CN114878668B - Full-automatic dry three-electrode electrochemiluminescence analyzer and application thereof in electrochemiluminescence detection - Google Patents

Full-automatic dry three-electrode electrochemiluminescence analyzer and application thereof in electrochemiluminescence detection Download PDF

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CN114878668B
CN114878668B CN202210518609.2A CN202210518609A CN114878668B CN 114878668 B CN114878668 B CN 114878668B CN 202210518609 A CN202210518609 A CN 202210518609A CN 114878668 B CN114878668 B CN 114878668B
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electrode
electrochemiluminescence
sheet
carrier sheet
detection
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CN114878668A (en
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章春笋
赖伟
周欣亚
石延阳
钟金彪
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South China Normal University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract

The invention discloses a full-automatic dry three-electrode electrochemiluminescence analyzer, which comprises a dry three-electrode electrochemiluminescence chip and a full-automatic electrochemiluminescence analyzer, wherein the dry three-electrode electrochemiluminescence chip comprises a detection sheet and a shell, the shell comprises an upper cover and a base, the detection sheet is arranged between the upper cover and the base and comprises a bottom plate, an electrode sheet and a carrier sheet, the electrode sheet is adhered below the bottom plate, the carrier sheet is adhered above the bottom plate, a counter electrode, a reference electrode and a working electrode are arranged on the substrate of the electrode sheet, an electrode contact is positioned in a substrate electric connection area, a hydrophilic area is arranged on the carrier sheet, a reaction reagent corresponding to a biomarker to be detected is fixed in a drying manner in the hydrophilic area, the upper cover is provided with a sample inlet and an open slot, the sample inlet coincides with the hydrophilic area, and the open slot corresponds to the electric connection area; the full-automatic electrochemiluminescence analyzer comprises a data processing and instrument control unit, an imaging detection unit, an electrochemical reaction excitation unit, an open detection window unit, a chargeable power supply, a display screen and a shell.

Description

Full-automatic dry three-electrode electrochemiluminescence analyzer and application thereof in electrochemiluminescence detection
Technical Field
The invention belongs to the technical field of medical analysis instruments, and particularly relates to a full-automatic dry three-electrode electrochemiluminescence analyzer capable of realizing quantitative detection of glutamic pyruvic transaminase and glycosylated hemoglobin.
Background
Glutamic-pyruvic transaminase (ALT) is also known as glutamate transaminase, and when liver tissue is destroyed, ALT is released from cells, resulting in rapid increase of ALT content in blood, so that ALT is recommended by the world health organization as the most sensitive detection index of liver function damage. At present, conventional methods for ALT enzyme activity detection include spectrophotometry, colorimetry and chromatography, but the methods are generally low in sensitivity, strict in pretreatment conditions and high in detection purity requirements, and large-scale instruments (such as spectrophotometers, enzyme labels and biochemical analyzers) are required to be matched, and are high in price, high in professional degree, complex in operation, large in size, long in detection time and high in maintenance cost, so that basic medical institutions, basic blood stations and families cannot purchase and use the ALT enzyme activity detection method.
Glycosylated hemoglobin (HbA 1 c) is produced from red blood cells in blood and glucose in serum under non-enzymatic conditions. The reaction is present in red blood cells, and has the characteristic of being slow and irreversible, and the content of the reaction in red blood cells can reflect the average blood sugar level of three to four months in a human body. Compared with blood sugar with larger fluctuation, hbA1c is not influenced by fasting, insulin injection or blood sugar reducing medicine administration when detecting whether a human body has diabetes, and becomes the gold standard for controlling and monitoring diabetes internationally at present. Currently, a conventional method for detecting HbA1c in hospitals is to use a large-scale biochemical analyzer for liquid-phase spectroscopy detection, and most of detection schemes selected in laboratories are kit antigen detection. The method has the advantages of long single detection period, high cost, large detection instrument volume, high price and great need of professional operators for operation.
The instant detection refers to clinical detection carried out by medical staff which does not receive clinical training or the patient, has the characteristics of simplifying complex operation, miniaturizing a large instrument, activating a detection site, timely reporting results, sharing result information and the like, can be operated by non-professional inspectors, and can rapidly obtain the detection results. Currently, electrochemiluminescence analyzers on the market suffer from the following drawbacks: (1) The system is large in size and difficult to detect in multiple scenes in real time; (2) The integration degree is low, and the universality among the elements is poor; (3) The automation degree is low, and the electrochemiluminescence signal needs to be manually processed by specific analysis software; (4) Most of solution carriers to be detected are wet chemical detection units such as porous mesh plates, liquid phase quartz tubes and the like, and dry type instant detection is difficult to realize; (5) the detection cost is high and the detection process is complex.
Therefore, there is a need for a full-automatic dry electrochemiluminescence analyzer that can be applied in multiple scenarios (such as hospitals, laboratories, families, etc.), can effectively overcome the drawbacks of the current electrochemiluminescence analyzers, and can realize low-cost instant detection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a full-automatic dry three-electrode electrochemical luminescence analyzer which has the advantages of simple structure, convenient operation, accurate detection, simple assembly and quick detection without professional personnel.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a full-automatic dry three-electrode electrochemiluminescence analyzer comprises a dry three-electrode electrochemiluminescence chip and a full-automatic electrochemiluminescence analyzer;
the dry three-electrode electrochemiluminescence chip comprises a detection sheet and a shell, wherein the shell comprises an upper cover and a base, the detection sheet is arranged between the upper cover and the base, the detection sheet comprises a bottom plate, an electrode sheet and a carrier sheet, the electrode sheet is adhered below the bottom plate, the carrier sheet is adhered above the bottom plate, a counter electrode, a reference electrode and a working electrode are arranged on a substrate of the electrode sheet, electrode contacts of the counter electrode, the reference electrode and the working electrode are positioned in an electric connection area of the substrate, a hydrophilic area is arranged on the carrier sheet, multiple reaction reagents corresponding to a biomarker to be detected are fixed in the hydrophilic area in a drying manner, the upper cover is provided with a sample inlet and an open slot, the sample inlet coincides with the hydrophilic area of the carrier sheet, and the open slot corresponds to the electric connection area of the electrode sheet;
the full-automatic electrochemiluminescence analyzer comprises a data processing and instrument control unit, an imaging detection unit, an electrochemical reaction excitation unit, an open detection window unit, a chargeable power supply, a display screen and a shell, wherein the data processing and instrument control unit is respectively connected with the display screen, the imaging detection unit, the electrochemical reaction excitation unit and the chargeable power supply, and the chargeable power supply is respectively connected with the display screen, the electrochemical reaction excitation unit and the open detection window unit;
the open detection window unit is used for conveying the dry three-electrode electrochemical luminescence chip to the position right below the imaging detection unit, the electrochemical reaction excitation unit is used for exciting an object to be detected to generate electrochemical luminescence reaction and send optical signals, the imaging detection unit is used for converting the optical signals generated by the electrochemical luminescence into electric signals and transmitting the electric signals to the data processing and instrument control unit, the data processing and instrument control unit is used for processing the received electric signals and transmitting the results to the display screen, and the display screen is used for man-machine interaction, image preview and result display.
Further, the biomarker to be detected is glutamic pyruvic transaminase ALT or fructosyl valine FV.
Further, the biomarker to be detected is glutamic pyruvic transaminase ALT, and the desiccation fixing reaction reagent of the carrier sheet comprises luminol, L-alanine, alpha-ketoglutarate, pyruvic oxidase PYOD and magnesium chloride MgCl 2 And thiamine pyrophosphate TPP.
Further, the biomarker to be detected is fructosyl valine FV, and the drying and fixing reaction reagent of the carrier sheet comprises luminol and fructosyl valine oxidase FAOD.
Further, the carrier sheet is made of a hydrophilic fiber material, which can be dust-free paper.
Further, the substrate of the electrode plate adopts a hydrophobic fiber material, and the hydrophobic fiber material can be hydrophobic cloth.
Further, be equipped with the mounting in the upper cover, be equipped with the recess in the base, the test piece is placed in the recess, and the mounting is pressed on the test piece.
Further, a circular region is provided on the working electrode, and the circular region of the working electrode is provided corresponding to the hydrophilic region of the carrier sheet.
The application of the full-automatic dry three-electrode electrochemiluminescence analyzer in electrochemiluminescence detection, when the biomarker to be detected is glutamic pyruvic transaminase ALT, the preparation method of the carrier sheet comprises the following steps:
firstly, 30. Mu.L of 12.5mM luminol is added dropwise to the hydrophilic region of the carrier sheet, then it is placed in an oven at 34 ℃ for drying for 30 minutes, secondly, 30. Mu.L of a mixed solution of 700mM L-alanine and 10mM alpha-ketoglutarate is added dropwise to the hydrophilic region of the carrier sheetWater area, air-dried at room temperature, and finally 10. Mu.L of 1.25g/L PYOD and 2.5mM MgCl 2 And 2.5mM TPP were added dropwise to the hydrophilic region of the carrier sheet and dried at room temperature, thereby obtaining a carrier sheet having the reactant immobilized thereon.
The application of the full-automatic dry three-electrode electrochemiluminescence analyzer in electrochemiluminescence detection, when the biomarker to be detected is fructosyl valine FV, the preparation method of the carrier sheet comprises the following steps:
first, 10. Mu.L of 15mM luminol was dropped to the hydrophilic region of the carrier sheet, which was then left to dry in an oven at 34℃for 1 hour, and then 7.5. Mu.L of 50U/mL of the FAOD solution with the protective agent added thereto was dropped to the hydrophilic region of the carrier sheet, which was left to dry in a refrigerator at 4℃for 4 hours, thereby obtaining a reagent-immobilized carrier sheet.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention combines three-electrode electrochemiluminescence and dry chemical technology, adopts dust-free paper to pre-solidate the reaction reagent, so that the structure is simpler, the manufacture is easier, the sensitivity and the linear range are good, the clinical detection requirements of ALT and HbA1c are met, the detection cost performance is high, the operability is strong, and the application range is wide.
2. According to the invention, a vertical flow mode of the solution to be detected is adopted, the sample inlet is aligned with the hydrophilic area of the carrier sheet on the detection sheet, the substance to be detected can be immediately contacted with the chemical reagent on the carrier sheet, and the reaction is rapid, so that the detection error and the time cost of the conventional lateral flow test strip caused by the flow mode are greatly reduced.
3. The invention can realize detection by directly dripping the solution to be detected, greatly simplifies the detection steps of ALT and HbA1c, saves labor and time, has simple operation, does not need professional personnel, and is beneficial to application and popularization in occasions such as basic medical units, flowing blood collection vehicles, laboratories, families, health examination centers and the like.
Drawings
Fig. 1 is a schematic structural view of a dry three-electrode electrochemical light emitting chip.
FIG. 2 is an exploded view of the test strip.
Fig. 3 is a schematic view of the structure of an electrode sheet.
Fig. 4 is a schematic structural view of a carrier sheet.
Fig. 5 is a schematic structural view of the upper cover.
Fig. 6 is a schematic structural view of the base.
FIG. 7 is a schematic diagram of the structure of a fully automatic dry three-electrode electrochemiluminescence analyzer of the present invention.
FIG. 8 is a graph of ECL intensity versus ALT concentration for the electrochemiluminescence, wherein inset A is a graph of ECL intensity versus ALT concentration for ALT concentrations in the range of 5-50U/L; panel B is a graph of the linear relationship between ECL intensity values and ALT concentration for ALT concentrations in the range of 50-1000U/L.
FIG. 9 is a graph showing the relationship between ECL strength and fructosyl valine FV concentration, wherein panel A is a graph showing the relationship between ECL strength and FV concentration in the range of 0.05-0.5 mM; panel B is a linear plot of ECL intensity values versus FV concentration for FV concentrations in the range of 0.5-2 mM.
Reference numerals illustrate:
a detection sheet-1; a housing-2; and (3) an upper cover: 2-1; and (2) base: 2-2; a bottom plate-3; electrode sheet-4; carrier sheet-5; a counter electrode-6; a reference electrode-7; a working electrode-8; a circular area-9; an electrical connection region-10; a hydrophobic region-11; hydrophilic region-12; a sample inlet-13; an open slot-14; a fixing member 15; a groove-16; a dry three-electrode electrochemical luminescence chip-17;
a full-automatic electrochemiluminescence analyzer-18; data processing and instrument control unit: 18-1; imaging detection unit: 18-2; electrochemical reaction excitation unit: 18-3; open detection window unit: 18-4; rechargeable power: 18-5; and (3) a display screen: 18-6; a shell: 18-7; and (3) a detection platform: 18-4-1; transfer station: 18-4-2.
Detailed Description
The full-automatic dry three-electrode electrochemiluminescence analyzer of the present invention will be further described with reference to the accompanying drawings and specific examples.
Example 1
Referring to fig. 7, the invention discloses a full-automatic dry three-electrode electrochemiluminescence analyzer, which comprises a dry three-electrode electrochemiluminescence chip 17 and a full-automatic electrochemiluminescence analyzer 18, wherein the dry three-electrode electrochemiluminescence chip 17 is arranged in the full-automatic electrochemiluminescence analyzer 18.
Referring to fig. 1 and 2, the dry three-electrode electrochemical light emitting chip includes a detection sheet 1 and a housing 2, the housing 2 includes an upper cover 2-1 and a base 2-2, and the detection sheet 1 is disposed between the upper cover 2-1 and the base 2-2. The detection sheet 1 includes a base plate 3, an electrode sheet 4, and a carrier sheet 5, the electrode sheet 4 being adhered below the base plate 3, and the carrier sheet 5 being adhered above the base plate 3.
Referring to fig. 2, 3 and 4, the substrate of the electrode sheet 4 is provided with a counter electrode 6, a reference electrode 7 and a working electrode 8, the working electrode 8 is provided with a circular area 9, and electrode contacts of the counter electrode 6, the reference electrode 7 and the working electrode 8 are located in an electrical connection area 10 of the substrate. The carrier sheet 5 comprises a hydrophobic area 11 and a hydrophilic area 12, the hydrophilic area 12 of the carrier sheet 5 is dried and fixed with a plurality of reaction reagents, and the circular area 9 of the working electrode 8 is arranged corresponding to the hydrophilic area 12 of the carrier sheet 5.
Referring to fig. 1, 5 and 6, the upper cover 2-1 is provided with a sample inlet 13 and an open slot 14, wherein the sample inlet 13 coincides with the hydrophilic region 12 of the carrier sheet 5, and the open slot 14 corresponds to the electrical connection region 10 of the detection sheet 1. The upper cover 2-1 is internally provided with a fixing piece 15, the base 2-2 is internally provided with a groove 16, the detection sheet 1 is placed in the groove 16, the fixing piece 15 is pressed on the detection sheet 1, and the fixing piece 15 is matched with the groove 16 to fix the bottom plate 3, the electrode sheet 4 and the carrier sheet 5.
In ALT detection, the desiccation fixation reagent of the carrier sheet 5 comprises luminol, L-alanine, alpha-ketoglutarate, pyruvate oxidase (PYOD), magnesium chloride (MgCl) 2 ) And thiamine pyrophosphate (TPP). ALT in the solution to be detected is detected immediately, and the detection principle is as follows: the L-alanine and alpha-ketoglutarate form pyruvic acid and L-glutamic acid under ALT catalysis, and pyruvic acid is expressed in PYOD (MgCl) 2 And TPP is coenzyme factor) to generate hydrogen peroxide (H) 2 O 2 ) Acetyl phosphate and carbon dioxide, H 2 O 2 Further as enhancement of luminol ECL reaction systemALT concentration can be calculated from ECL intensity values.
In HbA1c measurement, the dried immobilized reagent of the carrier sheet 5 includes luminol and fructosyl valine oxidase (FAOD). By detecting Fructosyl Valine (FV) in the solution to be detected, hbA1c is indirectly and immediately detected, and the detection principle is as follows: after the red blood cells in the blood sample are lysed, one unit HbA1c is lysed to produce two units FV, which under the action of FAOD produce H 2 O 2 ，H 2 O 2 And as an enhancer of the luminol ECL reaction system, the HbA1c content can be obtained by calculation according to the ECL intensity value.
The substrate of the electrode sheet 4 is made of a hydrophobic fiber material, and the hydrophobic fiber material can be hydrophobic cloth. The carrier sheet 5 is made of a hydrophilic fibrous material, which may be dust-free paper. The dust-free paper is an organic combination of a natural wood pulp surface and a polyester surface, has the advantages of sufficient toughness, dissolution resistance, strong liquid absorption, good light transmittance and the like, and well meets the requirements of reagent drying, sensor assembly, solution absorption to be tested and ECL signal acquisition.
The invention skillfully combines three-electrode electrochemiluminescence with a dry chemical technology, adopts low-cost dust-free paper to pre-solidate reaction reagents in the whole reaction process, and fully integrates the functions of a plurality of layers of test paper (such as a reagent layer, an auxiliary layer, a light-transmitting layer or a diffusion layer and the like) in the traditional test paper strip into a single-layer dust-free paper, so that the structure is simpler, the manufacture is easier, and the quantitative detection of ALT and HbA1c is realized by using the single-layer paper technology.
According to the invention, a vertical flow mode of the solution to be detected is adopted, the sample inlet 13 of the upper cover 2-1 is aligned with the hydrophilic area 12 of the carrier sheet 5 of the detection sheet 1, the substance to be detected can be in immediate contact with the chemical reagent on the carrier sheet 5, the reaction is rapid, and the detection error and the time cost caused by the flow mode of the conventional lateral flow test strip are greatly reduced.
The manufacturing method of the dry three-electrode electrochemical light-emitting chip specifically comprises the following steps:
s1, designing an electrode plate 4 and a carrier plate 5 by utilizing Adobe Illustrator software, thereby manufacturing a corresponding screen printing screen, and further manufacturing the electrode plate 4 and the carrier plate 5 by a screen printing technology; the housing 2 was designed using SolidWorks software and manufactured via a 3D printing device.
S2, preparing a carrier sheet 5 for drying and fixing the reaction reagent.
The preparation method of the carrier sheet 5 corresponding to ALT detection comprises the following steps: first 30 μl of 12.5mM luminol was added dropwise to the hydrophilic area 12 of the carrier sheet 5, which was then dried in an oven at 34 ℃ for 30 minutes; secondly, 30. Mu.L of a mixed solution of 700mM L-alanine and 10mM alpha-ketoglutaric acid was dropped onto the hydrophilic region 12 of the carrier sheet 5, which was left to stand at room temperature for air-drying; finally 10. Mu.L of 1.25g/L PYOD, 2.5mM MgCl 2 The mixed solution of 2.5mM TPP was dropped to the hydrophilic region 12 of the carrier sheet 5 and dried at room temperature, thereby obtaining a carrier sheet 5 having the reaction reagent immobilized thereon.
The preparation method of the carrier sheet 5 corresponding to HbA1c detection comprises the following steps: first 10 μl of 15mM luminol was added dropwise to the hydrophilic zone 12 of the carrier sheet 5, which was then dried in an oven at 34 ℃ for 1 hour; then, 7.5. Mu.L of the FAOD solution with the protective agent added thereto in 50U/mL was dropped onto the hydrophilic region 12 of the carrier sheet 5, and the resultant was left to stand in a refrigerator at 4℃for 4 hours, thereby obtaining a carrier sheet 5 with the reactant immobilized thereon.
S3, aligning the prepared electrode plate 4 with the lower side of the bottom plate 3 and adhering the electrode plate, adhering the prepared carrier plate 5 to a 2mm area vacated by the upper side of the bottom plate 3, aligning the center of a circular area 9 of the working electrode 8 with the center of a circular hydrophilic area 12 of the carrier plate 5, and obtaining a detection plate 1 of the dry three-electrode electrochemical luminescence chip 17; the detection sheet 1 is placed in the groove 16 of the base 2-2 of the housing 2, the upper cover 2-1 is assembled with the base 2-2, and the sample inlet 13 of the upper cover 2-1 is aligned with the circular hydrophilic area 12 of the carrier sheet 5 on the detection sheet 1, so that the dry three-electrode electrochemical luminescence chip 17 is obtained.
The invention realizes the dry three-electrode electrochemiluminescence technology for the first time and the application of the technology in the instant detection of ALT and HbA1c, provides the three-electrode electrochemiluminescence technology with the advantages of stability, reliability, high reaction speed, less reagent consumption, wide linear range, high sensitivity and low cost, and the on-site quantitative detection method of ALT and HbA1c, and provides a potential high cost performance detection method for medical institutions, blood stations of basic level, laboratories, families and the like.
Referring to fig. 7, the full-automatic electrochemiluminescence analyzer 18 includes a data processing and instrument control unit 18-1, an imaging detection unit 18-2, an electrochemiluminescence reaction excitation unit 18-3, an open detection window unit 18-4, a chargeable power supply 18-5, a display screen 18-6 and a housing 18-7, wherein the data processing and instrument control unit 18-1 is respectively connected with the display screen 18-6, the imaging detection unit 18-2, the electrochemiluminescence reaction excitation unit 18-3 and the chargeable power supply 18-5, and the chargeable power supply 18-5 is respectively connected with the display screen 18-6, the electrochemiluminescence reaction excitation unit 18-3 and the open detection window unit 18-4.
The data processing and instrument control unit 18-1 is equipped with a raspberry pi microprocessor, and controls the instrument and processes and analyzes the image signals by software. The imaging detection unit 18-2 includes a CMOS camera, an objective lens mounted on the camera, and a holder on which the camera is fixed. The electrochemical reaction excitation unit 18-3 includes a three-electrode electrochemiluminescence excitation circuit for exciting an electrochemiluminescence reaction. The open detection window unit 18-4 includes a transfer table, a detection table, on which the dry three-electrode electrochemiluminescence chip 17 is placed, and an electrode contact, which is connected to the electrical connection area 10 of the electrode sheet 4 on the detection sheet 1.
The detection operation steps of the full-automatic dry three-electrode electrochemiluminescence analyzer are as follows:
before the experiment starts, the rechargeable power supply 18-5 is turned on, the dry three-electrode electrochemical luminescence chip 17 is fixed on the detection table 18-4-1 of the open detection window unit 18-4, and the detection table 18-4-1 is located on the conveying table 18-4-2. The solution to be measured is dripped from the sample inlet 13 of the upper cover 2-1, and is rapidly adsorbed on the hydrophilic area 12 of the carrier sheet 5 in a vertical flow mode and reacts with the drying reagent fixed on the carrier sheet 5. At the same time, the hydrophilic region 12 of the carrier sheet 5 is closely adsorbed by the solution to the counter electrode 6, the reference electrode 7, and the working electrode 8 of the electrode sheet 4. The transfer table 18-4-2 is automatically transferred to the position right below the objective lens of the imaging detection unit 18-2 through a motor, an electrode contact in the open detection window unit 18-4 is connected with the electric connection area 10 in the detection sheet 1, after waiting for a few minutes, the electrochemical reaction excitation unit 18-3 is started, the coordinate of the electrochemical luminescence area is automatically positioned by the data processing and instrument control unit 18-1, the maximum electrochemical luminescence intensity value is automatically obtained and displayed on the display screen 18-6, and then the data is recorded and saved for further analysis.
The invention applies the dry three-electrode electrochemiluminescence technology to ALT and HbA1c detection for the first time, has good sensitivity and linear range, meets the clinical detection requirements of ALT and HbA1c, has high cost performance, strong operability and wide application range, greatly simplifies the detection steps of ALT and HbA1c, saves labor and time, is simple to operate, can realize detection without professional, and is beneficial to application and popularization in occasions such as basic medical units, mobile blood collection vehicles, laboratories, families, health physical examination centers and the like.
The invention realizes the mass production of the dry three-electrode electrochemiluminescence chip, can realize the quantitative instant detection of different targets (blood sugar, uric acid, lactic acid, hbA1c, ALT and the like) based on the enzyme substrate detection principle, adopts a low-cost full-automatic electrochemiluminescence analyzer, has simple assembly, high integration level, small volume, accurate detection and wide application prospect, and can achieve the detection effect of sample inlet/outlet only by simple operation.
Example 2
The embodiment is an application of a full-automatic dry three-electrode electrochemiluminescence analyzer in detecting ALT, and specifically comprises the following steps:
1. preferred experimental parameters include exposure time, excitation voltage, L-alanine concentration, alpha-ketoglutarate concentration, luminol concentration, PYOD concentration, and reaction time. Wherein the exposure time ranges from 100 to 600ms, preferably 400ms; the excitation voltage ranges from 0.5 to 2.5V, preferably 1.5V; the L-alanine concentration is in the range of 400 to 900mM, preferably 700mM; the concentration of alpha-ketoglutarate ranges from 2.5 to 20mM, preferably 10mM; the concentration of luminol ranges from 5 to 15mM, preferably 12.5mM; the concentration of PYOD is in the range of 0.25-2 g/L, preferably 1.25g/L; the reaction time ranges from 5 to 14 minutes, preferably 10 minutes.
2. And manufacturing a dry three-electrode ECL chip and adjusting a matched full-automatic electrochemiluminescence analyzer according to the preferred experimental parameters.
3. Several experimental groups were set, ALT concentrations were set to several different values: 0U/L, 5U/L, 10U/L, 20U/L, 50U/L, 100U/L, 200U/L, 500U/L, 1000U/L.
4. The procedure of the detection operation was the same as in example 1, and the detection result was shown in FIG. 8.
As can be seen from FIG. 8, the ECL intensity value increases with increasing ALT concentration at ALT concentrations in the range of 0-1000U/L. Furthermore, when the ALT concentration varies within the range of 5-50U/L, the ECL intensity value (expressed as Y) and the ALT concentration (expressed as X) show a linear relationship, and the linear equation can be expressed as Y=0.748X-0.558 (R) 2 = 0.9928) (insert a); when ALT concentration varies in the range of 50-1000U/L, ECL intensity values (expressed as Y) and ALT concentration logarithm (expressed as X) exhibit a linear relationship, which linear equation can be expressed as Y= 41.651X-34.114 (R) 2 = 0.9856) (insert B). Finally, the ALT detection limit calculated by using the linear equation is estimated to be 1.7015U/L. The method for calculating the detection limit comprises the following steps: y is Y L ＝Y b +3S b Wherein Y is b Mean ECL intensity value at blank, S b Standard deviation of the blank (five replicates) was used to obtain Y L And calculating the corresponding ALT concentration by the value to obtain the detection limit.
Example 3
The embodiment is an application of a full-automatic dry three-electrode electrochemiluminescence analyzer in HbA1c detection, and specifically comprises the following steps:
1. preferred experimental parameters include exposure time, excitation voltage, reaction time, buffer pH, luminol concentration, and FAOD concentration. Wherein the exposure time ranges from 100 to 1000ms, preferably 400ms; the excitation voltage ranges from 0.5 to 2.5V, preferably 1V; the reaction time ranges from 0.5 to 5 minutes, preferably 3 minutes; the pH range of the buffer solution is 8-12, preferably 9.5; the concentration of luminol ranges from 5 to 30mM, preferably 20mM; FAOD concentration is in the range of 10 to 100U/mL, preferably 50U/mL.
2. And manufacturing a dry three-electrode ECL chip and adjusting a matched full-automatic electrochemiluminescence analyzer according to the preferred experimental parameters.
3. Several experimental groups were set and HbA1c concentrations were set to several different values: 0mM, 0.05mM, 0.1mM, 0.2mM, 0.3mM, 0.5mM, 0.75mM, 1mM, 2mM.
4. The test procedure was the same as in example 1, except that the pretreatment step for the solution to be tested was added: the measurement result of adding the measurement solution containing HbA1c to a test tube containing a cell lysate to lyse HbA1c and generate free FV is shown in FIG. 9.
As can be seen from FIG. 9, the ECL strength value increases with increasing FV concentration in the range of 0-2 mM. In addition, when FV concentration varies in the range of 0.05-0.5mM, there is a good linear relationship between ECL intensity value (expressed as Y) and FV concentration (expressed as X), and the linear equation can be expressed as Y=7.339X+0.063 (R) 2 =0.9990) (insert a); when FV concentration varies in the range of 0.5-2mM, there is a good linear relationship between ECL intensity value (expressed as Y) and FV concentration (expressed as X), and the linear equation can be expressed as Y=1.3832X+2.992 (R) 2 = 0.9746) (insert B). Finally, the FV limit calculated using the linear equation was estimated to be 0.0022mM. The method for calculating the detection limit comprises the following steps: y is Y L ＝Y b +3S b Wherein Y is b Mean ECL intensity value at blank, S b Standard deviation of the blank (five replicates) was used to obtain Y L And calculating the corresponding FV concentration by the value to obtain the detection limit. Finally, the content and the detection limit of HbA1c in the solution to be detected can be obtained through the corresponding proportion relation between HbA1c and FV.
The foregoing description is directed to the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the invention, and all equivalent changes or modifications made under the technical spirit of the present invention should be construed to fall within the scope of the present invention.

Claims (10)

1. A full-automatic dry three-electrode electrochemiluminescence analyzer is characterized in that: comprises a dry three-electrode electrochemiluminescence chip and a full-automatic electrochemiluminescence analyzer;
the dry three-electrode electrochemiluminescence chip comprises a detection sheet and a shell, wherein the shell comprises an upper cover and a base, the detection sheet is arranged between the upper cover and the base, the detection sheet comprises a bottom plate, an electrode sheet and a carrier sheet, the electrode sheet is adhered below the bottom plate, the carrier sheet is adhered above the bottom plate, a counter electrode, a reference electrode and a working electrode are arranged on a substrate of the electrode sheet, electrode contacts of the counter electrode, the reference electrode and the working electrode are positioned in an electric connection area of the substrate, a hydrophilic area is arranged on the carrier sheet, multiple reaction reagents corresponding to a biomarker to be detected are fixed in the hydrophilic area in a drying manner, the upper cover is provided with a sample inlet and an open slot, the sample inlet coincides with the hydrophilic area of the carrier sheet, and the open slot corresponds to the electric connection area of the electrode sheet;
the full-automatic electrochemiluminescence analyzer comprises a data processing and instrument control unit, an imaging detection unit, an electrochemical reaction excitation unit, an open detection window unit, a chargeable power supply, a display screen and a shell, wherein the data processing and instrument control unit is respectively connected with the display screen, the imaging detection unit, the electrochemical reaction excitation unit and the chargeable power supply, and the chargeable power supply is respectively connected with the display screen, the electrochemical reaction excitation unit and the open detection window unit;
the open detection window unit is used for conveying the dry three-electrode electrochemical luminescence chip to the position right below the imaging detection unit, the electrochemical reaction excitation unit is used for exciting an object to be detected to generate electrochemical luminescence reaction and send optical signals, the imaging detection unit is used for converting the optical signals generated by the electrochemical luminescence into electric signals and transmitting the electric signals to the data processing and instrument control unit, the data processing and instrument control unit is used for processing the received electric signals and transmitting the results to the display screen, and the display screen is used for man-machine interaction, image preview and result display.
2. The fully automatic dry three-electrode electrochemiluminescence analyzer of claim 1, wherein the biomarker to be measured is glutamic pyruvic transaminase ALT or fructosyl valine FV.
3. The fully automatic dry three electrode electrochemiluminescence of claim 2The analyzer is characterized in that the biomarker to be detected is glutamic pyruvic transaminase ALT, and the desiccation fixing reaction reagent of the carrier sheet comprises luminol, L-alanine, alpha-ketoglutarate, pyruvic oxidase PYOD and magnesium chloride MgCl 2 And thiamine pyrophosphate TPP.
4. The full-automatic dry three-electrode electrochemiluminescence analyzer of claim 2, wherein the biomarker to be detected is fructosyl valine FV, and the drying and fixing reaction reagent of the carrier sheet comprises luminol and fructosyl valine oxidase FAOD.
5. The fully automatic dry three electrode electrochemiluminescence analyzer of claim 1 wherein the carrier sheet is a hydrophilic fiber material, which may be a dust free paper.
6. The fully automatic dry three-electrode electrochemiluminescence analyzer of claim 1, wherein the substrate of the electrode sheet is a hydrophobic fiber material, which may be a hydrophobic cloth.
7. The fully automatic dry three electrode electrochemiluminescence analyzer of claim 1 wherein a fixture is provided in the upper cover, a recess is provided in the base, the test strip is placed in the recess, and the fixture is pressed against the test strip.
8. The fully automatic dry three electrode electrochemiluminescence analyzer of claim 1 wherein the working electrode is provided with a circular region, the circular region of the working electrode being disposed in correspondence with the hydrophilic region of the carrier sheet.
9. The use of a fully automatic dry three electrode electrochemiluminescence analyzer according to any of claims 1 to 8 in electrochemiluminescence detection, wherein when the biomarker to be detected is glutamate pyruvate transaminase ALT, the preparation method of the carrier sheet is as follows:
first 30. Mu.L of 12.5mM luminol was added dropwise to the hydrophilic areas of the carrier sheet, which was then placedDrying in 34 deg.C oven for 30 min, dripping 30 μl of mixed solution of 700mM L-alanine and 10mM alpha-ketoglutaric acid into hydrophilic region of carrier sheet, air drying at room temperature, and air drying 10 μl of 1.25g/L PYOD and 2.5mM MgCl 2 And 2.5mM TPP were added dropwise to the hydrophilic region of the carrier sheet and dried at room temperature, thereby obtaining a carrier sheet having the reactant immobilized thereon.
10. The use of a fully automatic dry three electrode electrochemiluminescence analyzer according to any of claims 1 to 8 in electrochemiluminescence detection, wherein when the biomarker to be detected is fructosyl valine FV, the preparation method of the carrier sheet is:
first, 10. Mu.L of 15mM luminol was dropped to the hydrophilic region of the carrier sheet, which was then left to dry in an oven at 34℃for 1 hour, and then 7.5. Mu.L of 50U/mL of the FAOD solution with the protective agent added thereto was dropped to the hydrophilic region of the carrier sheet, which was left to dry in a refrigerator at 4℃for 4 hours, thereby obtaining a reagent-immobilized carrier sheet.
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