceptibility to environmental disturbances, and the need for professional personnel. For example, conventional optical detection means requires a light source, a filter, a photon detector, and an accurate optical path design, which not only increases the complexity of the instrument, but also increases the cost. Electrochemical biosensing methods are also common sensing methods, but the electrodes thereof generally need to be polished and modified, resulting in poor stability and accuracy of the methods. To overcome these limitations, researchers are exploring more efficient and stable signal readout schemes. In recent years, pressure, distance, temperature, even odor, and the like are used as readout signals, and certain advantages are exhibited in the aspect of rapid field detection, but the stability and operability of signal readout methods are not good enough.

In biosensors, the biometric information can be converted into different readout signals using different physicochemical principles. For example, the gas production reaction catalyzed by the nano-enzyme can convert the biological identification information into signals such as air pressure, distance and the like; the heat generated during the enzymatic reaction can be converted into a temperature signal using a thermistor. The signals can be conveniently read by simple equipment such as a pressure gauge, a thermometer and the like, and the biosensor is particularly suitable for constructing a portable biosensor. However, physical parameters like air pressure and temperature are easily interfered by environment, which brings inconvenience to field operation; in addition, the generation of these signals is still dependent on chemical reactions, adding to some extent uncertainty to the results. Therefore, in the field rapid detection technology, an ideal read signal not only has extremely high anti-interference capability, but also has a direct quantitative relation with the biological recognition reaction.

Because the signal of telecommunication is like voltage, resistance, electric current etc. not only measurement is convenient, is difficult for receiving external environment's interference moreover, if can directly change biological identification information into electric current or voltage signal, can greatly reduced sensor's cost, can improve the interference killing feature of instrument simultaneously. Through retrieval, no report of on-site rapid detection technology of current or voltage change caused by biological recognition into a read signal is found at present.

Disclosure of Invention

The invention aims to solve the problems of high cost, easy interference and the like in the field rapid detection of the existing biosensor, provides a biosensing detection method which is low in cost, good in stability and strong in anti-interference capability and is based on the change of the resistance of a micro-channel caused by the concentration change of insulating microspheres, and also aims to provide a biosensing detection device and a microfluidic chip for biosensing detection.

The working principle of the method and the device provided by the invention is as follows:

in micron-sized channels, significant blocking effects occur as the insulating particles pass through. If a constant voltage or current is applied across the channel, the presence of the insulating particles results in a change in resistance, with the magnitude of the change being positively correlated with the concentration of the insulating particles. Therefore, the biological recognition molecules are firstly modified on the surfaces of the magnetic beads and the insulating microspheres respectively, then the target substance to be detected is added for incubation, and the concentration of the unreacted insulating microspheres in the solution is related to the concentration of the target substance through magnetic separation. The solution is placed in a conductive cell, constant voltage or current is applied, electroosmotic flow drives the insulating microspheres to flow through the microchannel, a detection instrument reads the current value or the voltage value, the current value or the voltage value is compared with a blank control group, a current difference value can be obtained, the difference value depends on the concentration of the insulating microspheres, and therefore the concentration of a target object is related, and quantitative analysis can be carried out.

Taking capillary microchannel as an example, the inner wall surface of the capillary microchannel has negative charges, and under the action of high voltage, the positive charges in the solution interact with the negative charges on the inner wall surface of the capillary to form an electric double layer, and the positive charges are solvated, so that the solution in the capillary microchannel is caused to move to the negative pole integrally. If insulating particles are present in the solution, they are driven by electroosmotic flow through the capillary microchannel, and because of the small size difference between the insulating microspheres and the capillary inner diameter, the insulating particles in the capillary cause a significant blocking effect, resulting in an increase in resistance, as shown below.

Resistance of the microchannel without insulating particles present:

resistance of the microchannel in the presence of insulating particles:

R^↑′＝(2arctan(((d/2))/(|((S/π)|(d/2)^↑2))))/(σπ|((S/π)|(d/2)^↑2)))+(l|d)/σS

where l represents the microchannel length, S represents the microchannel cross-sectional area, d represents the insulating particle diameter, and σ represents the conductivity of the solution.

In order to achieve the first object, the present invention provides a biosensing detection method based on changes in the resistance of a microchannel caused by changes in the concentration of insulating microspheres, the method comprising the steps of:

(1) respectively modifying the surfaces of the magnetic beads and the insulating microspheres with biological recognition molecules, and then adding a target object to be detected to perform biological reaction;

(2) carrying out magnetic separation on the reacted solution, and taking the separated solution, wherein the concentration of the unreacted insulating microspheres in the solution is related to the concentration of the target object to be detected;

(3) placing an anode electrode and a cathode electrode at two ends of a micro-channel and applying constant voltage or current to form a closed-loop circuit, enabling mixed liquid to flow through the micro-channel arranged between the anode electrode and the cathode electrode under the driving of electroosmotic flow, enabling insulating microspheres to generate a blocking effect when the insulating microspheres flow through the micro-channel to cause the resistance of the closed-loop circuit to rise, enabling the rising amplitude to be positively correlated with the concentration of the insulating microspheres, and calculating the concentration of the insulating microspheres and indirectly obtaining the content of a target object to be detected by detecting the change value of the current or the voltage of the closed-loop circuit;

wherein the micro-channel has an inner diameter of 10-500 μm and a length of 0.1-5 mm.

The biological recognition molecule is an antibody and an antigen thereof which can generate immune competitive reaction with a target object to be detected; or the biological recognition molecule is a coated antibody and a labeled antibody thereof which can generate double-antibody sandwich immunoreaction with a target object to be detected; or the biological recognition molecule is a pair of probes capable of generating DNA molecular hybridization reaction with the target object to be detected.

Preferably, the microchannel has an inner diameter of 75 μm and a length of 2 mm.

Preferably, the insulating microspheres are polystyrene microspheres or polybutadiene microspheres or polyisoprene microspheres.

Preferably, the particle size of the insulating microspheres is 0.1-50 μm, wherein when the particle size of the insulating microspheres is 1 μm, the mass ratio of the insulating microspheres to the magnetic beads is 1: 1; when the particle size of the insulating microspheres is 3 micrometers, the mass ratio of the insulating microspheres to the magnetic beads is 1: 3.

Preferably, the voltage applied to the closed loop circuit is 1-1000 v, preferably 200 v.

In order to achieve the second purpose, the invention provides a biosensing detection device based on microchannel resistance change caused by concentration change of insulating microspheres, which comprises a reaction device and a conductive pool which are communicated through a pipeline, wherein a magnetic separator is arranged outside the reaction device, a valve is arranged on the pipeline, the conductive pool is composed of a first conductive pool and a second conductive pool, a microchannel is arranged between the first conductive pool and the second conductive pool, the inner diameter of the microchannel is 10-500 mu m, the length of the microchannel is 0.1-5 mm, a positive electrode and a negative electrode are respectively arranged inside the first conductive pool and the second conductive pool, the positive electrode and the negative electrode are respectively connected with a positive electrode and a negative electrode of a direct current power supply through leads to form a closed loop circuit, and a detection instrument and a power supply output controller are arranged on the closed loop circuit.

Preferably, a piston is arranged on the reaction device.

The invention also provides another microfluidic chip for biosensing detection, which comprises a chip body, the chip body is provided with a reaction channel and a micro-channel, the inner diameter of the micro-channel is 10-500 μm, the length of the micro-channel is 0.1-5 mm, a separation tank is arranged between the reaction channel and the micro-channel, a magnetic separator is arranged outside the separation tank, a first conductive pool and a second conductive pool are arranged at the two ends of the micro-channel, the separation pool is communicated with the first conductive pool, the first conductive pool and the second conductive pool are respectively internally provided with a positive electrode and a negative electrode which are connected with a positive electrode and a negative electrode of a direct current power supply arranged outside the chip body through leads to form a closed loop circuit, still be equipped with detection instrument and power output controller on the closed loop circuit, reaction channel's end is equipped with two introduction ports, two introduction ports and the peristaltic pump intercommunication of establishing at the chip body outside.

The invention has the beneficial effects that:

1) simple operation and low equipment cost: the current or voltage is used as a read signal, the measurement is convenient, the anti-interference capability is strong, complex and expensive instruments are not needed, and the cost is extremely low (300-500 yuan);

2) the sensing principle is efficient and has good accuracy: the change of the current or the voltage is determined by the concentration of the insulating microspheres instead of being caused by chemical reaction, so that the detection result is accurate and stable;

3) the analysis speed is fast: the detection process only involves one-step biochemical reaction, namely the identification and combination of the target object, thereby greatly shortening the detection time;

4) high-throughput detection: because the electrode does not need to be modified, the next sample can be measured immediately after one sample is measured, the detection efficiency is extremely high, and the method is suitable for rapid analysis of a large number of samples;

5) the platform is highly integrated, and the battery supplies power, can realize on-the-spot quick detection.

Drawings

FIG. 1: schematic structure diagram of electrochemical biological sensing device based on microchannel.

FIG. 2: the structure schematic diagram of the microfluidic chip for electrochemical biosensing detection.

FIG. 3: the inner diameters of the micro-channels are respectively 75 micrometers, 100 micrometers and 150 micrometers, and the lengths are 2 mm; the diameter of the PS microspheres is 1 mu m; the correlation between the measured current difference (Δ I) and the log value of PS microsphere concentration was determined at an applied voltage of 200 v.

FIG. 4: the inner diameters of the micro-channels are respectively 75 micrometers, 100 micrometers and 150 micrometers, and the lengths are 2 mm; the diameter of the PS microspheres is 3 mu m; the correlation between the measured current difference (Δ I) and the log value of PS microsphere concentration was determined at an applied voltage of 200 v.

FIG. 5: the inner diameter of the micro-channel is 75 μm, and the lengths are 2, 3 and 4mm respectively; the diameter of the PS microspheres is 1 mu m; the correlation between the measured current difference (Δ I) and the log value of PS microsphere concentration was determined at an applied voltage of 200 v.

FIG. 6: the inner diameter of the micro-channel is 75 μm, and the lengths are 2, 3 and 4mm respectively; the diameter of the PS microspheres is 3 mu m; the correlation between the measured current difference (Δ I) and the log value of PS microsphere concentration was determined at an applied voltage of 200 v.

FIG. 7: the inner diameter of the micro-channel is 75 μm, and the length is 2 mm; the diameter of the PS microspheres is 1 mu m; the correlation between the measured current difference (Δ I) and the log PS microsphere concentration values was determined for applied voltages of 0, 60, 80, 100, 120, 140, 160, 180, 200v, respectively.

FIG. 8: the inner diameter of the micro-channel is 75 μm, and the length is 2 mm; the diameter of the PS microspheres is 3 mu m; the correlation between the measured current difference (Δ I) and the log PS microsphere concentration values was determined for applied voltages of 0, 60, 80, 100, 120, 140, 160, 180, 200v, respectively.

FIG. 9: the inner diameter of the micro-channel is 75 μm, and the length is 2 mm; the linear relation between the current difference (delta I) measured when the diameters of the PS microspheres are respectively 1 μm and 3 μm and the logarithm value of the concentration of the PS microspheres.

FIG. 10: correlation between the measured current difference (Δ I) and the log of chlorpyrifos concentration for different magnetic bead to PS microsphere ratios.

FIG. 11: and (5) detecting a working curve of the chlorpyrifos.

FIG. 12: and detecting the working curve of procalcitonin.

FIG. 13: working curve for detecting salmonella DNA.

Detailed Description

The present invention will be described in further detail with reference to specific examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Description of test materials and related terms

Carboxyl magnetic beads: particle size 1 μm, 10mg/mL, available from Ocean NanoTech, USA.

Carboxylated Polystyrene (PS) microspheres: particle size 1 μm, 3 μm, 50mg/mL were purchased from Bangs Laboratories, Inc.

Chlorpyrifos antibody (3.5mg/mL), Chlorpyrifos-BSA conjugate (5.7 mg/mL): purchased from Shandong blue-Du Biotech, Inc.

Chlorpyrifos standard: available from carbofuran technologies.

Procalcitonin (1.4mg/mL), procalcitonin capture antibody, procalcitonin detection antibody: purchased from abcam.

Salmonella DNA detection probes and capture probes: designed and synthesized by Biotechnology engineering (Shanghai) Inc.

1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC), N-hydroxythiosuccinimide active ester (sulfo-NHS): purchased from Shanghai Aladdin Biotechnology Ltd.

MES, MEST: respectively a 2- (N-morpholine) ethanesulfonic acid buffer solution and a 2- (N-morpholine) ethanesulfonic acid-Tween 20 buffer solution.

Example 1 modification of magnetic beads and PS microspheres with biorecognition molecules

The activation of magnetic beads and PS microspheres and their coupling to biorecognition molecules are performed using conventional methods well known in the art, as follows:

1. activation of magnetic beads

1) 2mg of magnetic beads (average diameter 1 μm) were placed in a centrifuge tube, washed 2 times with 500 μ L of MEST (10mM MES, 0.05% Tween20, pH 6.0), and the supernatant removed by magnetic separation;

2) 5mg/mL EDC solution and 5mg/mL NHS solution were prepared in 10mM MES (pH 6.0);

3) respectively adding 100 mu L EDC (5mg/mL) and 50 mu L NHS (5mg/mL) into a centrifuge tube filled with magnetic beads, uniformly mixing the mixture by using a vortex device to fully suspend the magnetic beads, diluting the mixture to 500 mu L by MES, placing the mixture on a rotary mixer, and activating the mixture for 30min at 37 ℃;

4) performing magnetic separation, removing supernatant, adding 500 mu L MEST, and transferring the magnetic beads to a new centrifuge tube;

5) magnetic separation, removing supernatant, washing with 500. mu.L MEST 2 times, magnetic separation, removing supernatant.

The carboxyl on the surface of the magnetic beads is activated through the steps.

2. Activation of PS microspheres

1) Taking 2mg PS microspheres (with the average diameter of 1-10 mu m) to be placed in a centrifuge tube, washing for 2 times by using 500 mu L MEST (10mM MES, 0.05% Tween20, pH 6.0), centrifuging (10000rpm, 6min), and removing supernatant;

2) 5mg/mL EDC solution and 5mg/mL NHS solution were prepared in 10mM MES (pH 6.0);

3) respectively adding 100 mu L EDC (5mg/mL) and 50 mu L NHS (5mg/mL) into a centrifuge tube filled with PS microspheres, uniformly mixing by using a vortex device to fully suspend the PS microspheres, diluting to 500 mu L by using MES, placing on a rotary mixer, and activating for 30min at 37 ℃;

4) centrifuging (10000rpm, 6min), removing supernatant, washing 3 times with 500. mu.L MEST;

5) centrifugation (10000rpm, 6min) was carried out to remove the supernatant.

Through the steps, the carboxyl on the surface of the PS microsphere is activated.

3. Coupling of magnetic beads to biorecognition molecules

The coupling process is illustrated by taking a chlorpyrifos antibody as an example, and other biological recognition molecules including procalcitonin antibody, amino-modified DNA probe and the like can be used in a similar way.

1) Adding 100 mu g of chlorpyrifos antibody into the centrifuge tube filled with the magnetic beads, adjusting the total volume to 500 mu L by PBST, and shaking gently to mix the magnetic beads and the antibody;

2) placing the mixture on a rotary blending machine, and reacting for 3h at 37 ℃;

3) performing magnetic separation, removing supernatant, adding 500 μ L PBST (pH 7.4) containing 1% BSA, resuspending magnetic beads, placing on a rotary mixer, and sealing at 37 deg.C for 30 min;

4) magnetic separation, removing supernatant, washing with 500 μ L PBST for 3 times;

5) magnetic separation, removal of supernatant, the resulting chlorpyrifos antibody modified magnetic beads using 1mL PBST (pH 7.4, containing 0.02% NaN)₃0.5% BSA) and stored at 4 ℃.

4. Coupling of PS microspheres to biorecognition molecules

The coupling process is illustrated by using a BSA-chlorpyrifos conjugate as an example, and other biological recognition molecules including procalcitonin antibodies, amino-modified DNA probes, and the like can be similarly used.

1) Adding 100 mu g of BSA-chlorpyrifos into the centrifuge tube filled with the PS microspheres, adjusting the total volume to 500 mu L by PBST, and uniformly mixing the PS microspheres and the BSA-chlorpyrifos by gentle shaking;

2) placing the mixture on a rotary blending machine, and reacting for 3h at 37 ℃;

3) centrifuging (10000rpm, 6min), removing supernatant, adding 1% BSA-containing PBST (pH 7.4)500 μ L, resuspending PS microspheres, placing on a rotary mixer, and sealing at 37 deg.C for 30 min;

4) centrifuging (10000rpm, 6min), removing supernatant, washing 3 times with 500. mu.L PBST;

5) centrifuging (10000rpm, 6min), removing supernatant, and subjecting the obtained BSA-chlorpyrifos-modified PS microspheres to 1mL of PBST (pH 7.4, containing 0.02% NaN)₃0.5% BSA) and stored at 4 ℃.

EXAMPLE 2 construction of biosensing detection device and microfluidic chip

Referring to fig. 1, the apparatus comprises a reaction apparatus 1 and a conducting cell 2.

The reaction device 1 is a place for carrying out biological reaction, a heat preservation and stirring device can be arranged, a magnetic separator 5 is arranged outside the reaction device 1 and used for separating magnetic beads after reaction, and a piston 6 is further arranged on the reaction device 1 in order to accelerate the outflow of reaction solution.

The conductive pool 2 is used for containing the separated solution after reaction and detecting an electric signal, is communicated with the reaction device 1 through a pipeline 7 arranged at the bottom, and is provided with a valve 8 on the pipeline 7.

The conductive pool 2 is composed of a first conductive pool 3 and a second conductive pool 4, the first conductive pool 3 is communicated with the second conductive pool 4 through a micro-channel 9 arranged at the bottom, the inner diameter of the micro-channel 9 is 10-500 mu m, the length of the micro-channel is 0.1-5 mm, a positive electrode 10 and a negative electrode 11 are respectively arranged inside the first conductive pool 3 and the second conductive pool 4, a direct current power supply 12 is arranged outside the first conductive pool 3 and the second conductive pool 4, the positive electrode 10 and the negative electrode 11 are respectively connected with the positive electrode and the negative electrode of the direct current power supply 12 through wires to form a closed loop circuit, and a detection instrument 14 is arranged on the closed loop circuit and used for detecting and displaying voltage, current or resistance on the circuit. And the closed loop circuit is also provided with a power output controller 13 for controlling the voltage or current output of the direct current power supply.

The device can also be realized by a micro-fluidic chip, the structure of which is shown in fig. 2, a schematic diagram of a chip body is shown in a dotted line in the diagram, a reaction channel 16 and a micro-channel 9 are arranged on the chip body, a separation pool 15 is arranged between the reaction channel 16 and the micro-channel 9, a magnetic separator 5 is arranged outside the separation pool 15, a first conductive pool 3 and a second conductive pool 4 are arranged at two ends of the micro-channel 9, the separation pool 15 is communicated with the first conductive pool 3, a positive electrode 10 and a negative electrode 11 are respectively arranged in the first conductive pool 3 and the second conductive pool 4, the positive electrode 10 and the negative electrode 11 are connected with a positive electrode and a negative electrode of a direct current power supply 12 arranged outside the micro-fluidic chip through leads to form a closed loop circuit, and a detection instrument 14 and a power output controller 13 are further arranged on the closed loop circuit. The tail end of the reaction channel 16 is provided with two sample inlets 17, and the two sample inlets 17 are communicated with a peristaltic pump 18 arranged outside the microfluidic chip. That is, the reaction channel 16, the separation pool 15, the magnetic separator 5 and the peristaltic pump 18 on the chip together form a reaction device.

Example 3 investigation of the quantitative relationship between PS microsphere concentration and Electrical resistance

Adding PS microsphere solutions with different concentrations and diameters into the first conductive pool, switching on a power supply, enabling the solutions to flow through microchannels with different inner diameters between the first conductive pool and the second conductive pool under the driving of electroosmotic flow, detecting currents of a closed-loop circuit under different voltages, and inspecting the relationship between the PS microsphere concentration and the resistance.

Run 1): the inner diameters of the micro-channels are respectively 75 micrometers, 100 micrometers and 150 micrometers, and the lengths are 2 mm; the PS microspheres have a diameter of 1 μm and a concentration of 10³、10⁴、10⁵、10⁶、10⁷、10⁸pg/mL; the applied voltage was the current value measured at 200 v. As can be seen from the results in FIG. 3, there is a clear positive correlation between the difference in current (Δ I) measured for the three inner diameter microchannels and the log of PS microsphere concentration, with the best results when the inner diameter of the microchannel is 75 μm.

Run 2): the inner diameters of the micro-channels are respectively 75 micrometers, 100 micrometers and 150 micrometers, and the lengths are 2 mm; the PS microspheres have a diameter of 3 μm and a concentration of 10³、10⁴、10⁵、10⁶、10⁷、10⁸pg/mL; the applied voltage was the current value measured at 200 v. As can be seen from the results in FIG. 4, there is a clear positive correlation between the difference in current (Δ I) measured for the three inner diameter microchannels and the log of PS microsphere concentration, with the best results when the inner diameter of the microchannel is 75 μm.

Run 3): the inner diameter of the micro-channel is 75 μm, and the lengths are 2, 3 and 4mm respectively; the PS microspheres have a diameter of 1 μm and a concentration of 10³、10⁴、10⁵、10⁶、10⁷、10⁸pg/mL; the applied voltage was the current value measured at 200 v. As can be seen from the results of FIG. 5, the three lengthsThere was a clear positive correlation between the current difference (Δ I) measured in the microchannel and the log PS microsphere concentration, with the best results when the length of the microchannel was 2 mm.

Run 4): the inner diameter of the micro-channel is 75 μm, and the lengths are 2, 3 and 4mm respectively; the PS microspheres have a diameter of 3 μm and a concentration of 10³、10⁴、10⁵、10⁶、10⁷、10⁸pg/mL; the applied voltage was the current value measured at 200 v. As can be seen from the results in FIG. 6, there is a clear positive correlation between the difference in current (Δ I) measured for the three lengths of microchannel and the log PS microsphere concentration, with the best results being obtained when the microchannel length is 2 mm.

Run 5): the inner diameter of the micro-channel is 75 μm, and the length is 2 mm; the PS microspheres have a diameter of 1 μm and a concentration of 10³、10⁴、10⁵、10⁶、10⁷、10⁸pg/mL; the applied voltages were current values measured at 0, 60, 80, 100, 120, 140, 160, 180, 200v, respectively. From the results of fig. 7, it can be seen that there is a clear positive correlation between the difference in current (Δ I) measured in the microchannel and the log of PS microsphere concentration when different voltages are applied, with the best results when 200v is applied.

Run 6): the inner diameter of the micro-channel is 75 μm, and the length is 2 mm; the PS microspheres have a diameter of 3 μm and a concentration of 10³、10⁴、10⁵、10⁶、10⁷、10⁸pg/mL; the applied voltages were current values measured at 0, 60, 80, 100, 120, 140, 160, 180, 200v, respectively. From the results of fig. 8, it can be seen that there is a clear positive correlation between the difference in current (Δ I) measured in the microchannel and the log of PS microsphere concentration when different voltages are applied, with the best results when 200v is applied.

According to the test results, the PS microsphere concentration and the resistance of the micro-channel have good correlation, and the PS microsphere concentration can be obtained through detecting electric signals (including current and voltage) of a circuit, so that a theoretical basis is laid for the application of the invention. Wherein the effect is best when the microchannel has an inner diameter of 75 μm and a length of 2mm and the applied voltage is 200v, as shown in FIG. 9,under the condition, the current difference (delta I) has a good linear relation with the concentration logarithm value of the PS microspheres. In addition, R is relative to 1 μm PS microspheres (Y ═ 3.86X-10.40, R²0.99), when 3 μm PS microspheres were used (Y5.61X-16.22, R²0.99), the sensitivity is higher.

Example 4 detection of chlorpyrifos

Before the chlorpyrifos is detected, the concentrations of the PS microspheres modified by the chlorpyrifos-BSA conjugate and the magnetic beads modified by the chlorpyrifos antibody are optimized. Specifically, the inner diameter of the microchannel is 75 μm, and the length is 2 mm; the diameters of the PS microspheres are 1 and 3 mu m respectively; the applied voltage was 200 v; magnetic beads: the mass ratios of the PS microspheres are 1:2, 1:1, 2:1, 3:1 (the diameter of the PS microspheres is 1 μm) and 1:1, 2:1, 3:1 and 4:1 (the diameter of the PS microspheres is 3 μm). As can be seen from the results of fig. 10, the use of different ratios of PS microspheres and magnetic beads has a significant effect on the correlation between the current difference (Δ I) and the chlorpyrifos concentration, wherein when the diameter of the PS microspheres is 1 μm, the magnetic beads: the PS microspheres have the best effect when the ratio is 1: 1; and when the diameter of the PS microsphere is 3 mu m, the magnetic bead: the PS microspheres have the best effect when the ratio is 3: 1.

The chlorpyrifos in the citrus is detected, and the specific process is as follows:

1) diluting magnetic beads modified by chlorpyrifos antibody to 300 mu g/mL by PBS;

2) preparing 1mg/mL chlorpyrifos standard stock solution by using ethanol, and preparing standard working solutions with the concentrations of 0, 0.01, 0.1, 1, 10, 100 and 1000ng/mL respectively;

3) respectively adding 100 mu L of magnetic bead diluent (300 mu g/mL) and 100 mu L of chlorpyrifos standard solution (0, 0.01, 0.1, 1, 10, 100 and 1000ng/mL) into the reaction device, and incubating for 5-20 min;

4) diluting the PS microspheres modified by BSA-chlorpyrifos to 100 mu g/mL by using PBS;

5) adding 100 mu L of PS microsphere (diameter is 3 mu m) diluent (100 mu g/mL) into a reaction device, and incubating for 5-20 min;

6) applying a magnetic field outside the reaction device to adsorb the magnetic beads after reaction, simultaneously pressing a piston and opening a valve to enable the unbound PS microspheres-BSA-chlorpyrifos to enter a first conductive pool;

7) adding 300 mu L of PBS solution into the second conductive cell, controlling the output of a direct current power supply, applying 200V direct current voltage to the electrode, moving the solution to the cathode through the micro-channel (with the inner diameter of 75 mu m and the length of 2mm) integrally under the action of electroosmotic flow, wherein the PS microspheres and the micro-channel have the same order of magnitude, and when passing through, obvious blocking effect is generated, so that the resistance is increased, and at the moment, a detector can be used for recording current, and the current difference value can be obtained by comparing with a blank group;

8) taking the logarithmic value of the chlorpyrifos concentration as the abscissa and the current difference value as the ordinate, and making a standard curve as shown in fig. 11;

9) chlorpyrifos residues in citrus samples were tested using standard addition methods, as described above, with the results shown in table 1.

The principle of the chlorpyrifos detection method is as follows: in the reaction device, the magnetic beads modified by the chlorpyrifos antibody, the PS microspheres modified by the chlorpyrifos antigen and the chlorpyrifos in the sample are subjected to immune competition reaction, and when no chlorpyrifos exists in the sample, the magnetic beads modified by the chlorpyrifos antibody and the PS microspheres modified by the chlorpyrifos antigen are combined to generate a compound, so that the unreacted chlorpyrifos antigen modified PS microspheres in the solution are reduced; when the sample contains chlorpyrifos, the magnetic beads modified by the chlorpyrifos antibody, the PS microspheres modified by the chlorpyrifos antigen and the chlorpyrifos in the sample are competitively combined to generate a compound, so that the unreacted chlorpyrifos antigen-PS microspheres in the solution are increased. Therefore, the concentration of chlorpyrifos in the sample is positively correlated with the concentration of unreacted chlorpyrifos antigen-PS microspheres. And (3) carrying out magnetic separation on the compound, and indirectly obtaining the chlorpyrifos concentration in the sample by detecting the concentration of unreacted chlorpyrifos antigen-PS microspheres.

TABLE 1 results of chlorpyrifos in citrus using standard addition method

In addition, from the aspect of analysis performance, including sensitivity, stability, analysis time and the like, the detection method is compared with the traditional ELISA method, and the result is shown in Table 2, which shows that the method not only has higher sensitivity, but also has better stability, and the analysis time is shortened to be within 0.5 hour from 2-3 hours.

TABLE 2 comparison of the analytical Performance of the method and ELISA for Chlorpyrifos

Analysis of Performance	Detection limit (pg/mL)	RSD(％)	Analysis time (min)
				Method for producing a composite material	2.85	＜8.0	＜30
ELISA	250	＜12.0	＞120

Example 5 detection of Procalcitonin

In order to verify that the method is also applicable to macromolecules, an inflammation marker Procalcitonin (PCT) in human serum is detected, and the method comprises the following steps:

1) PCT-coated antibody-modified magnetic beads (2mg/mL) were diluted to 300. mu.g/mL with PBS;

2) preparing 1mg/mL PCT standard stock solution by using PBS (phosphate buffer solution), and preparing PCT standard solution with the concentration of 0, 0.01, 0.1, 1, 10 and 100ng/mL respectively;

3) respectively adding 100 mu L of magnetic bead diluent (300 mu g/mL) and 100 mu L of PCT standard solution (0, 0.01, 0.1, 1, 10 and 100ng/mL) into the reaction device, and incubating for 5-20 min;

4) performing magnetic separation, removing supernatant, adding 100 mu L of PS microspheres (the diameter is 3 mu m, and the concentration is 100 mu g/mL) modified by the PCT labeled antibody, and incubating for 5-20 min;

5) magnetically separating, and simultaneously pressing a piston to enable the unbound PS microspheres to enter a first conductive pool;

6) adding 100 mu L of PBS solution into the second conductive pool, applying 200V direct current voltage to the first conductive pool and the second conductive pool, and simultaneously recording current;

7) taking the logarithm of the PCT concentration as the abscissa and the current difference as the ordinate, making a standard curve, as shown in fig. 12;

8) PCT in human serum samples was tested by standard addition as described above and the results are shown in Table 3.

The principle of the procalcitonin detection method is as follows: in the reaction device, magnetic beads modified by the PCT coating antibody, PS microspheres modified by the PCT labeled antibody and PCT in a sample are subjected to double-antibody sandwich immunoreaction to generate a magnetic bead-coating antibody-procalcitonin-labeled antibody-PS microsphere compound. And (3) carrying out magnetic separation on the compound after the unreacted PCT labeled antibody-PS microspheres in the solution are inversely related to the concentration of procalcitonin in the sample, and indirectly obtaining the concentration of the procalcitonin in the sample by detecting the concentration of the unreacted PCT labeled antibody-PS microspheres.

TABLE 3 results of PCT in human serum by standard addition method

Example 6 detection of DNA

The feasibility of the method in detecting DNA is illustrated by taking salmonella as an example.

The method adopts a micro-fluidic chip to detect the salmonella, and comprises the following specific processes:

1) two probes (probes 1 and 2) of the DNA corresponding to the salmonella DNA to be detected are designed and coupled on the surfaces of the magnetic particles and the PS microspheres respectively.

2) Extracting salmonella genome DNA according to a kit method, and amplifying according to the following system: 5.0. mu.L of 10 XPCR buffer, 5.0. mu.L of the above genomic DNA supernatant, 1.0. mu.L (10. mu.M) of each of the upstream and downstream primers, 1.0. mu.L of DNA polymerase (5U/. mu.L), 1.0. mu.L of dNTP (10mM), 3.0. mu.L of MgCl₂And 33. mu.L of ultrapure water. The reaction conditions were as follows: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 1min, and amplification for 30 cycles; extension at 72 ℃ for 10 min.

3) Probe 1 modified magnetic beads (2mg/mL) were diluted to 300 μ g/mL with PBS;

4) mix 100. mu.L of magnetic bead-probe 1 (300. mu.g/mL), 100. mu.L of PS microsphere-probe 2 (100. mu.g/mL), 10. mu.L of sodium citrate solution, 40. mu.L of sterile water; mixing 100 mul of salmonella DNA sample, 10 mul of sodium citrate solution and 40 mul of sterile water; placing the two mixed solutions in 100 deg.C water bath for 10min, and rapidly placing in ice bath for 10 min;

5) injecting the two mixed solutions into a reaction channel through two sample inlets on the micro-fluidic chip respectively, and reacting for 30min at 50 ℃;

6) after the reaction solution is subjected to magnetic separation, the unbound PS microspheres-probes 2 enter a first conductive pool, PBS is added into a second conductive pool, 200v direct current voltage is applied to the first conductive pool and the second conductive pool, and current is recorded at the same time;

7) taking the logarithmic value of the salmonella concentration as the abscissa and the current difference as the abscissa, making a standard curve, as shown in fig. 13;

8) the standard addition method was used to detect salmonella in ham sausages, the procedure was the same, and the results are shown in table 4.

The principle of the salmonella detection method is as follows: in the reaction device, the magnetic beads modified by the probe 1, the PS microspheres modified by the probe 2 and the salmonella DNA in the sample are subjected to DNA molecular hybridization reaction to generate a compound. And (3) after the reaction, the concentration of the PS microspheres modified by the unreacted probe 2 in the solution is in negative correlation with the concentration of the salmonella DNA in the sample, the compound is subjected to magnetic separation, and the concentration of the salmonella DNA in the sample can be indirectly obtained by detecting the concentration of the PS microspheres modified by the unreacted probe 2.

TABLE 4 results of Salmonella detection in ham sausage by the Standard addition method

The current-mode sensing analysis method can also be a voltage-mode sensing method, and the specific principle is as follows: in micron-sized channels, significant blocking effects occur when the insulating particles pass through. If a constant current is applied across the channel, the presence of the insulating particles leads to an increase in resistance, with the magnitude of the increase being positively correlated with the concentration of the insulating particles. According to the principle, biological recognition molecules are firstly modified on the surfaces of magnetic beads and Polystyrene (PS) microspheres respectively, then a target object to be detected is added for incubation, and the concentration of the PS microspheres remaining in the solution is related to the concentration of the target object through magnetic separation. A certain amount of the solution is placed in a conductive cell, current is applied, electroosmotic flow drives the solution to flow through a microchannel, a voltmeter reads a voltage value, the voltage value is compared with a blank control group, a voltage difference value can be obtained, the size of the difference value depends on the concentration of the PS microspheres, and therefore the difference value is related to the concentration of a target object, and quantitative analysis can be carried out.

The applicant states that the technical solutions of the present invention are explained by the above embodiments, but the present invention is not limited to the above embodiments, that is, the present invention does not mean that the present invention must depend on the above specific embodiments to be implemented. Any modification of the invention or equivalent substitution of the materials for the invention chosen by the skilled person is within the scope of protection of the patent.