CN116643043A - Dry three-electrode electrochemiluminescence lateral flow immunosensor and detection application thereof - Google Patents
Dry three-electrode electrochemiluminescence lateral flow immunosensor and detection application thereof Download PDFInfo
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Abstract
The invention discloses a dry three-electrode electrochemiluminescence lateral flow immunosensor and detection application thereof, belonging to the technical field of microfluidic chips, wherein the sensor comprises a detection chip, and the detection chip comprises a sample adding pad, a combination pad, at least one detection pad, a quality control pad, an absorption pad, an electrode pad and a bottom plate; the electrode pad is provided with a common working electrode, a counter electrode and a reference electrode, the counter electrode is provided with a detection area counter electrode and a quality control area counter electrode, and the reference electrode is provided with a detection area reference electrode and a quality control area reference electrode; the sample adding pad, the combining pad, the detecting pad, the quality control pad and the absorbing pad are all provided with hydrophilic areas, the sample adding pad, the combining pad, the detecting pads, the quality control pad and the absorbing pad are sequentially arranged at the top of the electrode pad, the hydrophilic areas are sequentially communicated to form a single flow path, the hydrophilic areas of the detecting pad are respectively positioned right above the corresponding detecting area counter electrode and the detecting area reference electrode, and the hydrophilic areas of the quality control pad are positioned right above the quality control area counter electrode and the quality control area reference electrode.
Description
Technical Field
The invention relates to the technical field of microfluidic chips, in particular to a dry three-electrode electrochemiluminescence lateral flow immunosensor and detection application thereof.
Background
With the development of ECL detection technology, the electrodes used for the excitation reaction extend from the original two-electrode system to the three-electrode system. Compared with the traditional double-electrode system, the three-electrode system has stable electrode potential in the reaction process due to small driving voltage, has been successfully applied in the electrochemical and ECL fields, and becomes a research hot spot
A conventional three-electrode system includes a working electrode, a reference electrode, and a counter electrode. When a driving voltage is applied to the working electrode, current flows through the working electrode and the counter electrode, no current passes through the working electrode and the reference electrode as feedback loops, and potential difference is constant, so that the potential on the working electrode is kept constant in the reaction process, voltage drop caused by the reaction is avoided, and the current in a loop formed by the working electrode and the counter electrode is more stable.
ECL is an emerging detection means, and is gradually becoming a detection means for various markers due to the advantages of simple operation, low background signal, no need of using isotopes, high-flux detection and the like. The microfluidic three-electrode ECL biosensor combining the three-electrode system with ECL has good analysis performance, has been successfully applied to detection of markers such as genes, metal ions, protease, cells and the like at present, but still has some problems. For example, the electrode materials used for the sensor internal chip are expensive materials such as silver/silver chloride, gold, platinum carbon and the like; chip processing equipment is expensive; the chip modification process is complex; the conventional three-electrode ECL contains only one reaction cell, and it is difficult to achieve lateral flow required for immunodetection. These all greatly limit the application of three-electrode systems in the field of lateral flow immunodetection.
The dry chemical analysis technique is an analytical detection technique based on a dry chemical analysis technique. Compared with wet chemistry, dry chemistry can pre-dry the required reactants in the reaction area, simplifying the detection process. At present, the immune reagent dry plate developed based on the immune chromatography technology of the specific combination of the lateral flow and the antigen antibody utilizes fluorescent markers, gold nano-particle markers and the like to realize the detection of various biomarkers. The immunoreagent dry plate has the advantages of low price, simple and convenient use, rapid detection or no need of expensive instruments, and the like, overcomes some defects of the traditional wet ECL microfluidic biosensors, but is difficult to quantitatively detect, or is difficult to detect with high flux or needs a relatively expensive detector. Therefore, they are difficult to meet the need for quantitative, inexpensive, high throughput detection in one immunoassay.
In conclusion, the microfluidic biosensor has the advantages of quantification, low cost, rapidness, simplicity and convenience, is suitable for multi-scene instant detection, and can overcome the technical problem that the traditional three-electrode system is difficult to realize dry type immunodetection, which is to be solved by the technicians in the field.
Disclosure of Invention
The invention aims to solve the technical problems of the prior art, and the invention aims to provide a dry three-electrode electrochemiluminescence lateral flow immunosensor.
The invention also aims to provide a detection application of the dry three-electrode electrochemiluminescence lateral flow immunosensor.
In order to achieve the above purpose, the present invention provides a dry three-electrode electrochemiluminescence lateral flow immunosensor, comprising a detection chip, wherein the detection chip comprises a sample adding pad, a combination pad, at least one detection pad, a quality control pad, an absorption pad, an electrode pad and a bottom plate; the top of the electrode pad is provided with a common working electrode, the electrode pads at two sides of the common working electrode are respectively provided with a counter electrode and a reference electrode, one ends of the common working electrode, the counter electrode and the reference electrode in the same direction are all used as electric contact areas, the other end of the counter electrode is sequentially provided with a detection area counter electrode and a quality control area counter electrode which are equal in number and in one-to-one correspondence with the detection pads, and correspondingly, the other end of the reference electrode is sequentially provided with a detection area reference electrode and a quality control area reference electrode which are equal in number and in one-to-one correspondence with the detection pads, and the detection area counter electrode, the quality control area reference electrode and the quality control area reference electrode face the common working electrode; the sample adding pad is provided with a sample adding pad hydrophilic area, the combining pad is provided with a combining pad hydrophilic area, the detecting pad is provided with a detecting pad hydrophilic area, the quality control pad is provided with a quality control pad hydrophilic area, the absorbing pad is provided with an absorbing pad hydrophilic area, the sample adding pad, the combining pad, each detecting pad, the quality control pad and the absorbing pad are sequentially arranged at the top of the electrode pad, the sample adding pad hydrophilic area is sequentially communicated with the combining pad hydrophilic area, each detecting pad hydrophilic area, the quality control pad hydrophilic area and the absorbing pad hydrophilic area to form a single flow path, each detecting pad hydrophilic area is respectively positioned right above a corresponding detecting area counter electrode and a detecting area reference electrode, and the quality control pad hydrophilic area is positioned right above a quality control area counter electrode and a quality control area reference electrode.
As a further improvement, the electrode pad is made of a hydrophobic fiber material serving as a substrate and is made of a conductive material through screen printing, and the sample adding pad, the bonding pad, the detection pad, the quality control pad and the absorption pad are made of a hydrophilic fiber material serving as a substrate and form a hydrophobic area and a hydrophilic area on the substrate through screen printing of hydrophobic ink.
Further, the electrode pad is adhered to the top of the bottom plate, the middle parts of the sample adding pad, the combining pad, the detecting pad, the quality control pad and the absorbing pad are all clung to the electrode pad, and the two ends of the sample adding pad, the combining pad, the detecting pad, the quality control pad and the absorbing pad are adhered to the bottom plate.
Further, overlapping areas are arranged between the sample adding pad and the combining pad, between the combining pad and the detecting pad, between the detecting pad and the quality control pad, and between the quality control pad and the absorbing pad.
Further, the device also comprises an upper cover and a lower cover, wherein a mounting groove for mounting the detection chip is formed in the lower cover, the upper cover covers the detection chip, and the upper cover is respectively provided with a sample adding hole aligned with the hydrophilic areas of the sample adding pad, detection area observation windows aligned with the hydrophilic areas of the detection pads one by one, quality control area observation windows aligned with the hydrophilic areas of the quality control pads and an opening groove aligned with the electric contact area.
Further, the hydrophilic areas of the sample adding pad and the absorbent pad are treated by Tween; and drying and labeling the antibody after the hydrophilic region of the binding pad is treated by Tween.
Further, the detection pad hydrophilic region immobilizes a modified biomarker coated antibody.
Further, the hydrophilic region of the quality control pad is used for fixing and modifying the quality control coated antibody.
Further, the hydrophilic region of the detection pad and the hydrophilic region of the quality control pad are subjected to fixation modification by chitosan and glutaraldehyde.
In order to achieve the second purpose, the invention provides a detection application of a dry three-electrode electrochemiluminescence lateral flow immunosensor, which comprises the following steps:
s1, dropwise adding a sample solution to be detected containing a biomarker into a hydrophilic region of a sample adding pad of a sensor, wherein the sample solution to be detected flows through a hydrophilic region of a binding pad from the hydrophilic region of the sample adding pad, and the biomarker is specifically combined with a labeled antibody dried in the hydrophilic region of the binding pad to form a labeled antibody-biomarker complex; the complex further flows through each detection pad hydrophilic region and is combined with the biomarker coated antibody fixed on the detection pad hydrophilic region to form a sandwich-type complex of labeled antibody-biomarker-coated antibody immunization;
s2, after the immune reaction of the biological marker is finished, dropwise adding a buffer solution into the hydrophilic region of the sample adding pad, and removing the unbound labeled antibody in the hydrophilic region of the detection pad;
s3, starting the ECL analyzer, placing the sensor on an inlet and outlet module of the ECL analyzer, conveying the sensor to the position right below the imaging detection module by the inlet and outlet module, and communicating an electric contact area of the electrode pad with a constant potential module of the ECL analyzer;
s4, starting a detection button on the ECL analyzer, providing driving voltage through a constant potential module of the ECL analyzer, triggering the intramolecular reaction of the labeled antibody, generating an ECL signal, automatically collecting and analyzing the ECL signal by the ECL analyzer, and quantitatively detecting the biomarker according to the strength of the ECL signal.
Advantageous effects
Compared with the prior art, the invention has the advantages that:
1. according to the invention, the hydrophilic non-woven fabric and the hydrophobic electrode pad are skillfully used, each layer of pad can be respectively modified through a laminated structure, the problems of direct modification of the conventional dry plate electrode and mutual pollution of each region are avoided, lateral flow is combined, and three-electrode ECL immunodetection is creatively realized.
2. According to the design of the hydrophilic areas of the sample adding pad, the combining pad, the detecting pad, the quality control pad and the absorbing pad, the electrodes can enable current to form two parallel loops only after each layer of the chip is laminated and the flow path is full of the sample solution to be detected, so that the controllability of the chip triggering reaction is improved.
3. After the working electrode is communicated with the constant potential module of the analyzer, the detection loop and the quality control loop have different potentials and feedback signals on the premise of using the same working electrode, so that mutual interference between the detection area and the quality control area is avoided, and ECL reactions are independently carried out on the detection area and the quality control area respectively.
4. The dry three-electrode ECL lateral flow immunosensor provided by the invention uses a series flow path, so that the sensor can realize immunodetection by only one sample adding and washing, and the problem that the traditional three-electrode ECL lateral flow immunosensor needs multiple sample adding and washing when in wet immunodetection is solved.
5. The invention applies the dry three-electrode ECL lateral flow technology to the immunodetection for the first time, has good sensitivity and dynamic range, meets the requirement of the hypersensitive detection, and has low cost, convenient operation and short detection time compared with the conventional wet immunodetection.
6. According to the invention, the polylysine and ruthenium are combined by using the ruthenium compound, so that the polylysine and ruthenium are formed into an intramolecular co-reactant, and compared with the traditional ruthenium probe, the ECL signal intensity is greatly improved without adding the co-reactant, thereby improving the detection limit of the biomarker and expanding the dynamic detection range.
7. The biomarker detected by the invention can be replaced by changing the labeled antibody according to the requirement, thereby realizing the hypersensitive and quantitative detection of various biomarkers and greatly increasing the application range of the sensor.
8. The detection area and the quality control area of the sensor share the working electrode, and the multi-element immunodetection and quality control under a three-electrode system can be realized by simply prolonging the working electrode, the counter electrode and the reference electrode without increasing the total number of the electrodes.
9. The non-woven fabric used by the sensor is formed by combining natural wood pulp and artificial fibers, and has the advantages of being high in toughness, good in fluidity, high in light transmittance, convenient for ECL signals to pass through the detection pad and the quality control pad from the electrodes, and the like.
10. The invention uses the non-woven fabrics, greatly reduces the manufacturing cost of the sensor and simplifies the processing process of the sensor. This meets well the requirements of chip fabrication processing, reagent drying, solution flow and signal release.
11. The sensor provided by the invention is simple and convenient in use process, does not need professional operation, is beneficial to use in basic medical units or families, and has the value of multi-scene application.
Drawings
FIG. 1 is a schematic diagram of the structure of the present invention;
FIG. 2 is a schematic diagram of a laminated structure of a detection chip according to the present invention;
FIG. 3 is a schematic diagram of a structure of an electrode pad with only one detection area counter electrode according to the present invention;
FIG. 4 is a schematic diagram of a structure of an electrode pad with two detection area counter electrodes according to the present invention.
FIG. 5 is a schematic diagram showing the structure of a detection chip with only one detection area counter electrode in the present invention;
FIG. 6 is a schematic diagram of a detection chip with two detection area counter electrodes according to the present invention;
FIG. 7 is a graph showing the effect of exposure time on the ECL intensity values of dry three electrodes in accordance with the present invention;
FIG. 8 is a graph showing the effect of driving voltage on ECL strength of dry three electrodes in accordance with the present invention;
FIG. 9 is a graph showing the effect of labeled antibody dosage on the ECL intensity values of dry three electrodes in accordance with the present invention;
FIG. 10 is a graph showing the effect of glutaraldehyde amount on the dry three electrode ECL strength values in accordance with the present invention;
FIG. 11 is a graph showing the effect of cTnI coated antibody concentration on the ECL intensity values of dry three electrodes in the present invention;
FIG. 12 is a graph showing the effect of incubation time on the ECL intensity values of dry three electrodes in accordance with the present invention;
FIG. 13 is a graph of ECL intensity versus cTnI concentration for a dry three electrode in accordance with the present invention.
Wherein: 1-detection chip, 2-sample adding pad, 3-combination pad, 4-detection pad, 5-quality control pad, 6-absorption pad, 7-electrode pad, 8-common working electrode, 9-counter electrode, 10-reference electrode, 11-electric contact area, 12-detection area counter electrode, 13-quality control area counter electrode, 14-detection area reference electrode, 15-quality control area reference electrode, 16-sample adding pad hydrophilic area, 17-combination pad hydrophilic area, 18-detection pad hydrophilic area, 19-quality control pad hydrophilic area, 20-absorption pad hydrophilic area, 21-bottom plate, 22-upper cover, 23-lower cover, 24-sample adding hole, 25-detection area observation window, 26-quality control area observation window, 27-open slot, 28-detection area reaction tank and 29-quality control area reaction tank.
Detailed Description
The invention will be further described with reference to specific embodiments in the drawings.
Referring to fig. 1-13, a dry three-electrode electrochemiluminescence lateral flow immunosensor comprises a detection chip 1, wherein the detection chip 1 comprises a sample adding pad 2, a bonding pad 3, at least one detection pad 4, a quality control pad 5, an absorption pad 6 and an electrode pad 7.
The electrode pad is used for driving ECL reaction to be carried out, the top of the electrode pad 7 is provided with a common working electrode 8, the common working electrode 8 is an I-shaped electrode, and the electrode pads 7 at two sides of the common working electrode 8 are respectively provided with a counter electrode 9 and a reference electrode 10.
One end of the common working electrode 8, the counter electrode 9 and the reference electrode 10 in the same direction is used as an electric contact area 11. The other end of the counter electrode 9 is sequentially provided with detection area counter electrodes 12 and a quality control area counter electrode 13, wherein the number of the detection area counter electrodes is equal to that of the detection pads 4 and the detection area counter electrodes are in one-to-one correspondence. Correspondingly, the other end of the reference electrode 10 is sequentially provided with a detection area reference electrode 14 and a quality control area reference electrode 15, wherein the number of the detection area reference electrodes is equal to that of the detection pads 4, and the detection area reference electrodes are in one-to-one correspondence. The detection region counter electrode 12, the quality control region counter electrode 13, the detection region reference electrode 14 and the quality control region reference electrode 15 all face the common working electrode 8.
Preferably, the overall structure of the counter electrode 9, the reference electrode 10 is symmetrical with respect to the common working electrode 8.
The sample addition pad 2 is provided with a sample addition pad hydrophilic region 16, the bonding pad 3 is provided with a bonding pad hydrophilic region 17, the detection pad 4 is provided with a detection pad hydrophilic region 18, the quality control pad 5 is provided with a quality control pad hydrophilic region 19, and the absorption pad 6 is provided with an absorption pad hydrophilic region 20. The sample adding pad 2, the combining pad 3, each detecting pad 4, the quality control pad 5 and the absorbing pad 6 are sequentially arranged at the top of the electrode pad 7, and the sample adding pad hydrophilic region 16 is sequentially communicated with the combining pad hydrophilic region 17, each detecting pad hydrophilic region 18, the quality control pad hydrophilic region 19 and the absorbing pad hydrophilic region 20 to form a single flow path.
Preferably, overlapping areas are arranged between the sample adding pad 2 and the combining pad 3, between the combining pad 3 and the detecting pad 4, between the detecting pad 4 and the quality control pad 5 and between the quality control pad 5 and the absorbing pad 6, the width of the overlapping areas is 1-3 mm, preferably, the width of the overlapping areas is 2mm, and the sample adding pad hydrophilic area 16, the combining pad hydrophilic area 17, the detecting pad hydrophilic areas 18, the quality control pad hydrophilic area 19 and the absorbing pad hydrophilic area 20 can be conveniently communicated.
Specifically, as shown in fig. 2, the absorbent pad hydrophilic region 20 has an L-shaped structure, with a thinner hydrophilic region for connecting the quality control pad hydrophilic region 19 and a wider hydrophilic region for absorbing the surplus solution.
The hydrophilic region 19 of the quality control pad has a Z-shaped structure, and the hydrophilic region in the middle section of the Z shape is used for forming an ECL region, namely, a quality control region reaction tank 29 (a quality control region reaction tank 29 is formed between the quality control region counter electrode 13 and the common working electrode 8, and between the quality control region reference electrode 15 and the common working electrode 8), and forms a flow path together with the hydrophilic regions at the two ends of the Z shape, so as to connect the hydrophilic region 18 of the detection pad and the hydrophilic region 20 of the absorption pad.
The hydrophilic region 18 of the detection pad has a Z-shaped structure, and the hydrophilic region in the middle of the Z-shape is used to form an ECL region, namely, a detection region reaction tank 28 (a detection region reaction tank 28 is formed between the detection region counter electrode 12 and the common working electrode 8, and between the detection region reference electrode 14 and the common working electrode 8), and forms a flow path together with the hydrophilic regions at the two ends of the Z-shape, so as to connect the hydrophilic region 17 of the bonding pad and the hydrophilic region 19 of the quality control pad.
Each detection pad hydrophilic region 18 is located directly above the corresponding detection region counter electrode 12 and detection region reference electrode 14, and the quality control pad hydrophilic region 19 is located directly above the quality control region counter electrode 13 and quality control region reference electrode 15.
As shown in fig. 3 and 5, the number of detection pads 4 is one, and the number of corresponding detection zone reference electrodes 14 is one, that is, the number of detection pad hydrophilic regions 18 is one. As shown in fig. 4 and 6, the number of detection pads 4 is two, and the number of corresponding detection area reference electrodes 14 is two, that is, the number of detection pad hydrophilic areas 18 is two. If more hydrophilic areas 18 are needed, the number of detection pads 4 and the number of detection area counter electrodes 12 need only be increased accordingly.
The hydrophilic region 17 of the binding pad is in an I-shaped structure and is used for drying the labeled antibody and forming a flow path, connecting the hydrophilic region 16 of the sample adding pad with the hydrophilic region 18 of the detection pad, when the number of the hydrophilic regions 18 of the detection pad is greater than 1, the hydrophilic region 17 of the binding pad is connected with the hydrophilic region 16 of the sample adding pad and the first hydrophilic region 18 of the detection pad, each hydrophilic region 18 of the detection pad is sequentially connected, and the hydrophilic region 18 of the last detection pad is connected with the hydrophilic region 19 of the quality control pad.
The hydrophilic region 16 of the sample addition pad has an L-shaped structure, a thinner hydrophilic region is used for connecting the hydrophilic region 17 of the binding pad, and a wider hydrophilic region is used for adding the sample solution to be measured.
The ECL region of the common working electrode 8 is located at the portion where the "Z" -shaped midsection hydrophilic regions of the detection pad hydrophilic region 18 and the quality control pad hydrophilic region 19 overlap with the common working electrode 8.
The sample addition pad hydrophilic region 16, the conjugate pad hydrophilic region 17, the respective detection pad hydrophilic regions 18, the quality control pad hydrophilic region 19, and the absorbent pad hydrophilic region 20 are connected in series to form a single flow path.
A quality control loop is formed among the common working electrode 8, the quality control pad hydrophilic region 19 and the quality control region counter electrode 13, a detection loop is formed among the common working electrode 8, the detection pad hydrophilic region 18 and the detection region counter electrode 12, and the quality control loop and the detection loop are two parallel loops.
The electrode pad 7 is made of a hydrophobic fiber material serving as a substrate and is made of a conductive material through screen printing, for example, the conductive material is conductive carbon paste, the sample adding pad 2, the bonding pad 3, the detection pad 4, the quality control pad 5 and the absorption pad 6 are made of hydrophilic fiber materials serving as substrates, a hydrophobic area and a hydrophilic area are formed on the substrates through screen printing of hydrophobic ink, the hydrophilic fiber materials are made of polyester fiber non-woven fabrics, glass fibers and nitrocellulose membranes, and the hydrophobic ink is made of PP ink and a diluent.
The detection chip 1 further comprises a bottom plate 21, the bottom plate 21 is a hard sheet subjected to back glue treatment, such as any one of polyethylene terephthalate and polyvinyl chloride, the electrode pad 7 is adhered to the top of the bottom plate 21, the middle parts of the sample adding pad 2, the bonding pad 3, the detection pad 4, the quality control pad 5 and the absorption pad 6 are all clung to the electrode pad 7, and the two ends of the sample adding pad 2, the bonding pad 3, the detection pad 4, the quality control pad 5 and the absorption pad 6 are adhered to the bottom plate 21, namely the electrode pad 7, the absorption pad 6, the quality control pad 5, the detection pad 4, the bonding pad 3 and the sample adding pad 2 are sequentially laminated and adhered to the bottom plate 21.
The sensor further comprises an upper cover 22 and a lower cover 23, wherein a mounting groove for mounting the detection chip 1 is formed in the lower cover 23, the upper cover 22 covers the detection chip 1, the upper cover 22 is respectively provided with sample adding holes 24 aligned with the sample adding pad hydrophilic areas 16, detection area observation windows 25 aligned with the detection pad hydrophilic areas 18 one by one, quality control area observation windows 26 aligned with the quality control pad hydrophilic areas 19, and open grooves 27 aligned with the electric contact areas 11, and the open grooves 27 are used for powering the electric contact areas 11.
The hydrophilic area 16 of the loading pad is treated with tween. After Tween treatment is applied to the hydrophilic region 17 of the conjugate pad, the labeled antibody is dried, and the amount of the labeled antibody is 2 to 7. Mu.L, preferably 5. Mu.L. The hydrophilic region 18 of the detection pad is fixed with a modified biomarker coated antibody, the concentration of which is 40-140 mug mL -1 Preferably 100. Mu.g mL -1 . The hydrophilic region 19 of the quality control pad is fixedly modified with a quality control coated antibody, such as goat anti-chicken IgY, and the coated antibody is a secondary antibody irrelevant to a biomarker, so as to play a role in quality control. The hydrophilic areas 18 and 19 of the detection pad and the quality control pad are fixedly modified by chitosan and glutaraldehyde, and the dosage of glutaraldehyde is 3-6 mu L, preferably 4.5 mu L. Absorbent pad hydrophilic area 20 is treated with tween. The labeled antibody is a complex of terpyridyl ruthenium-intramolecular coreactant-detection antibody (Ru (II) -PLL-Ab).
The specific process of chip modification is as follows: firstly, 0.2% Tween solution is respectively dripped into a sample addition pad hydrophilic region 16, a synthetic pad hydrophilic region 17 and an absorption pad hydrophilic region 20, the dripping amounts of the sample addition pad hydrophilic region 16, the synthetic pad hydrophilic region 17 and the absorption pad hydrophilic region 20 are respectively 20, 5 and 13 mu L, and then the sample addition pad hydrophilic region 16, the synthetic pad hydrophilic region 17 and the absorption pad hydrophilic region 20 are placed in a 34 ℃ oven for drying for 30 minutes. Subsequently, 7 mu L of 2.5mg/mL chitosan and 4.5 mu L of 2.5% glutaraldehyde are sequentially modified in the detection pad hydrophilic region 18 and the quality control pad hydrophilic region 19, the detection pad hydrophilic region 18 and the quality control pad hydrophilic region 19 are placed in a constant temperature and humidity box with the temperature of 24 ℃ and the humidity of 50% for incubation for 30 minutes, the biomarker coated antibody is dripped into the detection pad hydrophilic region 18, the quality control coated antibody (IgY) is dripped into the quality control pad hydrophilic region 19, and finally the detection pad hydrophilic region 18 and the quality control pad hydrophilic region 19 are placed in a constant temperature and humidity box with the temperature of 24 ℃ and the humidity of 50% for incubation for 30 minutes, so that the biomarker coated antibody and the IgY are respectively fixed in the detection pad hydrophilic region 18 and the quality control pad hydrophilic region 19. Finally, 5. Mu.L of the labeled antibody was dropped on the hydrophilic region 17 of the conjugate pad, and it was dried in an oven at 34℃for 30 minutes to complete the modification of the internal chip.
The detection application of the dry three-electrode electrochemiluminescence lateral flow immunosensor comprises the following steps:
s1, dropwise adding a sample solution to be detected containing a biomarker from a sample adding hole 24 to a sample adding pad hydrophilic region 16 of a sensor, wherein the sample solution to be detected flows through a binding pad hydrophilic region 17 from the sample adding pad hydrophilic region 16, and the biomarker and a labeled antibody dried in the binding pad hydrophilic region 17 are specifically combined to form a labeled antibody-biomarker complex; the complex further flows through each detection pad hydrophilic region 18 and is combined with the biomarker coated antibody fixed on the detection pad hydrophilic region 18 to form a sandwich-type complex of 'labeled antibody-biomarker-coated antibody' immunization;
s2, after the immune reaction of the biological marker is finished, dropwise adding a buffer solution into the hydrophilic region 16 of the sample adding pad, and removing the unbound labeled antibody in the hydrophilic region 18 of the detection pad;
s3, starting the ECL analyzer, placing the sensor on an inlet and outlet module of the ECL analyzer, conveying the sensor to the position right below an imaging detection module by the inlet and outlet module, and communicating an electric contact area 11 of the electrode pad 7 with a constant potential module of the ECL analyzer;
s4, starting a detection button on the ECL analyzer, providing driving voltage through a constant potential module of the ECL analyzer, triggering the intramolecular reaction of the labeled antibody, generating ECL signals, automatically collecting and analyzing the ECL signals from a detection area observation window 25 and a quality control area observation window 26 by the ECL analyzer, and further quantitatively detecting the biomarker according to the strength of the ECL signals.
The exposure time of the CMOS camera in ECL analyzers is 100 to 600ms, preferably 400ms. The three-electrode ECL driving voltage is 2 to 4V, preferably 3.5V. The incubation time for the immune reaction is 2 to 8min, preferably 5min. The biomarker is cardiac troponin (cTnI), and the intramolecular coreactant is any one of polylysine, lysine and cysteine, preferably polylysine.
Detection application embodiment one
The detection process is as follows:
1. dripping a sample solution to be detected containing cTnI into the hydrophilic region 16 of the sample adding pad of the detection chip 1 through the sample adding hole 24 of the upper cover 22, and enabling the solution to rapidly flow through the hydrophilic region 17 of the binding pad and combine with the labeled antibody with specific affinity on the hydrophilic region to form a labeled antibody-cTnI complex;
the "labeled antibody-cTnI" complex further flows through the detection zone reaction cell 28 of the detection pad hydrophilic zone 18 and binds to the cTnI coated antibody of specific affinity thereon to form an "labeled antibody-cTnI coated antibody" immune sandwich type complex;
3. after waiting for a few minutes and after the immune reaction is finished, dropwise adding a buffer solution into the sample adding hole 24, and removing unbound labeled antibodies in the reaction tank 28 of the detection zone;
4. and capturing luminescence through an ECL analyzer, and analyzing the light intensity, so as to realize quantitative detection.
Detection application example two
Several important factors for influencing ECL intensity values in detection application example one: exposure time, driving voltage, amount of labeled probe, amount of glutaraldehyde, concentration of coated antibody, and incubation time (i.e., immunoreaction time) are preferred.
a) Preferred exposure time
1. The concentration of cTnI to be measured is 0.01ng mL -1 Exposure to lightThe time is undetermined, the driving voltage is 3V, the dosage of the labeled antibody is 5 mu L, the dosage of glutaraldehyde is 5 mu L, and the concentration of the cTnI coated antibody is 100 mu g mL -1 Incubation time was 5min.
2. Several experimental groups were set up: the exposure times were set to the following different values, respectively: 100ms, 200ms, 400ms, 500ms, 600ms.
3. The test procedure is the same as that of the test application example, and the experimental result is shown in fig. 7.
As can be seen from fig. 7, the ECL intensity value continuously increases and the signal-to-noise ratio gradually decreases as the exposure time increases from 100ms to 600ms. The reason for this phenomenon is: as the exposure time increases, the integral of ECL intensity values generated by the reaction tends to stabilize, and the integral of ECL intensity values generated by the background increases, so the total ECL intensity value increases and the signal to noise ratio decreases. In order to obtain both a higher ECL intensity value and a higher signal to noise ratio, 400ms is preferred as exposure time.
b) Preferably drive voltage
1. The concentration of cTnI to be measured is 0.01ng mL -1 The exposure time is 400ms, the driving voltage is undetermined, the dosage of the labeled antibody is 5 mu L, the dosage of glutaraldehyde is 5 mu L, and the concentration of the cTnI coated antibody is 100 mu g mL -1 Incubation time was 5min.
2. Several experimental groups were set up: the driving voltages are set to the following different values, respectively: 2V, 2.5V, 3V, 3.5V, 4V.
3. The test procedure is the same as that of the test application example, and the experimental result is shown in fig. 8.
As can be seen from fig. 8, the ECL intensity value continuously rises as the driving voltage increases from 2V to 3.5V; after 3.5V, ECL intensity values began to drop. The reason for this phenomenon may be: at high driving voltages background reactions (e.g. water oxidation) occur, which chemically and physically interfere with ECL emission. In order to obtain a higher ECL strength value, 3.5V is preferable as the driving voltage.
c) Preferably the amount of labeled antibody
1. The concentration of cTnI to be measured is 0.01ng mL -1 The exposure time is 400ms, the driving voltage is 3.5V, the dosage of the labeled antibody is undetermined, and glutaraldehydeThe amount of the cTnI coated antibody was 5. Mu.L, and the concentration of the cTnI coated antibody was 100. Mu.g mL -1 Incubation time was 5min.
2. Several experimental groups were set up: the amounts of labeled antibodies were set to the following different values: 2. Mu.L, 3. Mu.L, 4. Mu.L, 5. Mu.L, 6. Mu.L, 7. Mu.L.
3. The test procedure is the same as that of the test application example, and the experimental result is shown in fig. 9.
As can be seen from fig. 9, the ECL intensity value increases rapidly as the amount of labeled antibody increases from 2 μl to 5 μl; after 5 μl, ECL intensity values tended to be unchanged. The reason for this phenomenon may be: at a certain target concentration, the number of molecules of the labeled antibody gradually saturates. In order to obtain a higher ECL intensity value while controlling the cost, 5 μl is preferred as the amount of labeled antibody.
d) Preferably glutaraldehyde
1. The concentration of cTnI to be measured is 0.01ng mL -1 The exposure time is 400ms, the driving voltage is 3.5V, the dosage of the labeled antibody is 5 mu L, the dosage of glutaraldehyde is undetermined, and the concentration of the cTnI coated antibody is 100 mu g mL -1 Incubation time was 5min.
2. Several experimental groups were set up: glutaraldehyde amounts were set to the following different values: 3 μL, 4 μL, 4.5 μL, 5 μL, 5.5 μL, 6 μL.
3. The test procedure is the same as that of the test application example, and the experimental result is shown in fig. 10.
As can be seen from FIG. 10, the ECL strength value increased continuously as glutaraldehyde usage increased from 3. Mu.L to 4.5. Mu.L; after 4.5 μl, ECL intensity values began to drop. The reason for this phenomenon may be: excess glutaraldehyde affects the process of cTnI binding specifically to cTnI coated antibodies. In order to obtain higher ECL strength values, 4.5. Mu.L is preferred as glutaraldehyde amount.
e) Preferably cTnI coated antibody concentration
1. The concentration of cTnI to be measured is 0.01ng mL -1 The exposure time was 400ms, the driving voltage was 3.5V, the amount of labeled antibody was 5. Mu.L, the amount of glutaraldehyde was 4.5. Mu.L, the concentration of cTnI-coated antibody was pending, and the incubation time was 5min.
2. Several experimental groups were set up: coating resistThe body concentrations were set to the following different values, respectively: 40 μg mL -1 、60μg mL -1 、80μg mL -1 、100μg mL -1 、120μg mL -1 、140μg mL -1 。
3. The test procedure is the same as that of the test application example, and the experimental result is shown in fig. 11.
As can be seen from FIG. 11, when the concentration of cTnI coated antibodies was changed from 40. Mu.g mL -1 Increase to 100. Mu.g mL -1 When the ECL strength value is continuously increased; 100 μg mL -1 After that, ECL intensity value starts to decrease. The reason for this phenomenon may be: excess cTnI coated antibody affects the transfer of electrons from the electrode to the labeled antibody, preferably 100 μg mL for higher ECL intensity values -1 As the concentration of cTnI coated antibody.
f) Preferably incubation time
1. The concentration of cTnI to be measured is 0.01ng mL -1 The exposure time is 400ms, the driving voltage is 3.5V, the dosage of the labeled antibody is 5 mu L, the dosage of glutaraldehyde is 4.5 mu L, and the concentration of the cTnI coated antibody is 100 mu g mL -1 Incubation time was pending.
2. Several experimental groups were set up: the incubation times were set to the following different values: 2min, 3min, 4min, 5min, 6min, 8min.
3. The test procedure is the same as that of the test application example, and the experimental result is shown in fig. 12.
As can be seen from fig. 12, ECL intensity values increased continuously as the incubation time increased from 2min to 5 min; after 5min, ECL intensity values tended to stabilize. The reason for this phenomenon may be: the immune sandwich complex of the labeled antibody-biomarker-coated antibody is not generated after the immune reaction is finished. In order to obtain higher ECL intensity values while reducing the time required for detection, 5min is preferred as incubation time.
Detection application example III
The method comprises the following steps:
1. preferred parameters according to detection application example two: including exposure time 400ms; the driving voltage is 3.5V; the amount of the labeled probe is 5 mu L; glutaraldehyde volume 4.5 μl; cTnI bagThe concentration of the antibody is 100 mug mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The incubation time is 5min, and the dry three-electrode ECL lateral flow immunosensor is manufactured by adopting the detection chip 1 and only arranging one detection pad 4.
2. Several experimental groups were set up: the cTnI concentration in the sample solution to be tested was set to several different values.
3. The test procedure is the same as in the test application example, and the experimental result is shown in fig. 13.
As can be seen from the graph, the concentration of cTnI is between 0.5 and 1000pg mL -1 In the range, the ECL intensity value increases with the increase of the cTnI concentration value, the ECL intensity value (expressed by Y) and the cTnI concentration logarithmic value (expressed by X) have good linear relation, a linear equation can be expressed as Y=5.998+1.8011X, and the detection limit is estimated to be 0.494pg mL -1 . 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 for blank control (five replicates), using Y L And calculating the corresponding cTnI concentration by the value to obtain the detection limit.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and improvements can be made by those skilled in the art without departing from the structure of the present invention, and these do not affect the effect of the implementation of the present invention and the utility of the patent.
Claims (10)
1. The dry three-electrode electrochemiluminescence lateral flow immunosensor is characterized by comprising a detection chip (1), wherein the detection chip (1) comprises a sample adding pad (2), a combining pad (3), at least one detection pad (4), a quality control pad (5), an absorption pad (6), an electrode pad (7) and a bottom plate (21); the top of the electrode pad (7) is provided with a common working electrode (8), the electrode pads (7) at two sides of the common working electrode (8) are respectively provided with a counter electrode (9) and a reference electrode (10), one ends of the common working electrode (8), the counter electrode (9) and the reference electrode (10) in the same direction are all used as electric contact areas (11), the other ends of the counter electrodes (9) are sequentially provided with detection area counter electrodes (12) and a quality control area counter electrode (13) which are equal in number and in one-to-one correspondence with the detection pads (4), correspondingly, the other ends of the reference electrodes (10) are sequentially provided with detection area reference electrodes (14) and a quality control area reference electrode (15) which are equal in number and in one-to-one correspondence with the detection pads (4), and the detection area counter electrodes (12), the quality control area reference electrodes (13), the detection area reference electrodes (14) and the quality control area reference electrodes (15) are all oriented to the common working electrode (8); the sample adding pad (2) is provided with a sample adding pad hydrophilic region (16), the sample adding pad (3) is provided with a sample adding pad hydrophilic region (17), the sample detecting pad (4) is provided with a sample detecting pad hydrophilic region (18), the sample detecting pad (5) is provided with a sample detecting pad hydrophilic region (19), the sample absorbing pad (6) is provided with an sample absorbing pad hydrophilic region (20), the sample adding pad (2), the sample adding pad (3), each sample detecting pad (4), the sample detecting pad (5) and the sample absorbing pad (6) are sequentially arranged at the top of the electrode pad (7), the sample adding pad hydrophilic region (16) is sequentially communicated with the sample detecting pad hydrophilic region (17), each sample detecting pad hydrophilic region (18), the sample detecting pad hydrophilic region (19) and the sample absorbing pad hydrophilic region (20) to form a single flow path, each sample detecting pad hydrophilic region (18) is respectively located right above a corresponding sample detecting region counter electrode (12) and a sample detecting region reference electrode (14), and the sample detecting pad (6) is sequentially arranged right above the sample detecting region reference electrode (15).
2. The dry three-electrode electrochemiluminescence lateral flow immunosensor according to claim 1, wherein the electrode pad (7) is made of a hydrophobic fiber material as a substrate and is made of a conductive material through screen printing, and the sample addition pad (2), the bonding pad (3), the detection pad (4), the quality control pad (5) and the absorption pad (6) are made of a hydrophilic fiber material as substrates and form a hydrophobic area and a hydrophilic area on the substrates through screen printing of hydrophobic ink.
3. The dry three-electrode electrochemiluminescence lateral flow immunosensor according to claim 1, wherein the electrode pad (7) is adhered to the top of the bottom plate (21), the middle parts of the sample adding pad (2), the combining pad (3), the detecting pad (4), the quality control pad (5) and the absorbing pad (6) are all clung to the electrode pad (7), and two ends of the sample adding pad (2), the combining pad (3), the detecting pad (4), the quality control pad (5) and the absorbing pad (6) are all adhered to the bottom plate (21).
4. The dry three-electrode electrochemiluminescence lateral flow immunosensor of claim 1, wherein overlapping areas are arranged between the sample adding pad (2) and the bonding pad (3), between the bonding pad (3) and the detection pad (4), between the detection pad (4) and the quality control pad (5), and between the quality control pad (5) and the absorption pad (6).
5. The dry three-electrode electrochemiluminescence lateral flow immunosensor according to claim 1, further comprising an upper cover (22) and a lower cover (23), wherein a mounting groove for mounting the detection chip (1) is formed in the lower cover (23), the upper cover (22) covers the detection chip (1), and the upper cover (22) is respectively provided with a sample adding hole (24) aligned with the hydrophilic areas (16) of the sample adding pad, detection area observation windows (25) aligned with the hydrophilic areas (18) of each detection pad one by one, quality control area observation windows (26) aligned with the hydrophilic areas (19) of the quality control pad, and an opening groove (27) aligned with the electric contact area (11).
6. The dry three-electrode electrochemiluminescence lateral flow immunosensor of claim 1, wherein the sample addition pad hydrophilic region (16) and the absorbent pad hydrophilic region (20) are treated with tween; and drying and labeling the antibody after the hydrophilic area (17) of the binding pad is treated by adopting Tween.
7. A dry three electrode electrochemiluminescent lateral flow immunosensor according to claim 1, characterized in that the detection pad hydrophilic zone (18) is immobilized with a modified biomarker coated antibody.
8. The dry three-electrode electrochemiluminescence lateral flow immunosensor of claim 1, wherein the control pad hydrophilic region (19) immobilizes the modified control coating antibody.
9. The dry three-electrode electrochemiluminescence lateral flow immunosensor of claim 1, wherein the detection pad hydrophilic region (18) and the quality control pad hydrophilic region (19) are fixedly modified by chitosan and glutaraldehyde.
10. A detection application of a dry three-electrode electrochemiluminescent lateral flow immunosensor according to any one of claims 1 to 9, comprising the steps of:
s1, dropwise adding a sample solution to be detected containing a biomarker into a sample adding pad hydrophilic region (16) of a sensor, wherein the sample solution to be detected flows through a binding pad hydrophilic region (17) from the sample adding pad hydrophilic region (16), and the biomarker and a labeled antibody dried in the binding pad hydrophilic region (17) are specifically combined to form a labeled antibody-biomarker complex; the complex further flows through each detection pad hydrophilic region (18) and is combined with the biomarker coated antibody fixed on the detection pad hydrophilic region (18) to form a 'labeled antibody-biomarker-coated antibody' immune sandwich type complex;
s2, after the immune reaction of the biological marker is finished, dropwise adding a buffer solution into the hydrophilic region (16) of the sample adding pad, and removing the unbound labeled antibody in the hydrophilic region (18) of the detection pad;
s3, starting the ECL analyzer, placing the sensor on an inlet and outlet module of the ECL analyzer, conveying the sensor to the position right below the imaging detection module by the inlet and outlet module, and communicating an electric contact area (11) of the electrode pad (7) with a constant potential module of the ECL analyzer;
s4, starting a detection button on the ECL analyzer, providing driving voltage through a constant potential module of the ECL analyzer, triggering the intramolecular reaction of the labeled antibody, generating an ECL signal, automatically collecting and analyzing the ECL signal by the ECL analyzer, and quantitatively detecting the biomarker according to the strength of the ECL signal.
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