CN111830252B - Enzyme-linked immunosorbent assay sensor based on intelligent mobile terminal and use method - Google Patents
Enzyme-linked immunosorbent assay sensor based on intelligent mobile terminal and use method Download PDFInfo
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Abstract
The invention discloses an enzyme-linked immunosorbent assay sensor based on an intelligent mobile terminal and a using method thereof. An enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal, comprising: the result reading device comprises a darkroom for providing a light source, a light guide base and an intelligent mobile terminal with a light sensor, wherein the bottom of the darkroom is provided with a through hole for a light beam to pass through, the light guide base is provided with a light guide hole, and the light sensor, the light guide hole and the through hole are correspondingly arranged and are positioned on the same light path; the chromatography device comprises a sample injection layer, a binding layer, a chromatography barrel with a chromatography film and a water absorption layer which are sequentially connected, wherein the chromatography barrel is detachably connected with the binding layer and the water absorption layer, and the chromatography barrel after chromatography reaction is arranged on the light guide base conveniently. The instant detection device based on the smart phone combines an immunodetection process with the smart mobile terminal, and realizes interpretation of a detection result by means of the optical sensor of the smart mobile terminal.
Description
Technical Field
The invention relates to the technical field of rapid detection, in particular to an enzyme-linked immunosorbent assay sensor based on an intelligent mobile terminal and a using method thereof.
Background
In the detection field, including clinical detection, food safety detection, biological detection and the like, enzyme linked immunosorbent assay (ELISA) is one of the recognized gold standards, and the principle is that known antigens or antibodies are adsorbed on the surface of a solid phase carrier (polystyrene microplate), the antigens or antibodies labeled by Enzyme are combined on the carrier through immunoreaction, finally, the Enzyme substrate is catalyzed to develop color, and the OD value of specific wavelength light penetrating through the substrate solution is measured by an instrument to perform result interpretation. Has high sensitivity, relatively low cost and good stability, but requires special instruments and professionals for operation, and the detection time is longer, generally 2-4 hours. An Immunochromatographic Strip (ICS) detection technology is a rapid detection method which is widely used in recent years, and the principle is that Gold nanoparticles (AuNPs) with surfaces modified with antibodies 1 are sprayed on solid fibers (generally glass fibers), paired antibodies or antigens are fixed on a Test area (T line) of a Nitrocellulose (NC) membrane, secondary antibodies are fixed on a Control area (Control line, C line) of the NC membrane, the samples are firstly combined with the Gold-labeled antibodies 1 after being added, and then chromatography is carried out to respectively combine with the paired antibodies and the secondary antibodies through the NC membrane, so that the AuNPs are left on the NC membrane in a specific area to present a red line. Compared with ELISA, the detection time only needs 5-15 minutes, no special operator is needed, the price is lower, the sensitivity is much lower, the result is judged by naked eyes, and the error is larger. In many cases, especially in the face of public safety issues with local outbreaks, not only is the sensitivity and specificity of detection required, but also the rapidity, accuracy, cost and portability of detection are required, and immediate feedback of the detection results is required, so that conventional ELISA and ICS alone are often not satisfactory.
Disclosure of Invention
In order to solve the problems, the invention provides an enzyme-linked immunosorbent assay biosensor based on an intelligent mobile terminal and a use method thereof, the enzyme-linked immunosorbent assay biosensor (NLICA) is an instant detection device based on the intelligent mobile phone, combines an immunodetection process with the intelligent mobile terminal, can realize interpretation of a detection result by virtue of an ambient light sensor of the intelligent mobile terminal, does not need to depend on other detection equipment, does not need professional technical personnel for operation, and can realize the integration of simplification, portability and detection analysis of detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
an enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal, comprising:
the result reading device comprises a darkroom for providing a light source, a light guide base and an intelligent mobile terminal with a light sensor, wherein the bottom of the darkroom is provided with a through hole for light beams to pass through, the light guide base is provided with a light guide hole, and the light sensor, the light guide hole and the through hole are correspondingly arranged and are positioned on the same light path;
the chromatographic device comprises a sample introduction layer, a binding layer, a chromatographic barrel with a chromatographic membrane and a water absorption layer which are sequentially connected, wherein the chromatographic barrel is detachably connected with the binding layer and the water absorption layer, so that the chromatographic barrel after chromatographic reaction is conveniently placed on the light guide base;
the intelligent mobile terminal is used for detecting the light intensity of the color development liquid passing through the chromatography barrel through the optical sensor.
As a preferred embodiment of the enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal provided by the present invention, a light source assembly is disposed in the darkroom and is used for providing a light source; the light source assembly comprises a light-emitting light source and a light filter arranged in the light-emitting direction.
In the present invention, the light source is preferably a laser light source, and more preferably, is an LED lamp bead, such as an LED lamp bead with an excitation light wavelength of 450nm, but is not limited thereto.
In the present invention, the filter is a narrow-band filter with a bandwidth of 20nm and filters light of 450nm, but is not limited thereto.
In the present invention, the result reading device further comprises a control circuit, specifically, it comprises a battery, a charging interface, a switch, a resistor and a wire; wherein, the battery is preferably but not limited to a lithium battery, more preferably a lithium battery with specification of 32mm 26mm 3mm and capacity of 300 mAh; the specification of the charging interface is Micro-usb; the resistor specification is preferably in the range of 0-10K omega, which functions to adjust the light intensity of the light source.
Furthermore, a radiator is arranged above the light-emitting source, and is preferably but not limited to an aluminum-based multi-plate radiator, so that the working heat of the light-emitting source can be timely led out.
As a preferred embodiment of the enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal provided by the present invention, the result reading device further comprises a base table, on which a slot for limiting the light guide base is provided and an accommodating cavity for accommodating a side portion of the intelligent mobile terminal is provided below the slot, the base table is provided with a through hole or a through groove for allowing light to pass through; the darkroom is arranged above the clamping groove.
As a preferred embodiment of the enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal provided by the present invention, the light guide base moves laterally relative to the card slot, for example, the light guide base is slidably disposed in the card slot; or, the light guide base vertically moves relative to the card slot, and if the light guide base is directly clamped in the card slot.
As a preferred embodiment of the enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal provided by the invention, an optical fiber is arranged in the light guide hole.
As a preferred embodiment of the enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal provided by the present invention, the light guide base is provided with a plurality of barrel fixing grooves, and each barrel fixing groove is provided with the light guide hole. The light guide base moves transversely along the clamping groove.
So design can once bear a plurality of chromatography buckets through setting up a plurality of solid bucket grooves, realizes carrying out quick quantitative determination on-the-spot to a plurality of samples in succession. The chromatography barrel can be connected in the barrel fixing groove in a threaded mode, can also be connected in the barrel fixing groove in an interference fit mode, can also be connected in the barrel fixing groove in a concave-convex structure or a buckling mode, and is not limited to the above.
In the invention, the light guide base is preferably but not limited to a trapezoid strip, the light guide holes are distributed in the upper part of the light guide base in an array manner, a silica gel pad and the optical fiber which passes through the silica gel pad and transversely pass through the silica gel pad are arranged in each light guide hole, and when the positioning line on the light guide base moves to the indication scale, the optical fiber is superposed with the central point of the through hole below the darkroom.
Further, the light guide holes are preferably 9mm in diameter and 4mm in depth, the interval between the two light guide holes is 15mm, and shearing threads are arranged in the holes; the silica gel pad is preferably 9mm in diameter and 2mm in thickness and is used for sealing to prevent the color development liquid from seeping out; the optical fiber is preferably made of PMMA (polymethyl methacrylate), the diameter of the optical fiber is 1.2mm, the cutting length of the optical fiber is 4mm, and the optical fiber extends out of the bottom of the base by 1mm.
Correspondingly, the clamping groove is provided with a strip-shaped clamping groove, the groove structure of the clamping groove and the light guide base correspond to a trapezoidal structure, and the light guide base can conveniently slide into the clamping groove in the corresponding and transverse directions.
In the invention, the alignment part is arranged on the side edge of the base table and/or the outer side of the darkroom, and the optical sensor of the intelligent mobile terminal is inserted into the accommodating cavity opposite to the alignment part, so that the optical sensor corresponds to the bottom end of the optical fiber, namely, the darkroom through hole, the optical fiber and the optical sensor are positioned on the same optical path.
As a preferred embodiment of the enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal provided by the invention, the surface of the binding layer is sprayed with platinum nanoparticles modified by antibodies.
As a preferred embodiment of the enzyme-linked immunosorbent assay sensor based on the intelligent mobile terminal, the sample introduction layer, the binding layer, the chromatography barrel with the chromatography membrane and the water absorption layer are sequentially arranged along the vertical direction, and the sample introduction layer side is provided with a sample injection port.
In the invention, the sample introduction layer and the sample adding port are used as sample introduction layers and are positioned at the bottom of the chromatography device, the sample introduction layer is arranged at the bottom of the chromatography device, the sample adding port is arranged at the side edge of the bottom of the chromatography device, and the sample adding port is communicated with a sample pad of the sample introduction layer; a bonding pad is obliquely arranged in the bonding layer, and the bottom of the bonding pad is communicated with the sample pad; the chromatographic membrane is fixedly arranged on the barrel wall of the chromatographic barrel, and the bottom of the chromatographic membrane is communicated with the top of the combination pad; and the water absorption layer is internally provided with water absorption paper which is communicated with the top of the chromatographic membrane.
It is worth noting that a plane barrel wall can be arranged in the chromatography barrel, and a plane vertical plate can also be inserted into the chromatography barrel, so that the chromatography membrane can be conveniently and fixedly arranged. The structure of the chromatography barrel is not particularly limited, and the chromatography membrane can be fixedly arranged. In the invention, the inside of the chromatography barrel preferably adopts a D-shaped cross section, and one side of the plane of the chromatography barrel is used for fixedly arranging the chromatography membrane.
Before the chromatographic device is assembled, the sample pad and the combined pad are soaked in the sample pad treatment solution and the combined pad treatment solution respectively in advance and are dried; the antibody for detection is covalently coupled to the surface of platinum nanoparticles (PtNPs) through an intermediate compound; after the treatment of the bonding pad, the surface of the pad is sprayed with antibody-modified PtNPs through a three-position spraying platform. After a sample solution is dripped into the sample adding port, the solution enters the sample pad from the sample adding hole and then enters the combination pad through chromatography, so that the PtNPs on the combination pad enter the solution and then flow upwards together with the solution through chromatography, meanwhile, a target object in the solution and antibodies on the surfaces of the PtNPs generate immunoreaction to form PtNPs-antibody complexes, and then flow upwards through chromatography to enter the chromatographic membrane and generate immunoreaction with paired antibodies or antigens on the membrane, so that the PtNPs are left in a specific area on the NC membrane.
Further, the sample pad treatment solution formulation is preferably: 10mM PBS,0.5% ~2% Triton,0.05% NaN 3 The pH is from 7.0 to 8.5, but not limited thereto. The combined pad treatment fluid formula is preferably: 1% to 10% sucrose, 1% to 10% trehalose, 0.5% BSA,0.5% Tween-20,0.05% NaN 3 The pH is from 7.0 to 8.0, but not limited thereto.
It should be noted that, in the present invention, the base platform, the light guide base, the chromatography barrel, the sample injection layer, the bonding layer, and the water absorption layer bearing structure are preferably obtained by 3D printing, and the materials used in the present invention are photosensitive resin, polylactic acid, and the like, but are not limited thereto.
A use method of the enzyme-linked immunochromatographic sensor based on the intelligent mobile terminal comprises the following steps: dropwise adding a sample to the sample introduction layer, and waiting for the completion of chromatography; the chromatography barrel is disassembled and placed on the light guide base, and color development liquid is added into the chromatography barrel and stands in a dark place; then, the light guide base is placed below a darkroom, and a light sensor of the intelligent mobile terminal is placed below the light guide hole; and starting the light source, and detecting the light intensity of the color development liquid passing through the chromatography barrel by the intelligent mobile terminal through the light sensor to finally obtain the concentration of the detection object.
The intelligent mobile terminal is an intelligent terminal with an ambient light sensor, and preferably an Android operating system is an intelligent mobile phone or a tablet; more preferably, the operating system is a smartphone or tablet with an Android of 8.0.0 or more.
The detection principle of the invention is as follows: the final result of the nano enzyme-linked immunosorbent assay sensor is interpreted according to the intensity of monochromatic light after passing through the chromatography barrel, ptNPs left on a chromatography film in the chromatography barrel after immunochromatographic reaction can catalyze TMB substrate liquid (namely color development liquid) to develop color, the color development liquid has strong absorption to 450nm light, a switch of a reading device is turned on, the light emitted by an LED lamp bead (namely a light emitting source) is transmitted to the chromatography barrel through light transmission, the light is transmitted to an environment light sensor at the top of the intelligent mobile terminal through optical fibers in a light guide hole, the amount of the PtNPs is in direct proportion to the depth of the color and is in inverse proportion to the finally passed light intensity.
Compared with the prior art, the invention has the following advantages:
1. the invention relates to an instant detection device based on a smart phone, which combines an immunodetection process with the smart phone, can realize result interpretation by virtue of a smart phone ambient light sensor, can also upload a result to a network in real time, and can accurately analyze and feed back a detection result by virtue of big data;
2. the complicated washing and reaction steps of ELISA are completed by utilizing the chromatography process, and the complexity of ELISA operation is greatly reduced under the condition of little loss of detection sensitivity.
3. The invention utilizes the enzyme-catalyzed chromogenic system after chromatography, greatly improves the sensitivity compared with the common lateral chromatography, and has convenient operation and shorter time consumption compared with ELISA;
4. the invention uses Au @ PtNPs with peroxidase-like activity as nano-enzyme, and has the advantages of good thermal stability, convenient and quick synthesis, low preparation cost and the like;
5. all the supporting structures are formed by 3D printing, and the manufacturing cost is extremely low;
6. the LED lamp beads and the optical fibers are matched for use, so that the stability of signal transmission is improved, and the complexity of a system is reduced;
7. the invention has simple operation, low use cost, small and exquisite appearance, convenient carrying and short detection time, and is more suitable for families, basic medical institutions and detection sites compared with the traditional detection analysis method;
8. the nano enzyme-linked immunosorbent assay sensor based on the smart phone has wide application range, can be used for various detection items, such as food safety detection, environmental sanitation detection, clinical target detection and the like, is basically suitable for detection items of an immunochromatography method and an enzyme-linked immunosorbent assay, is suitable for different items, only needs to replace corresponding antibodies, and has strong applicability and plasticity.
Drawings
Fig. 1 is a real object diagram of a nano enzyme-linked immunochromatographic sensor device based on a smart phone according to the present invention.
Fig. 2 is an exploded view of the sensor device according to the present invention.
Fig. 3 is a side cross-sectional view of the sensor device structure of the present invention, wherein the lamp bead, the optical filter, the optical fiber, and the optical sensor of the mobile phone are located on the same center line.
FIG. 4 is a side cross-sectional view of a structure of a chromatographic portion of the sensor of the present invention.
FIG. 5 is a schematic view of the combination of the cartridge and the base of the sensor of the present invention.
Fig. 6 is a flow chart of the use of the sensor of the present invention.
FIG. 7 is a standard curve for detecting HCV core antigens in the use of the sensor of the present invention.
In the figure: 1. top cap, 2, battery, 3, variable resistor, 4, charging interface, 5, switch, 6, radiator, 7, excitation light source, 8, light filter, 9, chromatography bucket, 10, leaded light base, 11, draw-in groove, 12, holding chamber, 13, light sensor, 14, smart mobile phone, 15, optic fibre, 16, upper cover, 17, the layer that absorbs water, 18, the combination pad, 19, the binding layer, 20, the sample pad, 21, the layer of advancing a kind, 22, the pad that absorbs water, 23, the sample loading mouth, 24, reaction zone, 25, chromatographic film, 26, the location line, 27, the silica gel pad.
Detailed Description
In order to make those skilled in the art better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, shall fall within the protection scope of the present invention.
As shown in fig. 1, 2 and 3, the result reading device includes a top cover 1, a darkroom for providing a light source, a control circuit, a light guide base 10 and a base stand; the top cover 1, the darkroom, the light guide base 10 and the base platform are sequentially arranged from top to bottom, the top cover 1 covers the top of the darkroom, and the darkroom is fixedly arranged on the base platform. The light source part in the darkroom comprises an excitation light source 7 (such as an LED lamp bead), a radiator 6 and a light filter 8, wherein the radiator 6 is positioned above the excitation light source 7, and the light filter 8 is positioned below the excitation light source 7. The control circuit comprises a battery 2, a charging interface 4, a switch 5, a resistor 3 and a wire, wherein the switch 5, the resistor 3, the battery 2 and the excitation light source 7 form a loop through the wire, and the charging interface 4 is connected with the battery 2 so as to charge the battery 2 conveniently.
The light guide base 10 is a trapezoid strip, light guide holes are distributed in the upper portion of the light guide base 10 in an array mode, a silica gel pad 27 and an optical fiber 15 traversing through the silica gel pad 27 are arranged in each light guide hole, and when a positioning line 26 on the light guide base 10 moves to an indication scale on the base table, the optical fiber 15 coincides with the center point of a through hole in the bottom of a darkroom. The base platform is provided with a through groove for light to pass through, a clamping groove 11 is formed above the base platform, a containing cavity 12 is formed below the base platform, an aligning part is arranged on the containing cavity 12, the optical sensor 13 of the intelligent mobile terminal is conveniently aligned and placed into the containing cavity 12, after the optical sensor 13 is placed into the containing cavity 12, the optical sensor 13 corresponds to a through hole in the bottom of a darkroom, and after the light guide base 10 moves to an indication scale, the optical sensor 13, the optical fiber 15 and the through hole are located on the same light path.
The light guide base 10 is provided with a plurality of barrel fixing grooves, and the bottom of each barrel fixing groove is provided with the light guide hole. During the specific use, chromatography bucket 9 can be dismantled to each solid bucket groove and connect, light guide base 10 is followed draw-in groove 11 lateral shifting is convenient for slide and is detected one by one.
The result reading device is produced by the following steps:
(1) The main structure design and manufacture: respectively designing a base platform, a darkroom, a light guide base 10 and a circuit part structure through Solidworks software, exporting an STL file format, importing the STL file format into 3D printing software, and printing each part by using an FDM type 3D printer;
(2) Assembling a circuit and an optical path: welding the battery 2, the switch 5, the charging interface 4, the adjusting resistor 3 and the LED light source into a complete circuit by using a conducting wire, attaching the aluminum basal plane of the LED lamp bead to the radiator 6 by using a heat-conducting adhesive and fixing the radiator in a darkroom, and then sequentially assembling the objects into a printed structure according to the diagram;
(3) The light guide base 10 is structurally designed and manufactured: designing a structure shown in FIG. 6 through Solidworks software, exporting the structure to an STL file format, and printing the STL file format by using a DLP (digital light processing) type 3D printer;
(4) Assembling the light guide base 10: the silicone pad 27 is cut into a wafer with the same size as the light guide hole, and a hole of 0.8mm is drilled in the middle of the wafer and plugged into the light guide hole. The optical fiber 15 with the diameter of 1.2mm is cut into equal length, two ends of the optical fiber are polished by using sand paper with the mesh size of more than 3000, the final length is 4mm, the optical fiber is inserted into the silica gel pad 27, and one end of the optical fiber extends out of the bottom of the light guide base 10 by about 1mm.
The result reading device was used as follows:
(1) The chromatography barrel 9 is arranged in the light guide base 10, the color development liquid is added to avoid light and develop color, and the light guide base 10 is moved to the position line 26 to be aligned with the indication scale;
(2) The handset 14 is inserted into the receiving cavity 12 of the base station in an aligned manner, and then the associated light intensity detection software is turned on.
(3) The switch 5 is turned on to start the excitation light source 7, the light intensity of the light which penetrates through the chromatography barrel 9 is displayed on the screen of the mobile phone 14, the result is automatically recorded, and the concentration of the object to be detected can be obtained through conversion.
It should be noted that the light intensity detection software may be installed in a self-downloading manner for the smart mobile terminal, such as a light intensity meter app, and the software at least detects the light intensity.
As shown in fig. 4 and 5, the chromatography device includes: the sample injection layer 21, the binding layer 19, the chromatography barrel 9, the water absorption layer 17 and the upper cover 16 are sequentially spliced in an embedded manner, and the upper part of the chromatography barrel 9 is connected with the lower part of the water absorption layer 17 through threads. Wherein, the sample introduction layer 21 is arranged at the bottom of the chromatography device, the sample pad 20 is fixed at the bottom of the sample introduction layer 21, the sample adding port 23 is convexly arranged at the side of the sample introduction layer 21, and the sample adding port 23 is communicated with the sample pad 20 through a sample adding hole; the bonding pad 18 is obliquely arranged on the bonding layer 19 in a penetrating way; the inside of the chromatographic barrel 9 is D-shaped, and a chromatographic membrane 25 is fixed on one horizontal side of the chromatographic barrel; the water-absorbent layer 17 has a disc-like water-absorbent pad 22 at the uppermost portion thereof. The sample pad 20, the combination pad 18, the chromatographic membrane 25 and the absorbent pad 22 are sequentially connected end to end in a fitting manner. Wherein, before the chromatographic device is assembled, the sample pad 20 and the combination pad 18 are respectively soaked in the sample pad 20 treatment solution and the combination pad 18 treatment solution in advance and are dried; the antibody is firstly coupled to the surface of platinum nanoparticles (PtNPs) through an intermediate compound in a covalent way; after the combination pad 18 is soaked, the surface of the combination pad is sprayed with the antibody modified PtNPs through a three-position spraying point platform.
The working principle of the chromatography device is as follows: after the sample solution is dripped into the sample addition port 23, the solution enters the sample pad 20 from the sample addition hole and then enters the binding pad 18 through chromatography, so that the PtNPs on the binding pad 18 enter the solution and then carry out chromatography forward, meanwhile, a target object in the solution and an antibody on the surface of the PtNPs carry out immunoreaction to form a PtNPs-antibody complex, and then the PtNPs carry out chromatography upward to enter the chromatographic membrane 25 and carry out immunoreaction a conjugated antibody or antigen on the chromatographic membrane 25, so that the PtNPs are left in a reaction area 24 on the chromatographic membrane 25.
The chromatographic device is designed and manufactured by the following steps:
(1) The method is respectively designed by Solidworks software: the sample injection layer 21, the binding layer 19, the chromatography barrel 9, the water absorption layer 17 and the upper cover 16 are led out of the STL file format and printed by a DLP 3D printer, and the used material is photosensitive resin; the outer layer of the upper end of the chromatography barrel 9 is provided with a thread structure, the diameter is 9mm, the thread pitch is 0.32mm, and the depth is 2mm.
(2) Antibody coating: the antibody 1 was diluted to 0.5mg/ml with PBS, and then evenly painted on the middle of the nitrocellulose membrane by a three-dimensional spot-painting platform in an amount of 2 ul/cm.
(3) The material of the sample pad 20 is preferably a glass fiber film, more preferably a glass fiber film with the thickness of 0.8 mm; the bonding pad 18 material is preferably a glass fiber film, more preferably a glass fiber film with a thickness of 0.6 mm; the chromatographic membrane 25 is preferably a nitrocellulose membrane (NC membrane), more preferably an NC membrane with a pore size of 135 um; the absorbent paper 22 is preferably a cellulose paper, more preferably a cellulose paper having a thickness of 1mm.
Respectively soaking the sample pad 20 and the bonding pad 18 in a treatment solution for 1h, then drying at 65 ℃, and cutting into proper widths;
(4) PtNPs synthesis: ptNPs are prepared by using a method of reducing chloroplatinic acid by vitamin C, 100ml of pure water is stirred and heated to 90 ℃, 1ml of 100mM chloroplatinic acid and 1.2ml of 100mM vitamin C are added in sequence, the temperature is kept unchanged, and the PtNPs with the particle size of about 20nm can be prepared by stirring and heating for 30 min.
(5) Antibody labeling: taking 10ml of PtNPs solution, adding 0.5ml of H 3 PO 3 10 μ l of 1% tween-20, 100 μ l of 100mM MPEG-SH, spun at room temperature for 1h, 20 μ l of 2 mg/ml anti-HCV-core antibody 2 was added, spun at room temperature for 1h, added 1ml of 10% BSA, spun at room temperature for 1h,13000 rpm for 15min, resuspended to 1ml, homogenized with a three-dimensional spotting platformSprayed onto the treated bond pads 18.
The compound solution is as follows: 10mM PBS,5% sucrose, 0.1% PVP 3W,0.5% BSA,0.5% Tween-20,0.05% NaN 3 ，PH 7.4。
(6) Assembling the chromatographic device: the sample pad 20, the conjugate pad 18 and the absorbent pad 22 are cut into circular holes having a size corresponding to the structure by a puncher, the chromatographic carrier 25 is cut into strips having a width of 4mm by a slitter and the back side thereof is adhered to the horizontal wall of the chromatographic barrel 9 by an oil adhesive, and finally the components are sequentially assembled as shown in fig. 4.
The sensor is used for HCV-core detection by the following steps:
a. dripping the serum into the sample port 23 of the assembled chromatography device, standing for 10min at normal temperature, and waiting for the completion of chromatography;
b. taking out the chromatography barrel 9, screwing one end of the threaded port into the barrel fixing groove of the light guide base 10, adding the substrate color development solution into the chromatography barrel 9, and standing for 10min at normal temperature in a dark place;
c. the light guide base 10 is inserted into the card slot 11 of the base station, the positioning line 26 on the light guide base 10 is moved to coincide with the corresponding indicating scale of the card slot 11, and at this time, the excitation light source 7, the chromatography barrel 9, the optical fiber 15 and the optical sensor 13 are positioned on the same line, namely, on the same light path.
d. The smart phone 14 is inserted into the accommodating cavity 12 in a contraposition manner, light intensity measuring software at the end of the smart phone 14 is opened, and the light intensity penetrating through the chromatography barrel 9 is detected by the light sensing module of the smart phone 14, so that the concentration of the HCV-core in the blood serum can be converted (see fig. 6 and 7).
In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the invention and to simplify the description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and are not to be considered limiting of the invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless explicitly specified otherwise.
In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; may be mechanically coupled, may be electrically coupled or may be in communication with each other; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
It should be understood that the above-described embodiments are merely exemplary of some, and not all, embodiments of the present application, and that the drawings illustrate preferred embodiments of the present application without limiting the scope of the claims appended hereto. This application is capable of embodiments in many different forms and the embodiments are provided so that this disclosure will be thorough and complete. Although the present application has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that the present application may be practiced without modification or with equivalents of some of the features described in the foregoing embodiments. All equivalent structures made by using the contents of the specification and the drawings of the present application are directly or indirectly applied to other related technical fields and are within the protection scope of the present application.
Claims (6)
1. An enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal is characterized by comprising:
the result reading device comprises a darkroom for providing a light source, a light guide base and an intelligent mobile terminal with a light sensor, wherein the bottom of the darkroom is provided with a through hole for light beams to pass through, the light guide base is provided with a light guide hole, and the light sensor, the light guide hole and the through hole are correspondingly arranged and are positioned on the same light path;
the chromatographic device comprises a sample introduction layer, a binding layer, a chromatographic barrel with a chromatographic membrane and a water absorption layer which are sequentially connected, wherein the chromatographic barrel is detachably connected with the binding layer and the water absorption layer, so that the chromatographic barrel after chromatographic reaction is conveniently placed on the light guide base;
the intelligent mobile terminal is used for detecting the light intensity of the color development liquid passing through the chromatography barrel through an optical sensor;
the surface of the binding layer is sprayed with platinum nanoparticles modified by the antibody; a
The sample introduction layer, the binding layer, the chromatography barrel with the chromatography membrane and the water absorption layer are sequentially arranged along the vertical direction, and a sample introduction port is arranged on the side of the sample introduction layer;
the sample introduction layer and the sample adding port are used as sample introduction layers and are positioned at the bottom of the chromatography device, the sample introduction layer is arranged at the bottom of the chromatography device, the sample adding port is arranged at the side edge of the bottom of the chromatography device, and the sample adding port is communicated with a sample pad of the sample introduction layer; a bonding pad is obliquely arranged in the bonding layer, and the bottom of the bonding pad is communicated with the sample pad; the chromatographic membrane is fixedly arranged on the barrel wall of the chromatographic barrel, and the bottom of the chromatographic membrane is communicated with the top of the combination pad; the water absorption layer is internally provided with water absorption paper which is communicated with the top of the chromatographic membrane;
the result reading device also comprises a base platform, wherein a clamping groove for limiting the light guide base is arranged on the base platform, an accommodating cavity for accommodating the side part of the intelligent mobile terminal is arranged below the clamping groove, and the base platform is provided with a through hole or a through groove for facilitating light to pass through; the darkroom is arranged above the clamping groove;
the light guide base is provided with a plurality of barrel fixing grooves, and each barrel fixing groove is internally provided with the light guide hole.
2. The ELISA sensor based on the smart mobile terminal of claim 1, wherein the light guide base moves laterally relative to the card slot or the light guide base moves vertically relative to the card slot.
3. The ELISA sensor based on the intelligent mobile terminal of claim 1, wherein a light source component is arranged in the darkroom and used for providing a light source; the light source assembly comprises a light-emitting light source and a light filter arranged in the light-emitting direction.
4. The ELISA sensor based on the intelligent mobile terminal of claim 1, wherein an optical fiber is arranged in the light guide hole.
5. The ELISA sensor as claimed in claim 1, wherein the light guide base moves laterally along the card slot.
6. The use method of the intelligent mobile terminal-based enzyme-linked immunochromatographic sensor of claim 1, which is characterized in that:
dropwise adding a sample to the sample introduction layer, and waiting for the completion of chromatography; the chromatography barrel is detached and placed on the light guide base, color development liquid is added into the chromatography barrel, and the chromatography barrel is kept standing in a dark place; then, the light guide base is placed below a darkroom, and the optical sensor of the intelligent mobile terminal is placed below the light guide hole; and starting the light source, and detecting the light intensity of the color development liquid passing through the chromatography barrel by the intelligent mobile terminal through the optical sensor to finally obtain the concentration of the detected object.
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