CN115963262A - Rapid detection method of vibrio cholerae - Google Patents
Rapid detection method of vibrio cholerae Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection methods, and discloses a vibrio cholerae detection method based on a high-sensitivity GMR magnetic biochip, which comprises the following steps: s1, preparing a GMR biochip; s2, capturing vibrio cholerae on a GMR biochip; and S3, testing a GMR biochip and reading out vibrio cholerae information. The invention utilizes the GMR biochip technology based on the magnetic line concentrator, not only effectively improves the detection sensitivity of the GMR biochip to vibrio cholerae, but also the preparation process is compatible with the microfluidic technology, realizes the closed implementation of the whole biochip detection process, and improves the systematicness, the safety and the convenience of the GMR magnetic biochip. The chip technology has the advantages of miniaturization, low power consumption, good device uniformity and the like, and has wide application potential in the fields of biosafety, food quality inspection, disease diagnosis and the like.
Description
Technical Field
The invention relates to a vibrio cholerae detection method based on a high-sensitivity GMR magnetic biochip, and belongs to the technical field of biological detection methods.
Background
Vibrio cholerae belongs to the family Vibrionaceae, gram-negative bacteria. The ecological environment is located in coastal waters and estuaries, commonly associated with zooplankton and shellfish. At present, more than 200 kinds of Vibrio cholerae have been identified based on the variation of "O" antigen, and people are infected by ingesting water or food contaminated by Vibrio cholerae, most of ingested Vibrio cholerae is killed by gastric acid, and the living bacteria colonize in small intestine and produce cholera toxin, resulting in acute watery diarrhea. Despite the current improvements in water quality, hygiene and clinical treatment of cholera, the disease still causes about 10 million deaths worldwide each year. One analysis showed that in countries with an epidemic of cholera, approximately 2900 ten thousand cases and 95000 cases of death occur each year, with africa accounting for 60% and 68%, respectively, and almost all deaths occurring in developing countries. As the control of the cholera epidemic situation still faces a very severe situation in developing countries, it is very important to develop a rapid, accurate, highly specific, simple and highly sensitive detection method in order to improve the food and biological safety in China.
The detection of Vibrio cholerae is generally carried out by bacteriological detection, molecular biological detection and immunological detection. Bacteriological tests, as a more traditional test method, are technically mature but relatively complex and time-consuming to operate; the rapid and accurate detection of the vibrio cholerae can be realized by adopting a molecular biological detection method, but the required cost is higher; immunological detection methods allow rapid and simple detection, but such methods are often used as an aid to detection, and their specificity and sensitivity are relatively high.
The high-sensitivity rapid detection problem of the vibrio cholerae and the long-time preservation problem of the detected sample information. First, vibrio cholerae has a strong pathogenicity. Secondly, the high-sensitivity rapid detection is a key technology for effectively preventing infectious diseases, and the detection method capable of carrying out single-bit-order detection mainly comprises the traditional culture method and a nucleic acid detection method. The traditional culture method needs a long time, and the nucleic acid detection method needs expensive equipment and professional operation, which can not meet the requirements of high sensitivity, rapidness and convenience in detection. Finally, the existing vibrio cholerae detection technology based on the biosensor detection method has the problems of unstable detection signals, poor repeatability and the like, and can not detect again after the sample is detected. Therefore, in order to further improve the reliability and reproducibility of the detection of Vibrio cholerae, it is necessary to solve the problem of long-term preservation of sample information.
With the development of the interdiscipline, the biochip technology is developed, and the advantages of miniaturization, low power consumption, high integration and the like of the biochip make the biochip have huge application potential in the aspect of detecting biological targets such as bacteria, viruses and the like, while the magnetic biochip based on the giant magnetoresistance effect is one member of the biochip, the giant magnetoresistance effect (GMR) is an effect of changing the resistance value of a metal material under the action of an external magnetic field, electronic devices such as magnetic storage, magnetic sensors and the like prepared based on the effect have wide application prospects, the GMR biochip is mainly formed by combining a GMR magnetic sensor based on the giant magnetoresistance effect and an immunomagnetic bead technology, and compared with other biochip technologies (such as fluorescence, electrochemistry and the like), the biochip based on the magnetoelectronics has the advantages of low background noise, good uniformity, easy integration and the like, so that the biochip is very suitable for detecting vibrio cholerae, and the stability of the biochip based on the magnetic bead is good, the storage time is long, and the vibrio detection sample of the vibrio cholerae can be repeatedly detected for many times.
At present, the bacteria detection technology based on GMR biosensor has reached 100CFU/mL magnitude, but compared with other biosensors, such as fluorescent sensor, electrochemical sensor (10 CFU/mL magnitude) and the like, the detection sensitivity has larger difference, so that the application potential of GMR magnetic biochip in vibrio cholerae is limited, in order to solve the problem of long-term storage of vibrio cholerae detection sample information based on magnetic sensor chip, and have higher detection performance, it is necessary to develop a novel GMR biochip technology. The existing GMR biochip technology mainly utilizes a GMR sensor to detect a weak stray magnetic field of a magnetic label to obtain a biological detection function, but the method is limited in that the stray magnetic field generated by the used magnetic label is weak, and the detection with higher sensitivity cannot be realized, so that a new detection method needs to be developed to obtain higher detection capability.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a vibrio cholerae detection method based on a high-sensitivity GMR magnetic biochip, which aims to solve the problems in the prior art.
The technical scheme adopted by the invention is as follows: a vibrio cholerae detection method based on a high-sensitivity GMR magnetic biochip comprises the following steps:
s1, preparing a GMR biochip, wherein a biochemical reaction microfluidic channel is arranged on the GMR magnetic biochip, the biochemical reaction microfluidic channel is an S-shaped microchannel, and the pH value in the S-shaped microchannel is 8.8-9.0;
s2, capturing Vibrio cholerae on a GMR biochip, comprising the steps of:
s2.1, preparing a magnetic targeting label;
s2.2, magnetic modification of vibrio cholerae;
s2.3, performing functional modification on the surface of the GMR biochip;
s2.4, capturing Vibrio cholerae, injecting the Vibrio cholerae modified with the magnetic targeting label based on the antigen-antibody reaction to the surface of a GMR biochip through a microfluidic channel, and capturing the Vibrio cholerae marked by the magnetic label on the surface of the GMR biochip through the antigen-antibody reaction to obtain the GMR biochip after capturing the Vibrio cholerae by the antigen-antibody reaction;
and S3, testing a GMR biochip and reading out vibrio cholerae information.
Preferably, in the step S1, the GMR magnetic biochip includes a plate-shaped substrate, GMR lines are disposed on an upper end surface of the substrate, magnetic line collectors are disposed at positions of the upper end surface of the substrate on two sides of the GMR lines, respectively, and a magnetization direction between the two magnetic line collectors is perpendicular to the GMR lines; the tail parts of the GMR lines are respectively covered with an electrode, and the two electrodes are respectively electrically connected with electrode pins; the upper end surface of the substrate and the upper end surface of the magnetic force line collector are both provided with a silicon nitride protective film, and the silicon nitride protective film on the upper end surface of the GMR lines is also provided with SiO 2 A biochemical reaction film; the electrode bagThe method comprises the following steps of (1) including an electrode bottom layer and an electrode upper layer; the microfluidic channel is arranged on the upper end face of the substrate and positioned at the part around the GMR lines, and the GMR lines and the electrodes are positioned inside the microfluidic channel.
Preferably, the direction of the biochemical reaction microfluidic channel region is perpendicular to the GMR lines and parallel to the external magnetic field.
Preferably, the magnetic labels selected in step S2.1 are magnetic beads with a diameter equal to 1 μm.
Preferably, the GMR lines are of a meander type structure, the lines having a line width of 5 μm, a length of 500 μm and a gap of 15 μm.
Preferably, the magnetic line concentrator is a rectangular FeNi thin film with a thickness of 2 μm.
Preferably, the electrode bottom layer is a Cr/Cu electrode layer, and the thickness is 10nm/150nm.
Preferably, the electrode upper layer is an Au electrode layer and has a thickness of 150nm.
Preferably, the thickness of the silicon nitride protective film is 20nm.
Preferably, siO 2 The thickness of the biochemical reaction film is 10nm.
Preferably, the GMR biochip is fabricated as follows:
the method comprises the following steps: patterning GMR lines, and patterning the GMR lines on the surface of the substrate;
step two: manufacturing a magnetic line concentrator, and patterning the magnetic line concentrator on the surface of the substrate;
step three: manufacturing an electrode bottom layer, carrying out electrode patterning on a substrate, and then plating a Cr/Cu electrode layer;
step four: manufacturing a silicon nitride protective film, namely processing a silicon nitride protective film on the whole surface of a substrate, then patterning an electrode, and etching a silicon nitride layer on the surface of the electrode until a first layer of electrode is exposed;
step five: siO 2 2 Preparing a biochemical reaction film, namely preparing SiO in a graphical reaction area of a GMR line area by utilizing a sputtering process 2 A biochemical reaction membrane;
step six: manufacturing an upper electrode layer, patterning a biochemical reaction film on a GMR line region, patterning an electrode on a substrate, and taking an Au-plated electrode layer as an upper electrode layer;
step seven: and (3) manufacturing a microfluidic channel, and manufacturing the microfluidic channel in a GMR detection area on the surface of the substrate for detecting reaction.
Preferably, the GMR lines are fabricated using an ion beam etching process.
Preferably, the magnetic flux concentrator is prepared by a sputtering process, and the patterning is performed by a liftoff process.
Preferably, the bottom electrode layer is manufactured by a sputtering process, and the upper electrode layer is manufactured by a thermal evaporation process.
Preferably, the silicon nitride protective film is manufactured by a PECVD process.
Preferably, siO 2 The biochemical reaction film is prepared by a sputtering process.
Preferably, the microfluidic channel is prepared by a PDMS process.
Preferably, the step S3 includes the following steps: fixing the GMR biochip without biochemical reaction between the first solenoid and the second solenoid, switching on the GMR biochip and the detection circuit, supplying certain working current to the GMR biochip, applying current to the first solenoid and the second solenoid to enable the GMR region to generate a magnetic field, and recording the voltage value output by the GMR biochip without biochemical reaction; similarly, recording the voltage value output by the GMR biochip after the biochemical reaction; and judging the information of the biological target by the difference value of the two voltage values.
The invention has the beneficial effects that:
1. compared with the prior art, the method based on the magnetic field shunt detection method is mainly characterized in that a static magnetic field is formed in a GMR detection area by using a magnetic line concentrator, then the magnetic field is shunted by using a magnetic label, the magnetic field in the detection area is changed, and the signal change of a GMR device is brought, so that the detection of the magnetic label is realized. The more the number of the magnetic labels is, the more the shunted magnetic field is, the stronger the signal influence on the GMR device is, and the quantitative detection of the biological target is realized. Compared with the traditional GMR biochip, the GMR biochip has the capability of obviously improving the signal intensity, and on the detection limit, the GMR biochip can detect 25CFU/mL (the output value MR of GMR is 109 ppm) at the minimum, and the traditional GMR biochip can only detect 120CFU/mL (the output value MR of GMR is 121 ppm), so that the GMR biochip has the detection sensitivity which is improved by nearly 5 times compared with the traditional detection method, and also has the detection output signal intensity which is improved by nearly 4 times on the same concentration (such as 500CFU/mL concentration); the magnetic force line collector can reduce the external magnetic field required by GMR operation, and the power consumption of the detection system is reduced; secondly, the S-shaped microfluidic channel in the scheme can avoid the problem that vibrio cholerae is settled on the surface of the chip due to overlarge size, and the straight tube direction and the magnetic field excitation direction of the S-shaped microfluidic channel are the same, so that the aggregation of magnetic beads to magnetic lines of force is improved, the detection performance of a GMR biochip is improved, the sample contact is avoided, detection personnel are protected, and the application scene of the GMR biochip in the field of high-sensitivity biological information detection is expanded.
2. Compared with the prior art, the invention not only further improves the detection sensitivity of the GMR biochip, but also has the preparation process which is easily compatible with the existing IC circuit process, can realize the closed implementation of the monitoring process of the whole biochip, and improves the systematicness, the integration and the convenience of the GMR magnetic biochip. The chip technology has the advantages of miniaturization, low power consumption, good device uniformity and the like, and plays a great role in the fields of biosafety, food quality inspection, disease diagnosis and the like.
Drawings
FIG. 1 is a perspective view of a GMR magnetic biochip based on a magnetic line concentrator according to the present invention;
FIG. 2 is a schematic diagram of the process of capturing Vibrio cholerae according to the present invention;
FIG. 3 is a schematic diagram showing the detection of Vibrio cholerae in the present invention;
FIG. 4 is a comparison of the performance of GMR biochips of the invention with conventional GMR biochips.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments.
Reference numerals in the drawings of the specification include: GMR line 1, first magnetic force line collector 2a, second magnetic force line collector 2b, silicon nitride protective film 3a, siO 2 The device comprises a biochemical reaction film 3b, a first electrode pin 4a, a second electrode pin 4b, a microfluidic channel inlet 5a, a microfluidic channel outlet 5b, a microfluidic channel cavity 5c, a substrate 6, a vibrio cholerae antibody (primary antibody) 7, a magnetic label 8, a magnetic targeting label 9, a vibrio cholerae 10, a vibrio cholerae 11 modified with a magnetic targeting label, a GMR biochip 12, a self-assembled film 13, a capturing antibody (secondary antibody) 14 of the vibrio cholerae, a GMR biochip 15 after capturing the vibrio cholerae, a first solenoid 16a, a second solenoid 16b, a magnetic field 16c of a GMR region, a magnetic field 17 for shunting magnetic beads and a test instrument 18.
Example 1:
a vibrio cholerae detection method based on a high-sensitivity GMR magnetic biochip comprises the following steps:
s1, preparing a GMR biochip 12;
s2, capturing the vibrio cholerae 10 on a GMR biochip 12;
s3, testing the GMR biochip 12 and reading out the information of the vibrio cholerae 10.
Preferably, in step S1, the GMR magnetic biochip 12, as shown in fig. 1, includes a plate-shaped substrate 6 (gmrwarp), a GMR line 1 is disposed on an upper end surface of the substrate 6, a first magnetic force line collector 2a and a second magnetic force line collector 2b are respectively disposed at positions of the upper end surface of the substrate 6 on two sides of the GMR line 1, and a magnetization direction between the two magnetic force line collectors is perpendicular to the GMR line 1; the tail part of the GMR line 1 is respectively covered with an electrode, and the two electrodes are respectively and electrically connected with a first electrode pin 4a and a second electrode pin 4b; the upper end surface of the substrate 6 and the upper end surface of the magnetic force line collector are both provided with silicon nitride protective films 3a; a silicon dioxide biochemical reaction film 3b is arranged on the surface of the silicon nitride protection film 3a on the GMR line 1, and a biochemical reaction film 3b is arranged on the upper end surface of the GMR line 1; the electrode comprises an electrode bottom layer and an electrode upper layer; the area of the upper end surface of the substrate 6 around the GMR lines 1 is provided with a micro-fluidic channel cavity 5c, and the GMR lines 1 and the electrodes are positioned in the micro-fluidic channel cavity 5 c.
Wherein, GMR lines 1 are zigzag structure, GMR lines 1 have line width of 5 μm, length of 500 μm, and gap of 15 μm.
Wherein, the magnetic force line collector is a rectangular FeNi film with the thickness of 2 μm.
Preferably, the biochemical reaction microfluidic channel is an S-shaped microchannel, and the direction of a straight channel area of the S-shaped microchannel is perpendicular to the GMR line and parallel to the external magnetic field.
Wherein, the bottom electrode layer is a Cr/Cu electrode layer with the thickness of 10nm/150nm.
Wherein, the electrode upper layer is an Au electrode layer with the thickness of 150nm.
The thickness of the silicon nitride protective film 3a is 20nm.
Wherein, siO 2 The thickness of the biochemical reaction film 3b was 10nm.
The GMR biochip 12 is manufactured by the following steps:
the method comprises the following steps: patterning the GMR lines 1, and patterning the GMR lines 1 on the surface of the substrate 6;
step two: manufacturing a magnetic line concentrator, and patterning the magnetic line concentrator on the surface of the substrate 6;
step three: manufacturing an electrode bottom layer, carrying out electrode patterning on a substrate 6, and then plating a Cr/Cu electrode layer;
step four: manufacturing a silicon nitride protective film 3a, processing the silicon nitride protective film 3a on the whole surface of a substrate 6, then carrying out electrode patterning, and etching a silicon nitride layer on the surface of an electrode until a first layer of the electrode is exposed;
step five: siO 2 2 Preparing a biochemical reaction film, namely preparing SiO in a graphical reaction area of a GMR line area by utilizing a sputtering process 2 A biochemical reaction membrane;
step six: manufacturing an upper electrode layer, patterning a biochemical reaction film on the GMR line 1 region, patterning an electrode on the substrate 6, and taking an Au-plated electrode layer as an upper electrode layer;
step seven: and manufacturing a micro-fluidic channel cavity 5c, and manufacturing the micro-fluidic channel cavity 5c in a GMR detection area on the surface of the substrate 6 for detection reaction.
Wherein the GMR line 1 is fabricated using an ion beam etching process.
The magnetic force line collector is prepared through a sputtering process, and patterning is achieved through a liftoff process.
The bottom electrode layer is manufactured through a sputtering process, and the upper electrode layer is manufactured through a thermal evaporation process.
The silicon nitride protective film 3a is formed by a PECVD process.
Wherein, siO 2 The biochemical reaction film 3b is manufactured by a sputtering process.
Wherein, the micro-fluidic channel cavity 5c is prepared by PDMS technology.
Preferably, step S2 includes the following steps:
s2.1, preparing a magnetic targeting label 9, namely specifically combining biotin on the surface of a vibrio cholerae antibody (primary antibody) 7 modified with biotin with avidin on the surface of a magnetic label 8 modified with avidin to prepare the magnetic targeting label 9 of vibrio cholerae 10; according to the diameter of 0.2 μm, the length of about 0.5 μm of vibrio cholerae and the steric hindrance effect of immunoreaction, the selected magnetic label is a magnetic bead with the diameter equal to 200 nm;
s2.2, performing magnetic modification on the vibrio cholerae 10, namely performing antigen-antibody specific binding on a primary antibody on the surface of a magnetic targeting label 9 and the vibrio cholerae 10 to modify the magnetic targeting label 9 on the vibrio cholerae 10 to obtain the vibrio cholerae 11 which is based on antigen-antibody reaction and modified with the magnetic targeting label, and converting biological information of the vibrio cholerae 10 into magnetic information in the step;
s2.3, performing functional modification on the surface of the GMR biochip 12, and performing SiO (silicon dioxide) modification on the surface of the GMR biochip 12 2 Preparing a biological self-assembly film 13 on the biochemical reaction film 3b by using a chemical modification method, and modifying a capture antibody (secondary antibody) 14 of vibrio cholerae on the surface of the self-assembly film 13 so that the surface of the GMR biochip 12 has the capability of capturing vibrio cholerae 10;
s2.4, capturing Vibrio cholerae 10, injecting Vibrio cholerae 11 modified with a magnetic targeting label based on antigen-antibody reaction to the surface of a GMR biochip 12 through an inlet 5a of a microfluidic channel, keeping the pH value in the S-shaped channel at 8.8-9.0 according to the optimal living pH environment of the Vibrio cholerae, capturing the Vibrio cholerae 10 marked by the magnetic targeting label 9 on the surface of the GMR biochip 12 through the antigen-antibody reaction, then washing the microfluidic channel 5c by using PBS buffer (the pH value is 8.8), and allowing waste liquid to flow out through an outlet 5b of the microfluidic channel to obtain the GMR biochip 15 after capturing the Vibrio cholerae by using the antigen-antibody reaction.
In the embodiment, the capturing of the vibrio cholerae 10 on the surface of the GMR biochip 12 mainly comprises two steps, firstly, the GMR biochip 12 is subjected to surface functionalization modification, a mixed solution of sulfuric acid and hydrogen peroxide with a volume ratio of 7.
And then injecting 10 mmol/L3-aminopropyl-3-ethoxysilane ethanol solution into the chip through the inlet 5a of the microfluidic channel, reacting for 30min, performing amino silanization, rinsing the chip with ethanol for 2 times after the reaction is finished, and discharging the chip through the outlet 5b of the reaction cavity, and placing the chip in an oven at 100 ℃ for 45min. And after the reaction is finished, immersing the micro-fluidic channel cavity 5c into 10mmol/L terephthalaldehyde acetone solution through the inlet 5a, standing for 30min, reacting one aldehyde group and one amino group of the terephthalaldehyde, placing the other group on the surface for reaction, washing with deionized water after the reaction is finished, and discharging through the outlet 5b of the micro-fluidic channel.
Then, a second antibody PBS buffer solution with the concentration of 50uL and the concentration of 0.5mg/mL is injected into the micro-fluidic channel cavity 5c through the inlet 5a of the micro-fluidic channel, and the chip is placed for 4h under the constant temperature and humidity environment at 37 ℃ to fix the capture antibody (second antibody) 14 of the vibrio cholerae. Injecting a Tween-20PBS solution and a PBS buffer solution into a 5c through a 5a, oscillating at a constant temperature (37 ℃) for 5min respectively, removing capture antibodies (secondary antibodies) 14 of the non-fixed vibrio cholerae, discharging waste liquid through a 5b, injecting a 1mol/L trihydroxymethyl aminomethane (pH is 7.4) solution into a micro-fluidic channel cavity 5c through a micro-fluidic channel inlet 5a, placing for 30min, sealing other aldehyde groups in the 3-aminopropyl-3-ethoxysilane, washing by using the PBS buffer solution and deionized water, discharging through a micro-fluidic channel outlet 5b, removing the antibodies which are not fixed on the surface of the GMR biochip 12, and finishing the surface functionalization of the GMR biochip 12 for later use.
The method comprises the steps of mixing 100uL of a biotin-modified vibrio cholerae antibody (primary antibody) 7 and 0.5mg/mL of an avidin-modified magnetic label 8 (the magnetic label in the embodiment is a magnetic bead) at room temperature for 30 minutes to obtain a magnetic targeting label 9 of the biotin-modified vibrio cholerae antibody (primary antibody) 7 which is combined through the specific reaction of the avidin and the biotin, concentrating by using magnetic separation to remove the unbound biotin-modified vibrio cholerae antibody (primary antibody) 7, and supplementing a PBS solution (with a pH value of 8.8) to ensure that the magnetic targeting label 9 solution of the biotin-modified vibrio cholerae antibody (primary antibody) 7 which is combined through the specific reaction of the avidin and the biotin is 100uL.
Then 10uL of magnetic targeting label solution with avidin and biotin specificity reaction combined and modified with the biotin vibrio cholerae antibody (primary antibody) 7 and 1mL of vibrio cholerae 10 with the concentration of 10CFU/mL are mixed, and incubated for 20 minutes under the water bath condition of 37 ℃, antigen-antibody specificity combination is carried out by utilizing the primary antibody on the surface of the magnetic targeting label 9 of the biotin vibrio cholerae antibody (primary antibody) 7 modified with biotin through avidin and biotin specificity reaction combined and the vibrio cholerae 10, supersaturation magnetic modification is carried out on the vibrio cholerae 10, and the magnet is utilized for concentration, finally 20uL of concentrated vibrio cholerae 11 modified with the magnetic targeting label based on antigen-antibody reaction is obtained, injecting vibrio cholerae 11 modified with a magnetic targeting label based on antigen-antibody reaction into a micro-fluidic channel cavity 5c through a micro-fluidic channel 5a, performing specific reaction by using a vibrio cholerae capture antibody (secondary antibody) 14 on the surface of the chip and the vibrio cholerae 11 modified with the magnetic targeting label based on the antigen-antibody reaction, incubating in water bath at 37 ℃ for 20 minutes, then washing with PBS (with the pH value of 8.8) buffer solution for three times, discharging through a micro-fluidic channel 5b, obtaining a GMR biochip 15 after capturing vibrio cholerae by using the antigen-antibody reaction (specific combination of vibrio cholerae 10 and the secondary antibody), and performing next detection.
Preferably, step S3 includes the following steps: firstly fixing a GMR biochip 12 which does not generate biochemical reaction at the middle position between a first solenoid 16a and a second solenoid 16b, then switching on the GMR biochip 12 and a detection circuit, supplying a certain working current to the GMR biochip 12, then applying current to the solenoid a and the solenoid b to generate a magnetic field 16c of a GMR region, recording the voltage value output by the GMR biochip 12 at the moment, then fixing the GMR biochip 12 which generates biochemical reaction at the position of the GMR biochip 12 which does not generate reaction, then switching on the GMR biochip 12 and the detection circuit, supplying the same working current to the GMR biochip 12, then applying the same current to the solenoid a and the solenoid b to generate a magnetic field 16c of the same GMR region, and recording the voltage value output by the GMR biochip 12 at the moment through the detection circuit; the information of the detected biological target can be obtained through the difference value of the two voltage values.
In the embodiment, an antigen-antibody reaction (specific binding of vibrio cholerae 10 and a secondary antibody) is utilized to capture vibrio cholerae, and the whole process is realized through the test of a GMR biochip 15, the magnetic field 16c of the external GMR area of a first solenoid 16a and a second solenoid 16b is mainly utilized to excite the GMR biochip 12, the magnetic label 8 which is captured on the surface of the GMR biochip 12 and modified with avidin is magnetized, the magnetic label 8 modified with avidin can shunt the magnetic field 16c of the GMR area under the magnetization of the excitation magnetic field, so that the magnetic label area has a magnetic bead shunt magnetic field 17 which is stronger than the magnetic field 16c of the GMR area, the shunt can influence the effect of the excitation magnetic field on the GMR biochip 12, the number of the magnetic labels 8 modified with avidin can be obtained through reading data by a measuring instrument digital source table and the like, the number of the vibrio cholerae 10 can be obtained through calculating the number of the magnetic labels 8 modified with avidin, and the detection of the vibrio cholerae 10 can be realized.
In the present invention, the GMR biochip 12 test is carried out by magnetizing the GMR biochip 12 and the captured immune magnetic field with an external magnetic field. The magnetic bead is a superparamagnetic magnetic bead, can gather magnetic field 16c in the GMR region under the effect of external magnetic field, form stronger magnetic bead and shunt magnetic field 17, realized the reposition of redundant personnel to magnetic field 16c in the GMR region, the change of this reposition of redundant personnel can bring the resistance change based on GMR biological chip 12 of GMR magnetoelectronics, detect the change of resistance through testing instrument 18, alright obtain the quantity of the magnetic bead that awaits measuring, alright obtain the quantity of biological target through the quantity of magnetic bead, thereby realize the detection to biological target.
FIG. 4 is a graph showing the detection performance of the present invention compared to a conventional method, with the abscissa representing the concentration of bacteria (1 mL for all tests) and the ordinate representing the GMR output signal (the rate of change of GMR is represented by MR).
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of changes or substitutions within the technical scope of the present invention, and therefore, the scope of the present invention should be determined by the scope of the claims.
Claims (9)
1. A vibrio cholerae detection method based on a high-sensitivity GMR magnetic biochip is characterized by comprising the following steps:
s1, preparing a GMR biochip, wherein a micro-fluidic channel for biochemical reaction is arranged on the GMR magnetic biochip, the micro-fluidic channel is an S-shaped micro-channel, and the pH value in the S-shaped micro-channel is 8.8-9.0;
s2, capturing vibrio cholerae on a GMR biochip, comprising the following steps:
s2.1, preparing a magnetic targeting label;
s2.2, magnetic modification of vibrio cholerae;
s2.3, performing functional modification on the surface of the GMR biochip;
s2.4, capturing vibrio cholerae, namely injecting the vibrio cholerae modified with the magnetic targeting label based on the antigen-antibody reaction to the surface of a GMR biochip through a microfluidic channel, and capturing the vibrio cholerae marked by the magnetic label on the surface of the GMR biochip through the antigen-antibody reaction to obtain the GMR biochip after capturing the vibrio cholerae by utilizing the antigen-antibody reaction;
and S3, testing a GMR biochip and reading out vibrio cholerae information.
2. The method for detecting vibrio cholerae based on the high-sensitivity GMR magnetic biochip as claimed in claim 1, wherein the method comprises the following steps: in the step S1, the GMR magnetic biochip includes a plate-shaped substrate, GMR lines are disposed on an upper end surface of the substrate, magnetic force line collectors are respectively disposed at positions of the upper end surface of the substrate on two sides of the GMR lines, and a magnetization direction between the two magnetic force line collectors is perpendicular to the GMR lines; the tail parts of the GMR lines are respectively covered with an electrode, and the two electrodes are respectively and electrically connected with electrode pins; the upper end surface of the substrate and the upper end surface of the magnetic force line collector are both provided with silicon nitride protective films, and the silicon nitride protective films on the upper end surfaces of the GMR lines are also provided with SiO 2 A biochemical reaction film; the electrode comprises an electrode bottom layer and an electrode upper layer; the micro-fluidic channel is arranged on the upper end face of the substrate and positioned at the part around the GMR lines, and the GMR lines and the electrodes are positioned in the micro-fluidic channel.
3. The method for detecting vibrio cholerae based on the high-sensitivity GMR magnetic biochip as claimed in claim 2, wherein the method comprises the following steps: the direction of the biochemical reaction microfluidic channel area is vertical to the GMR line and parallel to the external magnetic field.
4. The method for detecting vibrio cholerae based on the high-sensitivity GMR magnetic biochip as claimed in claim 1, wherein the method comprises the following steps: the magnetic labels selected in step S2.1 are magnetic beads with a diameter equal to 1 μm.
5. The method for detecting vibrio cholerae based on the high-sensitivity GMR magnetic biochip as claimed in claim 2, wherein the method comprises the following steps: the GMR lines are of a zigzag structure, the line width of the lines is 5 mu m, the length of the lines is 500 mu m, and the gap of the lines is 15 mu m.
6. The method for detecting vibrio cholerae based on the high-sensitivity GMR magnetic biochip as claimed in claim 2, wherein the method comprises the following steps: the magnetic force line collector is a rectangular FeNi film with the thickness of 2 mu m.
7. The method for detecting vibrio cholerae based on the high-sensitivity GMR magnetic biochip as claimed in claim 2, wherein the method comprises the following steps: the GMR biochip is prepared by the following steps:
the method comprises the following steps: patterning the GMR lines, and patterning the GMR lines on the surface of the substrate;
step two: manufacturing a magnetic line concentrator, and patterning the magnetic line concentrator on the surface of the substrate;
step three: manufacturing an electrode bottom layer, carrying out electrode patterning on a substrate, and then plating a Cr/Cu electrode layer;
step four: preparing a silicon nitride protective film, namely processing the silicon nitride protective film on the whole surface of the substrate, then patterning an electrode, and etching the silicon nitride layer on the surface of the electrode until the first layer of electrode is exposed;
step five: siO 2 2 Preparing a biochemical reaction film, namely preparing SiO in a graphical reaction area of a GMR line area by utilizing a sputtering process 2 A biochemical reaction membrane;
step six: manufacturing an upper electrode layer, patterning a biochemical reaction film on a GMR line region, patterning an electrode on a substrate, and taking an Au-plated electrode layer as an upper electrode layer;
step seven: and (3) manufacturing a biochemical reaction microfluidic channel, and manufacturing the biochemical reaction microfluidic channel in a GMR detection area on the surface of the substrate for detecting reaction.
8. The method for detecting vibrio cholerae based on the high-sensitivity GMR magnetic biochip as claimed in claim 7, wherein the method comprises the following steps: the magnetic force line collector is prepared through a sputtering process, and patterning is achieved through a liftoff process.
9. The method for detecting vibrio cholerae based on the high-sensitivity GMR magnetic biochip as claimed in claim 1, wherein the method comprises the following steps: fixing the GMR biochip without biochemical reaction between the first solenoid and the second solenoid, switching on the GMR biochip and the detection circuit, supplying certain working current to the GMR biochip, applying current to the first solenoid and the second solenoid to make the GMR region generate a magnetic field, and recording the voltage value output by the GMR biochip without biochemical reaction; similarly, recording the voltage value output by the GMR biochip after the biochemical reaction; and judging the information of the biological target through the difference value of the two voltage values.
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