CN116539783A - Glycosylated hemoglobin detection device and method - Google Patents
Glycosylated hemoglobin detection device and method Download PDFInfo
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- CN116539783A CN116539783A CN202310568424.7A CN202310568424A CN116539783A CN 116539783 A CN116539783 A CN 116539783A CN 202310568424 A CN202310568424 A CN 202310568424A CN 116539783 A CN116539783 A CN 116539783A
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- 238000001514 detection method Methods 0.000 title claims abstract description 96
- 102000017011 Glycated Hemoglobin A Human genes 0.000 title claims abstract description 72
- 108010014663 Glycated Hemoglobin A Proteins 0.000 title claims abstract description 72
- 238000000034 method Methods 0.000 title claims abstract description 30
- 239000003480 eluent Substances 0.000 claims abstract description 143
- 238000013375 chromatographic separation Methods 0.000 claims abstract description 79
- 238000000926 separation method Methods 0.000 claims abstract description 51
- 238000002347 injection Methods 0.000 claims abstract description 30
- 239000007924 injection Substances 0.000 claims abstract description 30
- 238000010828 elution Methods 0.000 claims abstract description 26
- 239000000523 sample Substances 0.000 claims description 242
- 239000012488 sample solution Substances 0.000 claims description 45
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 32
- 238000012360 testing method Methods 0.000 claims description 26
- 238000004458 analytical method Methods 0.000 claims description 23
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 20
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 18
- 239000003729 cation exchange resin Substances 0.000 claims description 18
- 239000011780 sodium chloride Substances 0.000 claims description 16
- 238000002835 absorbance Methods 0.000 claims description 15
- 238000005342 ion exchange Methods 0.000 claims description 15
- 238000001228 spectrum Methods 0.000 claims description 14
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 10
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 10
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 10
- 230000003287 optical effect Effects 0.000 claims description 8
- 210000004369 blood Anatomy 0.000 claims description 7
- 239000008280 blood Substances 0.000 claims description 7
- 238000004364 calculation method Methods 0.000 claims description 5
- 238000004891 communication Methods 0.000 claims description 5
- 238000000227 grinding Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 230000005693 optoelectronics Effects 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 230000002949 hemolytic effect Effects 0.000 claims 1
- 230000007246 mechanism Effects 0.000 description 11
- 230000014759 maintenance of location Effects 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000004811 liquid chromatography Methods 0.000 description 6
- 238000012423 maintenance Methods 0.000 description 6
- 206010018910 Haemolysis Diseases 0.000 description 5
- 238000004140 cleaning Methods 0.000 description 5
- 230000008588 hemolysis Effects 0.000 description 5
- 238000005070 sampling Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 102000001554 Hemoglobins Human genes 0.000 description 4
- 108010054147 Hemoglobins Proteins 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 238000000835 electrochemical detection Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 150000001768 cations Chemical class 0.000 description 3
- 238000012864 cross contamination Methods 0.000 description 3
- 239000003219 hemolytic agent Substances 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- INGWEZCOABYORO-UHFFFAOYSA-N 2-(furan-2-yl)-7-methyl-1h-1,8-naphthyridin-4-one Chemical compound N=1C2=NC(C)=CC=C2C(O)=CC=1C1=CC=CO1 INGWEZCOABYORO-UHFFFAOYSA-N 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000005277 cation exchange chromatography Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- IXZISFNWUWKBOM-ARQDHWQXSA-N fructosamine Chemical compound NC[C@@]1(O)OC[C@@H](O)[C@@H](O)[C@@H]1O IXZISFNWUWKBOM-ARQDHWQXSA-N 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- DIRRKLFMHQUJCM-UHFFFAOYSA-N (2-aminophenyl)boronic acid Chemical group NC1=CC=CC=C1B(O)O DIRRKLFMHQUJCM-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000012459 cleaning agent Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000003819 low-pressure liquid chromatography Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000010339 medical test Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/16—Injection
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/16—Injection
- G01N30/20—Injection using a sampling valve
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/16—Injection
- G01N30/20—Injection using a sampling valve
- G01N2030/201—Injection using a sampling valve multiport valves, i.e. having more than two ports
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
- G01N2030/642—Electrical detectors photoionisation detectors
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a glycosylated hemoglobin detection device and a glycosylated hemoglobin detection method, wherein the glycosylated hemoglobin detection device comprises a sample injection unit, a separation unit and a detection unit; the sample injection unit comprises a syringe, a sample needle, a sample ring and a sample valve, wherein a plurality of ports are arranged in the sample valve, the syringe is connected with the sample needle through two ports of the sample valve, and the sample ring is connected to the two ports of the sample valve; the separation unit comprises eluent storage equipment, a high-pressure pump and a chromatographic separation column which are sequentially connected, the sample ring and the sample valve are further positioned between the high-pressure pump and the chromatographic separation column, and an outlet of the chromatographic separation column is connected with the detection unit. According to the invention, a separation unit is added on the basis of a sample injection and detection unit, different pipelines are communicated through adjustment of a plurality of ports in a sample injection valve, and the different pipelines are separated; the method adopts eluent to carry out elution separation, so as to be convenient for separating different components in glycosylated hemoglobin and improve the detection accuracy; the device has simple structure, lower cost and wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biological index detection, and relates to a glycosylated hemoglobin detection device and a glycosylated hemoglobin detection method.
Background
In a modern clinical examination laboratory, a general liquid chromatography analysis device is generally composed of a sample adding system, a cleaning system and a detection system, wherein the sample adding system is generally connected with a sample needle by an independent injector, and samples are driven by the suction and discharge actions of the injector to finish the sample adding action; the cleaning system is also provided with a plurality of independent injectors or cleaning pumps, and the cleaning liquid is driven to clean the pipeline, so that more parts such as the pumps and the injectors exist in the system, the structure is complex, and the maintenance cost is high.
In clinical medical tests, various biological substances in the body can be detected by liquid chromatography, for example, monitoring indexes related to diabetes include blood sugar, fructosamine and glycosylated hemoglobin, wherein the glycosylated hemoglobin is a product of combining hemoglobin in red blood cells with glucose, and the glycosylated hemoglobin comprises 3 sugar-containing components, namely HbA1a, hbA1b and HbA1c, wherein the HbA1c accounts for at most about 70%, and the structure is relatively stable, and the monitoring indexes are clinically used as monitoring indexes for controlling diabetes, so that different components in the glycosylated hemoglobin need to be separated, and the detection accuracy is improved.
When glycosylated hemoglobin is measured by liquid chromatography, the following methods are broadly classified: a measurement method using cation exchange chromatography, wherein various hemoglobins are fractionated by using a filler to which an ion exchange substance is immobilized and the respective hemoglobins are measured by using different charges; an affinity chromatography-based measurement method using a filler to which an aminophenylboronic acid group having a high affinity for sugar is immobilized. Among them, the cation exchange chromatography can be classified into low pressure liquid chromatography and high performance liquid chromatography according to the magnitude of system pressure, the latter being an internationally recognized standard for detecting glycosylated hemoglobin HbA1c in clinical practice.
CN 114200070a discloses a liquid chromatography device, which comprises a sample adding system and a detecting system, wherein the sample adding system comprises an electromagnetic valve group, an injector, a sample adding needle, a hemolysis tank and a solution tank; the solution tank is internally provided with a hemolysis agent or a cleaning agent; the syringe, the sample adding needle, the solution tank and the hemolysis tank are all connected with the electromagnetic valve group through pipelines, and the syringe is communicated with the sample adding needle, the syringe is communicated with the hemolysis tank or the syringe is communicated with the solution tank through switching of the electromagnetic valve group. The device reduces the number of the syringes and simplifies the structure of the device through the arrangement of the electromagnetic valve group, but the detection and analysis process of specific substances are not mentioned, in particular the chromatographic analysis of the substances to be detected which need to be separated is not involved.
CN 114166906a discloses a multi-item detection integrated machine, which comprises an electrochemical detection mechanism, a high-pressure liquid chromatography mechanism and a sampling mechanism, wherein the electrochemical detection mechanism is used for detecting the contents of blood sugar and fructosamine, and the high-pressure liquid chromatography mechanism is used for detecting the content of glycosylated hemoglobin; the sample adding mechanism is connected with the high-pressure liquid chromatography mechanism and is used for conveying the sample solution to the high-pressure liquid chromatography mechanism and dripping the sample solution into the electrochemical detection mechanism; the device emphasizes the integration of an electrochemical detection mechanism and a liquid chromatography structure, so that three detection items can be carried out on the same instrument platform; although the high pressure liquid chromatography mechanism includes a glycosylated hemoglobin accurate measurement device, how to elute hemoglobin to separate different glycosylated hemoglobin does not clearly improve the detection accuracy, and the problem of cross contamination of the eluent is not involved.
In summary, for the device and the method for detecting the glycosylated hemoglobin HbA1c by adopting the liquid chromatography, the elution and separation of different glycosylated hemoglobin in the sample to be detected are also needed according to the structure of the detection device, so as to ensure high separation degree, reduce cross contamination, simplify the structure of the instrument, and reduce the running and maintenance cost of the instrument.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide a glycosylated hemoglobin detection device and a glycosylated hemoglobin detection method, by arranging a sample injection unit, a separation unit and a detection unit in the detection device, particularly by eluting a sample solution to be detected by adopting an eluent through the separation unit, the glycosylated hemoglobin detection device and the glycosylated hemoglobin detection method are convenient for high-efficiency separation and accurate detection of different components in glycosylated hemoglobin, and can reduce cross contamination of the eluent; the device has the advantages of simple structure, low operation and maintenance cost, strong detection repeatability and wide application range.
To achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the present invention provides a glycosylated hemoglobin detection apparatus, the detection apparatus comprising a sample injection unit, a separation unit, and a detection unit; the sample injection unit comprises a syringe, a sample needle, a sample ring and a sample valve, wherein a plurality of ports are arranged in the sample valve, the syringe is connected with the sample needle through two ports of the sample valve, and two ends of the sample ring are respectively connected to one port of the sample valve; the separation unit comprises eluent storage equipment, a high-pressure pump and a chromatographic separation column which are sequentially connected, the sample ring and the sample valve are further positioned on a connecting pipeline between the high-pressure pump and the chromatographic separation column in the separation unit, and an outlet of the chromatographic separation column is connected with an inlet of the detection unit.
According to the main component of glycosylated hemoglobin and the existing detection method, the invention adds a separation unit, in particular a sample ring and a sample valve as common components of the sampling unit and the separation unit on the basis of the original sampling unit and the detection unit by improving the structure of the detection device, and can be communicated with different pipelines, such as sampling of sample solution, separation and detection of the sample solution and the like, and the pipelines with different process conditions can be separated to avoid mutual influence by adjusting a plurality of ports in the sampling valve; the eluent is adopted to carry out elution and separation on the sample to be detected, so that different components in the glycosylated hemoglobin can be conveniently separated, particularly, the eluent with different concentrations is adopted to carry out ion exchange gradient elution according to the difference of charge properties of each component, thereby being beneficial to the full separation of different components and improving the detection accuracy of each component; the detection device has the advantages of simple structure, simple and convenient operation, strong repeatability, lower operation and maintenance cost and wide application prospect.
The following technical scheme is a preferred technical scheme of the invention, but is not a limitation of the technical scheme provided by the invention, and the technical purpose and beneficial effects of the invention can be better achieved and realized through the following technical scheme.
As a preferred technical scheme of the invention, the sample injection unit further comprises a sample pool, wherein the sample pool stores sample solution.
Preferably, the sample cell is located below the sample needle.
Preferably, the syringe is connected to a first port of the sample valve and then to the sample needle through a second port of the sample valve.
Preferably, a first passage is connected between the first port and the second port.
Preferably, two ends of the sample ring are connected to a third port and a fourth port of the sample valve, respectively, forming a loop.
Preferably, a second passage is connected between the third port and the second port, and a third passage is connected between the fourth port and the first port.
In the invention, besides the feeding of the sample solution, the sample injection unit can be connected with a pipeline on the basis of the existing sample injection unit equipment in the processes of generating the sample solution, cleaning the sample needle and the like, and an electromagnetic valve group can be arranged, so that different pipelines are communicated through the arrangement and adjustment of a plurality of electromagnetic valves, and the independent operation of each operation process is ensured.
As a preferred embodiment of the present invention, the eluent storage device at least comprises two eluent storage devices, preferably three eluent storage devices, arranged in parallel.
Preferably, the eluent storage device comprises a first eluent storage device, a second eluent storage device and a third eluent storage device arranged in parallel.
Preferably, the first control valve, the second control valve and the third control valve are correspondingly arranged on the connecting pipeline of each eluent storage device and the high-pressure pump.
Preferably, the chromatographic separation column is packed with a cation exchange resin.
In the invention, the glycosylated hemoglobin and the non-glycosylated hemoglobin with different charges in the sample solution are adsorbed on the surface of the resin, and then the eluent with different concentrations is used for exchange elution.
Preferably, the outlet of the high pressure pump is connected to a fifth port of the sample valve, and the inlet of the chromatographic separation column is connected to a sixth port of the sample valve.
Preferably, a fourth passage is connected between the fifth port and the fourth port of the sample valve, a fifth passage is connected between the sixth port and the third port of the sample valve, and the high-pressure pump, the fifth port, the fourth port, the sample ring, the third port, the sixth port and the chromatographic separation column are in passages.
In the invention, the separation unit is responsible for conveying the sample to be detected from the sample ring to the chromatographic separation column, and then conveying different eluents to the chromatographic separation column for ion exchange elution separation.
As a preferable technical scheme of the invention, the detection unit comprises a flow-through chamber and an electronic component.
Preferably, the electronic components include an LED light source, a photodiode, and an optical pickup board.
In the invention, the electronic components are matched for use and are used for detecting photoelectric signals, wherein the LED light source is used for generating light, and the light source irradiates the components to be detected to generate optical signals; the photodiode converts an optical signal into an electrical signal, and is a semiconductor device; the optical detection board card comprises a plurality of capacitance resistors, filtering circuits, related amplifying circuits and the like, and amplifies and filters the electric signals converted by the diodes to eliminate interference signals and obtain real signals of components to be detected.
In the invention, the detection unit is responsible for detecting and collecting photoelectric signals of different haemoglobin separated by elution.
Preferably, the detection device further comprises a control unit, and the control unit is connected with the sample injection unit, the separation unit and the detection unit.
Preferably, the control unit includes a hardware component including an optoelectronic signal analyzer and a test result display device, and a software component including control analysis software in the test result display device.
In the invention, the control unit is responsible for controlling each device according to a certain logic requirement, analyzing the collected signals and displaying the results.
In another aspect, the present invention provides a method for detecting glycosylated hemoglobin using the above-described detecting apparatus, the method comprising the steps of:
(1) Controlling the operation of the sample needle, the syringe and the sample valve to enable the sample ring to be communicated with the pipelines of the sample needle and the syringe, and conveying the sample solution to be tested into the sample ring;
(2) After the step (1) is finished, controlling the operation of a sample valve to ensure that a sample ring is communicated with a pipeline of a high-pressure pump and a chromatographic separation column, controlling the operation of the high-pressure pump, conveying sample solution in the sample ring to the chromatographic separation column, and conveying eluent with different concentrations to the chromatographic separation column for eluting and separating;
(3) And (3) conveying the different components eluted and separated in the step (2) to a detection unit for collecting and analyzing photoelectric signals to obtain a test analysis result of the glycosylated hemoglobin.
As a preferable technical scheme of the invention, the sample solution to be tested in the step (1) is formed by mixing a blood sample and a hemolysis agent.
Preferably, the sample needle of step (1) is moved to a sample collection site for collection of a sample solution.
Preferably, when the sample loop of step (1) is in communication with the sample needle and the tubing of the syringe, the sample needle, the second port, the second passageway, the third port, the sample loop, the fourth port, the third passageway, the first port, and the syringe form a passageway.
In the method for detecting glycosylated hemoglobin, the sample injection operation of the sample solution comprises the following steps: controlling the sample needle to run to a sample collection position; controlling the sample valve to operate so that the sample loop is communicated with the sample needle and the syringe pipeline; the syringe is controlled to operate for sample collection and the sample is transported into the sample loop.
As a preferable technical solution of the present invention, when the sample ring in step (2) is in communication with the high-pressure pump and the pipeline of the chromatographic separation column, the high-pressure pump, the fifth port, the fourth passage, the fourth port, the sample ring, the third port, the fifth passage, the sixth port, and the chromatographic separation column are in passage.
Preferably, the operating pressure of the high-pressure pump in the step (2) is 4 to 15MPa, for example, 4MPa, 6MPa, 8MPa, 10MPa, 12MPa, 14MPa or 15MPa, etc., but the operating pressure is not limited to the values listed, and the other values in the range are the values listed.
Preferably, the high pressure pump in step (2) delivers the eluent to the chromatographic separation column through the sample loop and delivers the sample solution to the chromatographic separation column in advance using fluid flow dynamics.
Preferably, the chromatographic separation column in the step (2) is filled with a cation exchange resin, and the cation exchange resin comprises a styrene cation exchange resin and an acrylic cation exchange resin according to the type of the matrix.
Preferably, the glycosylated hemoglobin and the non-glycosylated hemoglobin with different charges in the sample solution in the step (2) are adsorbed on the surface of the cation exchange resin, and then ion exchange elution separation is performed by using eluents with different concentrations.
As a preferable technical scheme of the invention, the eluent in the step (2) comprises three eluents with different concentrations, and the elution sequence is from low to high, namely a first eluent, a second eluent and a third eluent.
Preferably, the composition of the eluent in step (2) comprises sodium dihydrogen phosphate, sodium chloride and sodium azide.
Preferably, the concentration of sodium dihydrogen phosphate in the eluent of step (2) is independently 30-40 mmol/L, such as 30mmol/L, 32mmol/L, 34mmol/L, 36mmol/L, 38mmol/L or 40mmol/L, etc.; the concentration of sodium azide is independently 2 to 4mmol/L, such as 2mmol/L, 2.5mmol/L, 3mmol/L, 3.5mmol/L, 4mmol/L, etc.; however, the present invention is not limited to the above-mentioned values, and other values within the above-mentioned range are equally applicable.
Preferably, the different concentrations of the eluent in the step (2) are mainly different in concentration of sodium chloride, and the concentration of sodium chloride in the first eluent is 30-40 mmol/L, such as 30mmol/L, 32mmol/L, 34mmol/L, 36mmol/L, 38mmol/L, 40mmol/L, etc.; the concentration of sodium chloride in the second eluent is 50-60 mmol/L, such as 50mmol/L, 52mmol/L, 54mmol/L, 56mmol/L, 58mmol/L or 60mmol/L, etc.; the concentration of sodium chloride in the third eluent is 150-180 mmol/L, such as 150mmol/L, 155mmol/L, 160mmol/L, 165mmol/L, 170mmol/L, 175mmol/L or 180mmol/L, etc.; however, the present invention is not limited to the above-mentioned values, and other values within the respective ranges are equally applicable.
In the invention, the eluent is an inorganic salt buffer solution with wide pH range, and the Na ion has higher exchange capacity on cation exchange resin than amino positive ions on protein, so that the elution can be realized.
As a preferred embodiment of the present invention, the residence time of the single eluent in the chromatographic separation column in the step (2) is 10 to 25s, for example, 10s, 12s, 14s, 15s, 16s, 18s, 20s, 22s, 24s or 25s, etc., but not limited to the values recited, and other values within the range of values are equally applicable.
In the present invention, the eluent continuously flows and is eluted while flowing through the chromatographic separation column, and the retention time of the eluent in the chromatographic separation column is determined according to the concentration of the eluent and the separation degree of the chromatographic separation column.
Preferably, the eluent in the step (2) enters a chromatographic separation column and then is subjected to ion exchange elution, and the component to be detected in the sample solution enters the eluent.
Preferably, the components eluted by the different eluents in the step (2) are different, the high-pressure pump sequentially and continuously sends the different eluents with the concentration from low to high into the chromatographic separation column for eluting and separating, and the different components are sequentially and continuously separated for detection.
In the invention, after sample solution injection is completed, a sample valve is controlled to operate, so that a sample ring is communicated with a high-pressure pump and a chromatographic separation column pipeline; controlling the high-pressure pump and the first control valve to operate, conveying the samples in the sample ring to the chromatographic separation column, and conveying the first eluent to the chromatographic separation column for eluting separation; controlling the high-pressure pump and the second control valve to operate, and conveying the second eluent to the chromatographic separation column for elution and separation; and controlling the high-pressure pump and the third control valve to operate, and conveying the third eluent to the chromatographic separation column for eluting and separating.
In a preferred embodiment of the present invention, the eluent containing the eluent component in step (3) is transported to a flow-through chamber of the detection unit, and the detection is completed during the flow process.
Preferably, the collecting of the photoelectric signal in the step (3) includes: detecting the absorbance of the component to be detected, converting the absorbance into a corresponding electric signal according to the intensity of the absorbance, and converting the electric signal into a chromatographic signal spectrum for analysis.
Preferably, after the photoelectric signal in the step (3) is converted into a chromatographic signal, analysis and calculation are performed by adopting self-grinding algorithm software, and a map is generated by the analysis and calculation result and is displayed by display equipment.
According to the invention, through elution and separation, the operation of a high-pressure pump is controlled, different eluted components are conveyed into a flow chamber of a detection unit for collecting photoelectric signals, and then the photoelectric signals are analyzed and processed by a control unit to generate a map, and a test result is displayed through a display device.
The method for analyzing the photoelectric signal comprises the following steps: filtering the acquired original photoelectric signals by using electronic noise and optical noise, and converting the filtered signals into original chromatographic signals; acquiring a baseline value of the detection unit according to the original chromatographic signal; subtracting the original chromatographic signal values at different time points from the baseline value to obtain the chromatographic signal relative values (relative chromatographic signals) at different time points; obtaining different order derivatives of the chromatographic signal relative values at different time points, and obtaining a time starting point, a time ending point and a peak time point of a chromatographic peak through preset judging conditions; separating the chromatographic signal of the corresponding chromatographic peak from the relative chromatographic signal according to the time start and time end of the chromatographic peak.
According to the calculation formula A i ＝∫h t dt calculates the corresponding areas of different chromatographic peaks, wherein A i Represents the peak area, h, of the separated chromatographic peak t Represents the relative value of the chromatographic signal at time t. The content of the component to be measured is calculated based on the areas of the different chromatographic peaks.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, through structural improvement of the detection device, a separation unit is added on the basis of an original sample injection unit and a detection unit, particularly a sample ring and a sample valve are used as common components of the sample injection unit and the separation unit, different pipelines can be communicated through adjustment of a plurality of ports in the sample injection valve, and the pipelines under different process conditions can be separated, so that mutual influence is avoided;
(2) According to the method, the eluent is adopted to carry out elution and separation on the sample to be detected, so that different components in glycosylated hemoglobin can be separated conveniently, particularly, the eluent with different concentrations is adopted, and ion exchange gradient elution is carried out according to the difference of charge properties of each component, so that the full separation of different components is facilitated, and the detection accuracy of each component is improved;
(3) The detection device provided by the invention has the advantages of simple structure, simplicity and convenience in operation, strong repeatability, lower running and maintenance cost and wide application prospect.
Drawings
FIG. 1 is a schematic diagram showing the structure of a glycosylated hemoglobin measurement apparatus according to embodiment 1 of the present invention;
FIG. 2 is a test analysis result showing the glycosylated hemoglobin detection method according to example 3 of the present invention;
FIG. 3 is a test analysis result showing the glycosylated hemoglobin detection method according to example 4 of the present invention;
FIG. 4 is a test analysis result showing the glycosylated hemoglobin detection method according to example 5 of the present invention;
wherein, 1-syringe, 2-sample needle, 3-sample cell, 4-sample valve, 41-first port, 42-second port, 43-third port, 44-fourth port, 45-fifth port, 46-sixth port, 5-sample loop, 6-eluent storage device, 61-first eluent storage device, 62-second eluent storage device, 63-third eluent storage device, 71-first control valve, 72-second control valve, 73-third control valve, 8-high pressure pump, 9-chromatography separation column, 10-detection unit, 11-test result display device.
Detailed Description
For better illustrating the present invention, the technical scheme of the present invention is convenient to understand, and the present invention is further described in detail below. The following examples are merely illustrative of the present invention and are not intended to represent or limit the scope of the invention as defined in the claims.
The following are exemplary but non-limiting examples of the invention:
example 1:
the embodiment provides a glycosylated hemoglobin detection apparatus, the structural schematic diagram of which is shown in fig. 1, and the glycosylated hemoglobin detection apparatus includes a sample injection unit, a separation unit, and a detection unit 10; the sample injection unit comprises a syringe 1, a sample needle 2, a sample ring 5 and a sample valve 4, wherein a plurality of ports are arranged in the sample valve 4, the syringe 1 is connected with the sample needle 2 through two ports of the sample valve 4, and two ends of the sample ring 5 are respectively connected to one port of the sample valve 4; the separation unit comprises eluent storage equipment 6, a high-pressure pump 8 and a chromatographic separation column 9 which are sequentially connected, the sample ring 5 and the sample valve 4 are further positioned on a connecting pipeline between the high-pressure pump 8 and the chromatographic separation column 9 in the separation unit, and an outlet of the chromatographic separation column 9 is connected with an inlet of a detection unit 10.
The sample injection unit also comprises a sample pool 3, wherein a sample solution is stored; the cuvette 3 is located below the sample needle 2.
The syringe 1 is connected to a first port 41 of the sample valve 4 and then to the sample needle 2 via a second port 42 of the sample valve 4.
A first passage is connected between the first port 42 and the second port 42.
The two ends of the sample ring 5 are connected to a third port 43 and a fourth port 44 of the sample valve 4, respectively, forming a loop.
A second passage is connected between the third port 43 and the second port 42, and a third passage is connected between the fourth port 44 and the first port 41.
The eluent storage device 6 comprises three eluent storage devices 6 which are arranged in parallel, namely a first eluent storage device 61, a second eluent storage device 62 and a third eluent storage device 63.
The first control valve 71, the second control valve 72, and the third control valve 73 are provided in the connection line between each eluent reservoir 6 and the high-pressure pump 8.
The chromatographic separation column 9 is filled with a styrene cation exchange resin.
The outlet of the high pressure pump 8 is connected to a fifth port 45 of the sample valve 4 and the inlet of the chromatographic separation column 9 is connected to a sixth port 46 of the sample valve 4.
A fourth passage is connected between the fifth port 45 and the fourth port 44 of the sample valve 4, a fifth passage is connected between the sixth port 46 and the third port 43 of the sample valve 4, and the high-pressure pump 8, the fifth port 45, the fourth port 44, the sample ring 5, the third port 43, the sixth port 46, and the chromatographic separation column 9 form a passage.
The detection unit 10 comprises a flow-through chamber and electronic components; the electronic components comprise an LED light source, a photodiode and an optical detection board card.
The detection device further comprises a control unit, and the control unit is connected with the sample injection unit, the separation unit and the detection unit 10.
The control unit comprises hardware components and software components, the hardware components comprise an optoelectronic signal analyzer and a test result display device 11, and the software components comprise control analysis software in the test result display device 11.
Example 2:
the embodiment provides a glycosylated hemoglobin detection apparatus, which comprises a sample injection unit, a separation unit and a detection unit 10; the sample injection unit comprises a syringe 1, a sample needle 2, a sample ring 5 and a sample valve 4, wherein a plurality of ports are arranged in the sample valve 4, the syringe 1 is connected with the sample needle 2 through two ports of the sample valve 4, and two ends of the sample ring 5 are respectively connected to one port of the sample valve 4; the separation unit comprises eluent storage equipment 6, a high-pressure pump 8 and a chromatographic separation column 9 which are sequentially connected, the sample ring 5 and the sample valve 4 are further positioned on a connecting pipeline between the high-pressure pump 8 and the chromatographic separation column 9 in the separation unit, and an outlet of the chromatographic separation column 9 is connected with an inlet of a detection unit 10.
The sample injection unit also comprises a sample pool 3, wherein a sample solution is stored; the cuvette 3 is located below the sample needle 2.
The syringe 1 is connected to a first port 41 of the sample valve 4 and then to the sample needle 2 via a second port 42 of the sample valve 4.
A first passage is connected between the first port 42 and the second port 42.
The two ends of the sample ring 5 are connected to a third port 43 and a fourth port 44 of the sample valve 4, respectively, forming a loop.
A second passage is connected between the third port 43 and the second port 42, and a third passage is connected between the fourth port 44 and the first port 41.
The eluent storage device 6 comprises four eluent storage devices 6 which are arranged in parallel, namely a first eluent storage device 61, a second eluent storage device 62, a third eluent storage device 63 and a fourth eluent storage device in sequence.
The first control valve 71, the second control valve 72, the third control valve 73 and the fourth control valve are provided in the connection line between each eluent reservoir 6 and the high-pressure pump 8.
The chromatographic separation column 9 is packed with an acrylic cation exchange resin.
The outlet of the high pressure pump 8 is connected to a fifth port 45 of the sample valve 4 and the inlet of the chromatographic separation column 9 is connected to a sixth port 46 of the sample valve 4.
A fourth passage is connected between the fifth port 45 and the fourth port 44 of the sample valve 4, a fifth passage is connected between the sixth port 46 and the third port 43 of the sample valve 4, and the high-pressure pump 8, the fifth port 45, the fourth port 44, the sample ring 5, the third port 43, the sixth port 46, and the chromatographic separation column 9 form a passage.
The detection unit 10 comprises a flow-through chamber and electronic components; the electronic components are an LED light source, a photodiode and an optical detection board card.
The detection device further comprises a control unit, and the control unit is connected with the sample injection unit, the separation unit and the detection unit 10.
The control unit comprises hardware components and software components, the software components comprise an optoelectronic signal analyzer and a test result display device 11, and the software components comprise control analysis software in the test result display device 11.
Example 3:
the present embodiment provides a glycosylated hemoglobin detection method, which is performed using the apparatus of embodiment 1, comprising the steps of:
(1) Controlling the operation of the sample needle 2, the injector 1 and the sample valve 4 to enable the sample ring 5 to be communicated with the pipelines of the sample needle 2 and the injector 1, forming a passage by the sample needle 2, the second port 42, the second passage, the third port 43, the sample ring 5, the fourth port 44, the third passage, the first port 41 and the injector 1, and conveying a sample solution to be tested into the sample ring 5, wherein the sample solution to be tested is formed by mixing a blood sample and a hemolytic agent, and the sample needle 2 is operated to a sample collecting position, namely, a position above the sample pool 3 for collecting the sample solution;
(2) After the step (1) is finished, controlling the operation of the sample valve 4 to enable the sample ring 5 to be communicated with the high-pressure pump 8 and the pipeline of the chromatographic separation column 9, forming a passage by the high-pressure pump 8, the fifth port 45, the fourth passage, the fourth port 44, the sample ring 5, the third port 43, the fifth passage, the sixth port 46 and the chromatographic separation column 9, controlling the operation of the high-pressure pump 8, conveying sample solution in the sample ring 5 into the chromatographic separation column 9, wherein the operation pressure of the high-pressure pump 8 is 10MPa, filling styrene cation exchange resin in the chromatographic separation column 9, adsorbing glycosylated hemoglobin and non-glycosylated hemoglobin in the sample solution on the surface of the cation exchange resin, conveying three eluents with different concentrations into the chromatographic separation column 9 respectively, and performing ion exchange elution separation by using eluents with different cation concentrations;
the eluent comprises sodium dihydrogen phosphate, sodium chloride and sodium azide, the three eluents are introduced sequentially from low to high in concentration, namely a first eluent, a second eluent and a third eluent, the concentration of the sodium dihydrogen phosphate in the three eluents is 32mmol/L, the concentration of the sodium azide is 3mmol/L, the concentration of the sodium chloride in the three eluents is from low to high in concentration, namely 35mmol/L, 55mmol/L and 160mmol/L, the retention time of the eluents in the chromatographic separation column 9 is 20s, the eluents enter the chromatographic separation column 9 and then are subjected to ion exchange elution, and the components to be detected in the sample solution enter the eluent;
(3) Conveying different components eluted and separated in the step (2) to a flow-through chamber of a detection unit 10 for collecting and analyzing photoelectric signals, wherein components eluted by eluents with different concentrations are different and detected respectively, and the collecting of the photoelectric signals comprises the following steps: the absorbance of the component to be detected is detected firstly, then the absorbance is converted into a corresponding electric signal according to the intensity of the absorbance, the electric signal is converted into a chromatographic signal spectrum, the chromatographic signal spectrum is analyzed by adopting self-grinding algorithm software, the analysis result generates a spectrum, and the spectrum is displayed by test result display equipment 11, so that the test analysis result of the glycosylated hemoglobin is obtained.
In this embodiment, the above method is used to detect the component of glycosylated hemoglobin, and the test analysis result is shown in fig. 2, and includes four parts from position 1 to position 4; position 1 can be seen for the NGSP value (%) and IFCC value (mmol/mol), hbF (%), hbA1 (%) of HbA1 c; position 2 can view sample information; position 3 can view the percentage content, retention time (S) and peak area of GHb 7 components (FP, A1A, A1B, F, LA c+, SA1C, A0), and also view the number of trays and total area of the current detection result; position 4 can be used for looking at the chromatogram of the sample result, and position 4 can be used for looking at a plurality of peaks, namely FP peak, A1A peak 4-1, A1B peak 4-2, F peak 4-3, la1C+ peak 4-4, SA1C peak 4-5 and A0 peak 4-6, in sequence, and the peaks in the darkened shadow part are SA1C peaks. The GHb represents glycosylated hemoglobin, and HbA1c represents glycosylated hemoglobin, the latter being one of the former.
According to the test analysis result and the eluent adopted respectively, the main components eluted by the first eluent are A1A and A1B, the main components eluted by the second eluent are LA1C+ and SA1C, the main component eluted by the mixed solution of the first eluent and the second eluent is F, and the main component eluted by the third eluent is A0.
In this example, the accuracy of HbA1c component content measurement in glycosylated hemoglobin can reach 99.5%.
Example 4:
the present embodiment provides a glycosylated hemoglobin detection method, which is performed using the apparatus of embodiment 1, comprising the steps of:
(1) Controlling the operation of the sample needle 2, the injector 1 and the sample valve 4 to enable the sample ring 5 to be communicated with the pipelines of the sample needle 2 and the injector 1, forming a passage by the sample needle 2, the second port 42, the second passage, the third port 43, the sample ring 5, the fourth port 44, the third passage, the first port 41 and the injector 1, and conveying a sample solution to be tested into the sample ring 5, wherein the sample solution to be tested is formed by mixing a blood sample and a hemolytic agent, and the sample needle 2 is operated to a sample collecting position, namely, a position above the sample pool 3 for collecting the sample solution;
(2) After the step (1) is finished, controlling the operation of the sample valve 4 to enable the sample ring 5 to be communicated with the high-pressure pump 8 and the pipeline of the chromatographic separation column 9, forming a passage by the high-pressure pump 8, the fifth port 45, the fourth passage, the fourth port 44, the sample ring 5, the third port 43, the fifth passage, the sixth port 46 and the chromatographic separation column 9, controlling the operation of the high-pressure pump 8 to convey the sample solution in the sample ring 5 to the chromatographic separation column 9, wherein the operation pressure of the high-pressure pump 8 is 5MPa, the chromatographic separation column 9 is filled with cation exchange resin, glycosylated hemoglobin and non-glycosylated hemoglobin in different charges in the sample solution are adsorbed on the surface of the cation exchange resin, and then conveying three eluents with different concentrations to the chromatographic separation column 9 respectively, and carrying out ion exchange elution separation by using eluents with different cation concentrations;
the eluent comprises sodium dihydrogen phosphate, sodium chloride and sodium azide, the three eluents are introduced sequentially from low to high in concentration, namely a first eluent, a second eluent and a third eluent, the concentration of the sodium dihydrogen phosphate in the three eluents is 36mmol/L, the concentration of the sodium azide is 2mmol/L, the concentration of the sodium chloride in the three eluents is from low to high in concentration, namely 40mmol/L, 60mmol/L and 150mmol/L, the retention time of the eluents in the chromatographic separation column 9 is 25s, the eluents enter the chromatographic separation column 9 and then are subjected to ion exchange elution, and the components to be detected in the sample solution enter the eluent;
(3) Conveying different components eluted and separated in the step (2) to a flow-through chamber of a detection unit 10 for collecting and analyzing photoelectric signals, wherein components eluted by eluents with different concentrations are different and detected respectively, and the collecting of the photoelectric signals comprises the following steps: the absorbance of the component to be detected is detected firstly, then the absorbance is converted into a corresponding electric signal according to the intensity of the absorbance, the electric signal is converted into a chromatographic signal spectrum, the chromatographic signal spectrum is analyzed by adopting self-grinding algorithm software, the analysis result generates a spectrum, and the spectrum is displayed by test result display equipment 11, so that the test analysis result of the glycosylated hemoglobin is obtained.
In this example, the same method was used to perform the component measurement of glycosylated hemoglobin, and the test analysis results shown in FIG. 3 are shown, and four portions are also included in FIG. 3, and the parameters that can be seen in this example are the same as those in example 3, including the NGSP value (%) and IFCC value (mmol/mol), hbF (%), hbA1 (%); sample information; percentage content of GHb 7 components, retention time (S) and peak area; the chromatogram of the sample result is shown in fig. 3.
In this example, the accuracy of measurement of HbA1c component content in glycosylated hemoglobin was 99.54%.
Example 5:
the present embodiment provides a glycosylated hemoglobin detection method, which is performed using the apparatus of embodiment 1, comprising the steps of:
(1) Controlling the operation of the sample needle 2, the injector 1 and the sample valve 4 to enable the sample ring 5 to be communicated with the pipelines of the sample needle 2 and the injector 1, forming a passage by the sample needle 2, the second port 42, the second passage, the third port 43, the sample ring 5, the fourth port 44, the third passage, the first port 41 and the injector 1, and conveying a sample solution to be tested into the sample ring 5, wherein the sample solution to be tested is formed by mixing a blood sample and a hemolytic agent, and the sample needle 2 is operated to a sample collecting position, namely, a position above the sample pool 3 for collecting the sample solution;
(2) After the step (1) is finished, controlling the operation of the sample valve 4 to enable the sample ring 5 to be communicated with the high-pressure pump 8 and the pipeline of the chromatographic separation column 9, forming a passage by the high-pressure pump 8, the fifth port 45, the fourth passage, the fourth port 44, the sample ring 5, the third port 43, the fifth passage, the sixth port 46 and the chromatographic separation column 9, controlling the operation of the high-pressure pump 8 to convey the sample solution in the sample ring 5 to the chromatographic separation column 9, wherein the operation pressure of the high-pressure pump 8 is 15MPa, the chromatographic separation column 9 is filled with cation exchange resin, glycosylated hemoglobin and non-glycosylated hemoglobin with different charges in the sample solution are adsorbed on the surface of the cation exchange resin, and then respectively conveying three eluents with different concentrations to the chromatographic separation column 9 for elution separation, and carrying out ion exchange elution separation by using eluents with different cation concentrations;
The eluent comprises sodium dihydrogen phosphate, sodium chloride and sodium azide, the three eluents are introduced sequentially from low to high in concentration, namely a first eluent, a second eluent and a third eluent, the concentration of the sodium dihydrogen phosphate in the three eluents is 40mmol/L, the concentration of the sodium azide is 4mmol/L, the concentration of the sodium chloride in the three eluents is from low to high in concentration, namely 30mmol/L, 50mmol/L and 180mmol/L, the retention time of the eluents in a chromatographic separation column is 15s, the eluents are subjected to ion exchange elution after entering the chromatographic separation column 9, and the components to be detected in the sample solution enter the eluent;
(3) Conveying different components eluted and separated in the step (2) to a flow-through chamber of a detection unit 10 for collecting and analyzing photoelectric signals, wherein components eluted by eluents with different concentrations are different and detected respectively, and the collecting of the photoelectric signals comprises the following steps: the absorbance of the component to be detected is detected firstly, then the absorbance is converted into a corresponding electric signal according to the intensity of the absorbance, the electric signal is converted into a chromatographic signal spectrum, the chromatographic signal spectrum is analyzed by adopting self-grinding algorithm software, the analysis result generates a spectrum, and the spectrum is displayed by test result display equipment 11, so that the test analysis result of the glycosylated hemoglobin is obtained.
In this example, the same method was used to perform the component measurement of glycosylated hemoglobin, and the test analysis results shown in FIG. 4 are shown, and four portions are also included in FIG. 4, and the parameters that can be seen in the same manner as in example 3 include the NGSP value (%) and IFCC value (mmol/mol), hbF (%), hbA1 (%) of HbA1c, respectively; sample information; percentage content of GHb 7 components, retention time (S) and peak area; the chromatogram of the sample result is shown in fig. 4.
In this example, the accuracy of measurement of HbA1c component content in glycosylated hemoglobin was 99.49%.
According to the embodiment, through structural improvement of the detection device, the separation unit is added on the basis of the original sample injection unit and the detection unit, particularly the sample ring and the sample valve are used as common components of the sample injection unit and the separation unit, different pipelines can be communicated through adjustment of a plurality of ports in the sample injection valve, and the pipelines under different process conditions can be separated, so that mutual influence is avoided; the method adopts the eluent to carry out elution and separation on the sample to be detected, is convenient for separating different components in glycosylated hemoglobin, particularly adopts the eluent with different concentrations, and respectively carries out ion exchange gradient elution according to the difference of charge properties of each component, thereby being beneficial to the full separation of different components and improving the detection accuracy of each component; the detection device has the advantages of simple structure, simple and convenient operation, strong repeatability, lower operation and maintenance cost and wide application prospect.
The present invention is described in detail by the above embodiments, but the present invention is not limited to the above detailed devices and methods, i.e., it does not mean that the present invention must be implemented by the above detailed devices and methods. It should be apparent to those skilled in the art that any modifications of the present invention, equivalent substitutions for the apparatus of the present invention, addition of auxiliary apparatus, selection of specific modes, etc., are within the scope of the present invention and the scope of the disclosure.
Claims (10)
1. The glycosylated hemoglobin detection apparatus is characterized by comprising a sample injection unit, a separation unit and a detection unit; the sample injection unit comprises a syringe, a sample needle, a sample ring and a sample valve, wherein a plurality of ports are arranged in the sample valve, the syringe is connected with the sample needle through two ports of the sample valve, and two ends of the sample ring are respectively connected to one port of the sample valve; the separation unit comprises eluent storage equipment, a high-pressure pump and a chromatographic separation column which are sequentially connected, the sample ring and the sample valve are further positioned on a connecting pipeline between the high-pressure pump and the chromatographic separation column in the separation unit, and an outlet of the chromatographic separation column is connected with an inlet of the detection unit.
2. The glycosylated hemoglobin detection apparatus according to claim 1, wherein said sample introduction unit further comprises a sample cell in which a sample solution is stored;
preferably, the sample cell is located below the sample needle;
preferably, the syringe is connected to a first port of the sample valve and then to the sample needle through a second port of the sample valve;
preferably, a first passage is connected between the first port and the second port;
preferably, two ends of the sample ring are respectively connected to a third port and a fourth port of the sample valve to form a loop;
preferably, a second passage is connected between the third port and the second port, and a third passage is connected between the fourth port and the first port.
3. The glycosylated hemoglobin detection apparatus according to claim 1 or 2, characterized in that the eluent storage device comprises at least two eluent storage devices, preferably three, arranged in parallel;
preferably, the eluent storage device comprises a first eluent storage device, a second eluent storage device and a third eluent storage device which are arranged in parallel;
preferably, a first control valve, a second control valve and a third control valve are correspondingly arranged on the connecting pipeline of each eluent storage device and the high-pressure pump;
Preferably, the chromatographic separation column is filled with cation exchange resin;
preferably, the outlet of the high pressure pump is connected to a fifth port of the sample valve, and the inlet of the chromatographic separation column is connected to a sixth port of the sample valve;
preferably, a fourth passage is connected between the fifth port and the fourth port of the sample valve, a fifth passage is connected between the sixth port and the third port of the sample valve, and the high-pressure pump, the fifth port, the fourth port, the sample ring, the third port, the sixth port and the chromatographic separation column are in passages.
4. The glycosylated hemoglobin detection apparatus according to any one of claims 1 to 3, wherein the detection unit comprises a flow-through chamber and an electronic component;
preferably, the electronic components comprise an LED light source, a photodiode and an optical detection board card;
preferably, the detection device further comprises a control unit, and the control unit is connected with the sample injection unit, the separation unit and the detection unit;
preferably, the control unit includes a hardware component including an optoelectronic signal analyzer and a test result display device, and a software component including control analysis software in the test result display device.
5. A method for detecting glycosylated hemoglobin using the detection apparatus as defined in any one of claims 1 to 4, wherein said method comprises the steps of:
(1) Controlling the operation of the sample needle, the syringe and the sample valve to enable the sample ring to be communicated with the pipelines of the sample needle and the syringe, and conveying the sample solution to be tested into the sample ring;
(2) After the step (1) is finished, controlling the operation of a sample valve to ensure that a sample ring is communicated with a pipeline of a high-pressure pump and a chromatographic separation column, controlling the operation of the high-pressure pump, conveying sample solution in the sample ring to the chromatographic separation column, and conveying eluent with different concentrations to the chromatographic separation column for eluting and separating;
(3) And (3) conveying the different components eluted and separated in the step (2) to a detection unit for collecting and analyzing photoelectric signals to obtain a test analysis result of the glycosylated hemoglobin.
6. The method of claim 5, wherein the sample solution to be tested in step (1) is formed by mixing a blood sample with a hemolyzing agent;
preferably, the sample needle in the step (1) is operated to a sample collection position to collect a sample solution;
preferably, when the sample loop of step (1) is in communication with the sample needle and the tubing of the syringe, the sample needle, the second port, the second passageway, the third port, the sample loop, the fourth port, the third passageway, the first port, and the syringe form a passageway.
7. The method of claim 5 or 6, wherein when the sample loop of step (2) is in communication with the high pressure pump, the line of the chromatographic separation column, the high pressure pump, the fifth port, the fourth port, the sample loop, the third port, the fifth port, the sixth port, and the chromatographic separation column are in communication;
preferably, the operating pressure of the high-pressure pump in the step (2) is 4-15 MPa;
preferably, the high-pressure pump in the step (2) conveys the eluent to the chromatographic separation column through a sample ring in advance by utilizing the dynamic force of liquid flow;
preferably, the chromatographic separation column of step (2) is packed with a cation exchange resin;
preferably, the glycosylated hemoglobin and the non-glycosylated hemoglobin with different charges in the sample solution in the step (2) are adsorbed on the surface of the cation exchange resin, and then ion exchange elution separation is performed by using eluents with different concentrations.
8. The method according to any one of claims 5-7, wherein the eluent in step (2) comprises three eluents of different concentrations, and the eluents are introduced in the order of from low to high concentrations, namely a first eluent, a second eluent and a third eluent;
Preferably, the composition of the eluent of step (2) comprises sodium dihydrogen phosphate, sodium chloride and sodium azide;
preferably, the concentration of the sodium dihydrogen phosphate in the eluents with different concentrations in the step (2) is independently 30-40 mmol/L, and the concentration of the sodium azide is independently 2-4 mmol/L;
preferably, the different concentrations of the eluent in the step (2) are mainly different in concentration of sodium chloride, the concentration of sodium chloride in the first eluent is 30-40 mmol/L, the concentration of sodium chloride in the second eluent is 50-60 mmol/L, and the concentration of sodium chloride in the third eluent is 150-180 mmol/L.
9. The method according to any one of claims 5 to 8, wherein the residence time of the single eluent in the chromatographic separation column in step (2) is from 10 to 25s;
preferably, the eluent in the step (2) enters a chromatographic separation column and then is subjected to ion exchange elution, and components to be detected in the sample solution enter the eluent;
preferably, in the step (2), the components eluted by the eluents with different concentrations are different, the high-pressure pump sequentially and continuously sends the different eluents with the concentrations from low to high into the chromatographic separation column for eluting and separating, and sequentially and continuously separates the different components for detection.
10. The method of any one of claims 5 to 9, wherein the eluent containing the eluting component in step (3) is delivered to a flow-through chamber of a detection unit;
preferably, the collecting the photoelectric signal in the step (3) includes: firstly detecting the absorbance of the component to be detected, converting the absorbance into a corresponding electric signal according to the intensity of the absorbance, and converting the electric signal into a chromatographic signal spectrum for analysis;
preferably, after the photoelectric signal in the step (3) is converted into a chromatographic signal, analysis and calculation are performed by adopting self-grinding algorithm software, and a map is generated by the analysis and calculation result and is displayed by test result display equipment.
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