CN111774111A - Micro-fluidic chip for detecting glycosylated hemoglobin and detection method thereof - Google Patents
Micro-fluidic chip for detecting glycosylated hemoglobin and detection method thereof Download PDFInfo
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- CN111774111A CN111774111A CN202010664767.XA CN202010664767A CN111774111A CN 111774111 A CN111774111 A CN 111774111A CN 202010664767 A CN202010664767 A CN 202010664767A CN 111774111 A CN111774111 A CN 111774111A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/723—Glycosylated haemoglobin
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/726—Devices
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- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0829—Multi-well plates; Microtitration plates
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- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
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- B01L2300/0887—Laminated structure
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
- G01N2035/00099—Characterised by type of test elements
- G01N2035/00158—Elements containing microarrays, i.e. "biochip"
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a micro-fluidic chip for detecting glycosylated hemoglobin and a detection method thereof. A sample introduction through hole and a waste liquid lead-out through hole are respectively arranged on the cover sheet layer. The sample detection unit comprises one or more sample inlets, a dilution chamber, an affinity chromatography channel, a detection chamber and a waste liquid pool. The sample introduction through hole is communicated with the sample introduction chamber, and the waste liquid guide through hole is communicated with the waste liquid pool. The invention integrates sample feeding, sample diluting and optical and electric detection into a whole through the microfluidic chip, greatly simplifies the operation process, reduces the consumption of the sample and the reagent, does not need to be provided with expensive instruments, greatly reduces the use cost, enables the on-site instant detection of the glycosylated hemoglobin to be possible, and has foreseeable great economic value and social value.
Description
Technical Field
The invention relates to the technical field of micro-fluidic chips, in particular to a micro-fluidic chip for detecting glycosylated hemoglobin and a detection method thereof.
Background
Diabetes is a metabolic disease characterized by hyperglycemia due to defective insulin secretion or impaired insulin action. At present, diabetes is mainly judged by detecting blood sugar parameters, but the blood sugar parameters can only represent the blood sugar level when blood is drawn, and the accurate diagnosis of the diabetes is limited. Recent medical studies have demonstrated that: the concentration of the glycosylated hemoglobin (GHb) in the blood is relatively stable, and the concentration value can accurately reflect the blood sugar level within the recent 2-3 months, so that doctors can diagnose the diabetes early and know the control condition of the diabetes.
At present, there are two main types of methods for detecting glycated hemoglobin in clinical laboratories: one class of methods is based on the difference in charge between glycated hemoglobin and non-glycated hemoglobin, such as ion chromatography, electrophoresis, and the like; another method is based on the structural features of the glycated group on hemoglobin, such as affinity chromatography, ion trapping, and immunization. In the above methods, although high performance liquid ion chromatography (HPLC) is known as a gold standard method, it has the disadvantages of complicated operation procedure, high instrument cost, and the like, and often requires a laboratory-level operation environment and professional technical operators, so that the method is not suitable for application and popularization of instant rapid detection of diabetes.
Microfluidic chips, also known as Lab-on-a-chips, refer to a biological or chemical laboratory built on a few square centimeters Chip. It integrates the basic operation units of sample introduction, reaction, separation, sorting, detection, etc. related in the biological and chemical fields into a very small chip, and the network is formed by micro-channels, so that the controllable fluid can penetrate through the whole system to implement various functions of conventional biological or chemical laboratories. The micro-fluidic chip technology has the characteristics of small sample volume, high integration level and easy realization of automatic control and high-throughput analysis, so that the biochemical reaction operation on the micro-fluidic chip is more convenient and faster than the conventional analysis sample pretreatment, and the cost is low.
Disclosure of Invention
The invention aims to provide a micro-fluidic chip for detecting glycosylated hemoglobin, which has the characteristics of convenient sample introduction, low sample consumption, high detection speed, low cost and the like and can realize full-automatic detection of the glycosylated hemoglobin.
Another object of the present invention is to provide a glycated protein assay method which has high reaction efficiency, short assay time, and significantly improved assay resolution and sensitivity.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
The invention provides a micro-fluidic chip for detecting glycosylated hemoglobin, which is sequentially provided with a cover plate layer, a fluid layer with a hollow channel structure and a substrate layer from top to bottom, wherein the hollow channel structure and the substrate layer jointly form a sample detection unit;
the cover plate layer is respectively provided with a sample introduction through hole for sample introduction of the sample introduction chamber and a waste liquid lead-out through hole for leading out waste liquid of the waste liquid pool;
the sample detection unit comprises one or more sample introduction chambers, a dilution chamber, an affinity chromatography channel, a detection chamber and a waste liquid pool, wherein the sample introduction through hole is communicated with the sample introduction chambers, and the waste liquid is led out and communicated with the waste liquid pool.
A method for detecting glycosylated hemoglobin, comprising the steps of:
s1, introducing the whole blood sample to be detected into a sample inlet chamber through a sample inlet through hole, and starting an external pump to enable the whole blood sample to enter the dilution chamber through a microfluidic channel;
s2, breaking the red blood cells in the whole blood sample under the action of the hemolysin reagent and releasing the proteins in the red blood cells;
s3, injecting a diluent into the sample introduction chamber from the sample introduction through hole, wherein the diluent flows into the dilution chamber through the sample introduction chamber to dilute the whole blood sample to obtain a diluted sample, and at the moment, the drain valve is in a closed state;
s4, increasing the pump pressure of an external pump, and introducing a mobile phase asparagine-linked immunosorbent assay buffer solution into a sample introduction chamber from a sample introduction through hole, wherein the mobile phase asparagine-linked immunosorbent assay buffer solution brings the diluted sample into the affinity chromatography channel through the sample introduction chamber and the dilution chamber;
s5, specifically binding glycated hemoglobin in the diluted sample with the aminophenylboronic acid agarose gel, and carrying non-glycated hemoglobin away by the mobile phase asparagine-linked immunosorbent buffer solution and leading the non-glycated hemoglobin out of the waste liquid outlet through hole;
s6, introducing a mobile phase sorbitol buffer solution into the sample introduction chamber from the sample introduction through hole, wherein the mobile phase sorbitol buffer solution flows to the affinity chromatography channel through the sample introduction chamber and the dilution chamber, and elutes the glycosylated hemoglobin specifically bound on the aminobenzene boronic acid agarose gel;
s7, the eluted glycosylated hemoglobin flows through the detection chamber, and detection of the glycosylated hemoglobin is performed through a microelectrode sensor or a photoelectric sensor.
The micro-fluidic chip for detecting the glycosylated hemoglobin has the advantages that:
1. the invention is based on a microfluidic chip technology platform, the microfluidic chip technology has the characteristics of small sample volume, high integration level and easy realization of automatic control and high-flux analysis, a sample detection unit is arranged on the microfluidic chip, and the sample detection unit comprises one or more sample chambers, a dilution chamber, an affinity chromatography channel, a detection chamber and a waste liquid pool. The micro-fluidic chip integrates sample feeding, sample dilution and optical and electrical detection, greatly simplifies the operation process, reduces the consumption of samples and reagents, does not need to be provided with expensive instruments, greatly reduces the use cost, enables the on-site instant detection of the glycosylated hemoglobin to be possible, and has foreseeable great economic value and social value.
2. Compared with the ion chromatography, the affinity chromatography is simple and rapid to operate, low in cost, strong in specificity, free of interference of abnormal hemoglobin, relatively insensitive to the influence of hemoglobin modified after translation and pathological hemoglobin, and not as strict as the ion exchange method on the storage time of a blood sample, so that the affinity chromatography detection of the glycosylated hemoglobin is realized on a microfluidic chip, the sample processing time is greatly shortened, the cost of reagents and instruments is greatly reduced, the detection resolution and sensitivity are remarkably improved, the on-site instant detection of the glycosylated hemoglobin is possible, and the method is expected to be widely popularized in the clinical detection of the glycosylated hemoglobin.
3. According to the invention, the drain valve is arranged between the dilution chamber and the affinity chromatography channel, and the solution is blocked or introduced from the dilution chamber to the affinity chromatography channel by the cooperation of the drain valve and the external pump. On one hand, the hemoglobin can be fully crushed and diluted in the diluting chamber, so that the condition that the content of the glycosylated hemoglobin in the affinity chromatography channel is lower because the hemoglobin is not completely crushed and enters the affinity chromatography channel is avoided, and the consumption of a sample and a reagent is reduced. On the other hand, in the pump pressure increasing process of the external pump, the flow velocity of the solution in the diluting chamber is correspondingly increased, and the contact between the whole blood sample and the hemolysin reagent is further increased, so that the red blood cells in the whole blood sample are fully crushed, the content of the glycosylated hemoglobin in the diluted sample is improved, and the accuracy of subsequent glycosylated hemoglobin detection is improved.
4. The waste liquid pool is communicated with the waste liquid lead-out through hole for leading out the waste liquid. Because the waste liquid can be timely derived from the waste liquid through hole, the cross contamination caused by the contact of the waste liquid and the subsequently added reagent is avoided, and the detection accuracy is ensured.
5. The invention can adopt a microelectrode sensor to detect or a photoelectric sensor to detect the glycosylated hemoglobin. The microelectrode sensor realizes the electrochemical detection of the glycosylated hemoglobin by directly preparing a gold electrode or a platinum electrode in a detection chamber of the microfluidic chip, and is convenient for the on-site instant detection of the microfluidic chip. In addition, the invention does not need to be equipped with laboratory-level operating environment and professional technical operators, not only has simple operating procedure, but also reduces the cost of the detecting instrument.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic plan view showing the construction of a microfluidic chip for assaying glycated hemoglobin according to example 1 of the present invention;
FIG. 2 is a schematic perspective view showing a microfluidic chip for glycated hemoglobin measurement according to example 1 of the present invention;
FIG. 3 is a schematic plan view showing the construction of a microfluidic chip for assaying glycated hemoglobin according to example 2 of the present invention;
FIG. 4 is a schematic plan view showing the structure of a microfluidic chip for assaying glycated hemoglobin according to example 3 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following is a detailed description of a microfluidic chip for detecting glycated hemoglobin according to an embodiment of the present invention.
Example 1
Referring to fig. 1 and 2, a microfluidic chip for detecting glycated hemoglobin provided in an embodiment of the present invention includes, in order from top to bottom, a cover sheet layer 1, a fluid layer 2 having a hollow channel structure, and a substrate layer 3, where the hollow channel structure and the substrate layer together form a sample detection unit.
And the cover plate layer is respectively provided with a sample introduction through hole 11 for sample introduction of the sample introduction chamber and a waste liquid lead-out through hole 12 for leading out waste liquid of the waste liquid pool.
The sample detection unit comprises one or more sample introduction chambers 21, a dilution chamber 22, an affinity chromatography channel 23, a detection chamber 24 and a waste liquid pool 25, wherein the sample introduction through hole 11 is communicated with the sample introduction chambers 21, and the waste liquid derivation through hole 12 is communicated with the waste liquid pool 25.
Further, in this embodiment, the microfluidic chip is formed by processing one or more of glass, polydimethylsiloxane, polymethyl methacrylate, and polycarbonate. The processing method of the hollow channel structure comprises laser etching, soft etching, photoetching, wet etching or CNC precision processing. The substrate layer 3 is a blank substrate layer.
Referring to fig. 1 and 2, further, in this embodiment, the sample inlet chamber 21, the dilution chamber 22, the affinity chromatography channel 23, the detection chamber 24 and the waste liquid pool 25 are sequentially connected in series via a microfluidic channel 26.
Further, in the present embodiment, a hemolysin reagent for breaking up red blood cells is disposed inside the dilution chamber 22, and the hemolysin reagent is fixed to the inner wall of the dilution chamber 22 by physical adsorption or chemical adsorption. The dosage of the hemolysin reagent is 10-50 mu L, and the reaction time of the hemolysin reagent and the whole blood sample is 3-8 minutes. The hemolysin reagent may be selected from Shanghaineo Biotech limited, and the like. After the whole blood enters the dilution chamber 22, the red blood cells are broken up by the hemolysin reagent to release hemoglobin, and then the hemoglobin is diluted to a certain multiple by introducing a dilution buffer.
Further, in this embodiment, a shut-off valve is disposed between the dilution chamber 22 and the affinity chromatography channel 23. The shut-off valve may be used to block or introduce the solution in the dilution chamber 22 into the affinity chromatography channel 23.
Further, in the present embodiment, the shutoff valve is a shutoff valve, a pneumatic valve, or a drain valve.
When the material of the micro-fluidic chip is elastic polydimethylsiloxane, a stop valve or a pneumatic valve can be adopted. When the shut-off valve is opened, the microfluidic channel 26 is put in flow and the solution is introduced from the dilution chamber 22 into the affinity chromatography channel 23. When the shut-off valve is closed, the microfluidic channel 26 is closed and the solution is blocked in the dilution chamber 22.
The pneumatic valve adopts a film with elasticity. When the pneumatic valve is opened, it is pressurized by the air pump, causing the elastic membrane of the pneumatic valve to expand, closing the passage, blocking the solution in the dilution chamber 22. When the pneumatic valve is closed, the air pump is closed to reduce the pressure, the elastic membrane of the pneumatic valve is restored to the original membrane state, and the channel is circulated, so that the solution is introduced into the affinity chromatography channel 23.
In this embodiment, a trap is used. Hydrophobic valves are created by preparing a hydrophobic surface in a specific region of the microfluidic channel 26 between the dilution chamber 22 and the affinity chromatography chamber 23. Under the action of surface tension, the solution is confined in front of the hydrophobic surface. When the pressure of the external pump is increased, the pressure in the microfluidic channel is increased, and the solution breaks through the hydrophobic surface, so that the solution is introduced into the affinity chromatography channel 23.
The affinity chromatography channel 23 is internally provided with aminophenylboronic acid sepharose for specific binding with hemoglobin. The aminophenylboronic acid agarose gel is used as a carrier of glycosylated hemoglobin affinity chromatography, and when the total glycosylated hemoglobin passes through the carrier, a cis-disaccharide alcohol part containing glucose on the surface of a stable glycosylated hemoglobin molecule is specifically combined with a boric acid gene on a carrier stationary phase in a coordination manner. The aminophenylboronic acid agarose gel may be selected from Shanghai Haishen industries, Inc., and the like.
Further, in the present embodiment, the aminophenylboronic acid agarose exists in the form of a packed column within the affinity chromatography channel 23, and fills the inside of the affinity chromatography channel 23.
Further, in this embodiment, the aminophenylboronic acid agarose is present in an open-column form within the affinity chromatography channel 23 and is coated on the inner surface of the affinity chromatography channel 23.
The aminophenylboronic acid agarose exists in a packed column form or an open column form in the affinity chromatography channel 23, so that the hemoglobin can be ensured to be fully contacted with the aminophenylboronic acid agarose, and then be specifically adsorbed on the aminophenylboronic acid agarose, and the detection accuracy is ensured.
Further, in the present embodiment, a microelectrode sensor for detecting glycated hemoglobin is provided in the detection chamber 24. The microelectrode sensor adopts an MEMS (micro-electromechanical systems) process, and a gold electrode or a platinum electrode is directly prepared in a detection chamber of the microfluidic chip, so that the electrochemical detection of the glycosylated hemoglobin is realized, and the on-site instant detection of the microfluidic chip is facilitated.
Further, in the present embodiment, a photoelectric sensor is disposed outside the detection chamber 24. The photoelectric sensor in this embodiment is selected from the group consisting of the micro silicon photocells of hamamatsu.
The method for detecting the glycosylated hemoglobin comprises the following steps:
s1, adding a whole blood sample of several microliters to tens of microliters into the sample inlet chamber 21 through the sample inlet through hole 11, and starting the external pump to enable the whole blood sample to enter the dilution chamber 22 through the microfluidic channel 26.
S2, and breaking down the red blood cells in the whole blood sample under the action of the hemolysin reagent and releasing the proteins in the red blood cells.
S3, injecting a diluent into the sample introduction chamber 21 from the sample introduction through hole 11, wherein the diluent flows into the dilution chamber 22 through the sample introduction chamber 21 to dilute the whole blood sample, so as to obtain a diluted sample, and at this time, the trap is in a closed state. Due to the restriction of the trap and the low pumping pressure of the external pump, both the whole blood sample and the diluent are confined in the dilution chamber 22 during dilution.
And S4, increasing the pump pressure of an external pump, and introducing a mobile phase asparagine-linked immunosorbent assay buffer solution into the sample inlet chamber 21 from the sample inlet through hole 11, wherein the mobile phase asparagine-linked immunosorbent assay buffer solution brings the diluted sample into the affinity chromatography channel 23 through the sample inlet chamber 21 and the dilution chamber 22.
S5, the glycosylated hemoglobin in the diluted sample is specifically combined with the aminophenylboronic acid agarose gel, and the non-glycosylated hemoglobin is carried away by the mobile phase asparaginyl amine buffer solution and is led out from the waste liquid outlet through hole 12.
S6, introducing a mobile phase sorbitol buffer solution into the sample inlet chamber 21 from the sample inlet through hole 11, wherein the mobile phase sorbitol buffer solution flows to the affinity chromatography channel 23 through the sample inlet chamber 21 and the dilution chamber 22, and elutes glycated hemoglobin specifically bound to the aminobenzeneboronic acid sepharose.
S7, the eluted glycated hemoglobin flows through the detection chamber 24, and detection of glycated hemoglobin is performed by a microelectrode sensor or a photosensor.
When the micro-fluidic chip for detecting the glycosylated hemoglobin is used, the whole blood, the diluent and the mobile phase are only required to be injected into the sample chamber 21 in sequence, so that full-automatic detection can be realized, and the detection of the glycosylated hemoglobin can be realized within 3-10 minutes. The invention has the advantages of convenient sample introduction, less sample consumption, high reaction efficiency, short detection time and the like.
Example 2
Referring to fig. 3, the present embodiment provides a microfluidic chip for detecting glycated hemoglobin, which is different from embodiment 1 in that:
the sample detection unit comprises two sample chambers 21, and the two sample chambers 21 are respectively connected with a dilution chamber 22 and used for introducing different samples into the dilution chamber 22. In use, a whole blood sample is introduced from the first sample chamber 211, and in step S2, a diluent is injected from the second sample chamber 212 and the test is performed according to the test procedure in example 1.
When the micro-fluidic chip for detecting the glycosylated hemoglobin provided by the embodiment is used, only the whole blood, the diluent and the mobile phase are required to be sequentially injected from the sampling chamber 21, so that full-automatic detection can be realized, and the glycosylated hemoglobin can be detected within 3-10 minutes. The embodiment has the advantages of convenient sample introduction, less sample consumption, high reaction efficiency, short detection time and the like.
Example 3
Referring to fig. 4, the present embodiment provides a microfluidic chip for detecting glycated hemoglobin, which is different from embodiment 1 in that:
the microfluidic chip of this embodiment includes three sample detection units. In the detection, three different whole blood samples are respectively added into the corresponding sample chambers 21, and the detection is performed according to the detection procedure in example 1, so that the detection results of the glycated hemoglobin of the three samples can be obtained within 10 minutes. The embodiment has the advantages of convenient sample introduction, less sample consumption, high reaction efficiency, short detection time and the like.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. The microfluidic chip for detecting the glycosylated hemoglobin is characterized in that a cover plate layer, a fluid layer with a hollow channel structure and a substrate layer are sequentially arranged on the microfluidic chip from top to bottom, and the hollow channel structure and the substrate layer jointly form a sample detection unit;
the cover plate layer is respectively provided with a sample introduction through hole for sample introduction of the sample introduction chamber and a waste liquid lead-out through hole for leading out waste liquid of the waste liquid pool;
the sample detection unit comprises one or more sample introduction chambers, a dilution chamber, an affinity chromatography channel, a detection chamber and a waste liquid pool, wherein the sample introduction through hole is communicated with the sample introduction chambers, and the waste liquid is led out and communicated with the waste liquid pool.
2. The microfluidic chip for detecting glycated hemoglobin according to claim 1, wherein the microfluidic chip is formed by one or more of glass, polydimethylsiloxane, polymethyl methacrylate and polycarbonate, the hollow channel is processed by laser etching, soft etching, photolithography, wet etching or CNC precision machining, and the substrate layer is a blank substrate layer.
3. The microfluidic chip for detecting glycated hemoglobin according to claim 1, wherein the sample chamber, the dilution chamber, the affinity chromatography channel, the detection chamber, and the waste reservoir are sequentially connected in series via a microfluidic channel.
4. The microfluidic chip for detecting glycated hemoglobin according to claim 1, wherein a hemolysin reagent for breaking red blood cells is disposed in the dilution chamber, the hemolysin reagent is immobilized on the inner wall of the dilution chamber by physical adsorption or chemical adsorption, the dose of the hemolysin reagent is 10-50 μ L, and the reaction time of the hemolysin reagent with the whole blood sample is 3-8 min; and amino phenyl boronic acid agarose gel for specific binding with glycosylated hemoglobin is arranged in the affinity chromatography channel.
5. The microfluidic chip for detecting glycated hemoglobin according to claim 4, wherein the aminophenylboronic acid agarose exists in the form of a packed column within the affinity chromatography channel and fills the inside of the affinity chromatography channel.
6. The microfluidic chip for detecting glycated hemoglobin according to claim 4, wherein the aminophenylboronic acid agarose exists in an open-column form within the affinity chromatography channel and is coated on the inner surface of the affinity chromatography channel.
7. The microfluidic chip for detecting glycated hemoglobin according to claim 1, wherein a shut-off valve is disposed between the dilution chamber and the affinity chromatography channel, and the shut-off valve is a shut-off valve, a pneumatic valve or a drain valve.
8. The microfluidic chip for detecting glycated hemoglobin according to claim 1, wherein a microelectrode sensor for detecting glycated hemoglobin is disposed in the detection chamber.
9. The microfluidic chip for detecting glycated hemoglobin according to claim 1, wherein a photosensor is disposed outside the detection chamber.
10. A method for detecting glycated hemoglobin, using the microfluidic chip according to any one of claims 1 to 9, comprising the steps of:
s1, introducing the whole blood sample to be detected into a sample inlet chamber through a sample inlet through hole, and starting an external pump to enable the whole blood sample to enter the dilution chamber through a microfluidic channel;
s2, breaking the red blood cells in the whole blood sample under the action of the hemolysin reagent and releasing the proteins in the red blood cells;
s3, injecting a diluent into the sample introduction chamber from the sample introduction through hole, wherein the diluent flows into the dilution chamber through the sample introduction chamber to dilute the whole blood sample to obtain a diluted sample, and at the moment, the drain valve is in a closed state;
s4, increasing the pump pressure of an external pump, and introducing a mobile phase asparagine-linked immunosorbent assay buffer solution into a sample introduction chamber from a sample introduction through hole, wherein the mobile phase asparagine-linked immunosorbent assay buffer solution brings the diluted sample into the affinity chromatography channel through the sample introduction chamber and the dilution chamber;
s5, specifically binding glycated hemoglobin in the diluted sample with the aminophenylboronic acid agarose gel, and carrying non-glycated hemoglobin away by the mobile phase asparagine-linked immunosorbent buffer solution and leading the non-glycated hemoglobin out of the waste liquid outlet through hole;
s6, introducing a mobile phase sorbitol buffer solution into the sample introduction chamber from the sample introduction through hole, wherein the mobile phase sorbitol buffer solution flows to the affinity chromatography channel through the sample introduction chamber and the dilution chamber, and elutes the glycosylated hemoglobin specifically bound on the aminobenzene boronic acid agarose gel;
s7, the eluted glycosylated hemoglobin flows through the detection chamber, and detection of the glycosylated hemoglobin is performed through a microelectrode sensor or a photoelectric sensor.
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