CN111751539A - High-flux vertical flow immune test paper analysis microarray - Google Patents
High-flux vertical flow immune test paper analysis microarray Download PDFInfo
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a high-flux vertical flow immune test paper analysis microarray which is used for simultaneously detecting various targets in a sample; the high-throughput vertical flow immunoassay test paper analysis microarray takes a porous PDMS resin chip as an inlet channel of a sample, and sequentially comprises the porous PDMS resin chip, a double-sided adhesive tape, a chromatography pad, an absorption pad, the double-sided adhesive tape and the PDMS resin chip from top to bottom; the high-throughput vertical flow immunoassay test paper analysis microarray has the advantages of low cost, easy preparation, high throughput, high sensitivity and high specificity, has wide application prospect in clinical examination of four inflammatory factors, virus and bacterial infection distinguishing and reasonable medical resource distribution realization, and can also provide effective support in the fields of food safety, environmental monitoring, public health detection and the like.
Description
Technical Field
The invention relates to a high-flux vertical flow immune test paper analysis microarray, which is used for the field of high-flux high-sensitivity instant detection.
Background
The vertical current immunodetection technology is used as a new measure for instant detection, the sample processing speed is high, the analysis time is short, the error of the judgment result of a user is effectively reduced, and the HOOK effect generated by false negative is avoided. However, the existing vertical flow test strip can only detect one or a few targets, the detection flux is low, the reproducibility of the detection result is poor, and the detection cost is high. In addition, the chromatographic pad in the vertical flow chromatography test strip at the present stage is generally a two-dimensional nitrocellulose membrane or cellulose acetate membrane, which is a random porous micro-nano structure. On one hand, the irregularity of the structure causes the uneven distribution of the metal nano particles in the test area, the fluctuation of Raman signals is large, and the repeatability and the accuracy of the measurement result are low; on the other hand, the chromatographic speed at different positions is also inconsistent, thereby affecting the detection performance.
Disclosure of Invention
The invention aims to provide a high-flux vertical flow immune test paper analysis microarray, which combines immune test paper with a three-dimensional ordered porous vertical double-channel structure with SERS (surface enhanced Raman scattering), can overcome the defects of the traditional disordered structure film, and enhances the SERS signal of a detection label, thereby further improving the detection sensitivity and reducing the detection limit. In addition, the distribution heights of micropores in the porous vertical double-channel structure are orderly, the columnar pore channels are parallel to each other, the density, the diameter and the depth of the pores can be adjusted, the thermal stability and the chemical stability are high, and the detection repeatability and the accuracy are greatly improved. The microarray structure of the 96/384 hydrophilic channel realized by wax spraying and printing is combined with a plurality of SERS labels, a plurality of clinical samples and a plurality of indexes can be detected simultaneously, and a novel method with low cost, less sample consumption and time saving is provided for clinical high-throughput detection.
The anodic aluminum oxide film is a three-dimensional ordered porous vertical double-channel structure, and the micropores are in a cylindrical or hexagonal structure and are directly communicated with each other in the vertical direction, so that the flow of the nano-label in the interior is smoother. The relatively large surface area and aspect ratio provides more nanoparticle binding sites than a two-dimensional structure. Therefore, the background signal can be effectively reduced, and the detection accuracy is improved.
The paper microfluidic chip is called paper chip for short, and uses paper (such as filter paper, chromatographic paper, nylon membrane, cellulose acetate membrane, cellulose nitrate membrane, etc.) as chip making material and is a microfluidic chip for biochemical analysis. Paper chips have a number of significant advantages over other microfluidic chips: the sample transport device has the advantages of simple and convenient manufacture, low cost, environmental friendliness, light weight and low sample consumption, and realizes sample transport by utilizing the capillary force provided by the porous structure of the sample transport device without external driving force. The paper chip is made of hydrophilic and hydrophobic materials: hydrophobic materials such as photoresist, Polydimethylsiloxane (PDMS), polystyrene, ink, wax, etc.; the hydrophilic material is the matrix material of the paper chip: such as filter paper, chromatography paper, cellulose acetate film, cellulose nitrate film, etc.
The method for constructing the microarray on the test paper mainly comprises a photoetching method, a drawing method, a printing method and the like. Among them, wax-spraying printing in the printing method is the most commonly used, and has significant advantages compared with other methods: the method can print a sheet of paper with the size of A4 paper (21 cm multiplied by 29.7 cm) in less than 5 min; the hydrophilic channel on the immune test paper is not contacted with photoresist or other polymers, so that the pollution of chemical substances to the hydrophilic channel is avoided; the minimum width of the micro-channel can reach 100 mu m, and only a few reagents and samples are consumed, so that the detection cost is further reduced; the microarray structure can simultaneously detect a plurality of samples and a plurality of indexes, and the high-flux detection is realized in a real sense.
The invention provides a high-throughput vertical flow immunoassay test paper analysis microarray, which takes a porous PDMS resin chip of 96/384 as an inlet channel of a sample, and sequentially distributes a porous PDMS resin chip, a double-sided adhesive tape, a 96/384 microarray chromatography pad isolated by wax, a 96/384 microarray absorption pad isolated by wax, a double-sided adhesive tape and a PDMS resin chip as substrates from top to bottom. The absorption pad and the PDMS resin chip are overlapped up and down through the double-sided adhesive tape. The size of the micropore array on the chromatography pad and the absorption pad is the same, and the chromatography pad and the absorption pad are overlapped up and down. The pore diameter of the micropore array on the chromatographic pad is the same as that of the bottom of the micropore array of the porous PDMS resin chip, and the micropore array and the bottom of the micropore array of the porous PDMS resin chip are overlapped up and down through a double-sided adhesive tape.
Wherein:
the preparation method of the porous PDMS resin chip comprises the following specific steps:
step 1: and preparing a truncated cone-shaped structure array on the silicon wafer through SU-8 photoresist to prepare the chip male die.
Step 2: pouring Polydimethylsiloxane (PDMS) on a chip male mold, wherein the pouring height is slightly lower than the height of a frustum column of the male mold to form a micropore array, and taking down the micropore array after molding to obtain a reverse mold with micropores;
and step 3: and pouring the agarose which is melted by heating on a reverse mold, cooling and forming, and taking down to obtain the porous PDMS resin chip.
The micropore of the porous PDMS resin chip is a truncated cone-shaped through hole, the diameter of the upper bottom surface of the through hole is 2-4mm, and the diameter of the lower bottom surface of the through hole is 1-2 mm; the height of the through hole is 3-6 mm; the distance between the upper surfaces of the adjacent through holes is 0.4-1mm, and the distance between the lower bottom surfaces is 1-2 mm. The number of microwells was 96, rectangular (8 rows and 12 columns), or 384, rectangular (16 rows and 24 columns).
The chromatography pad is a chromatography pad with a vertical double-pass three-dimensional ordered structure, the diameter of the hole of the three-dimensional ordered structure is 100-1000nm, and the depth of the hole is 30-200 mu m; the chromatographic pad has protein adsorption capacity, is made of one of alumina, polystyrene, silica, polymethyl methacrylate, polyethylacrylate, polyethylene, cellulose nitrate, cellulose acetate, nylon or polyvinylidene fluoride, and has a length of 36-68mm and a width of 25-36 mm.
The microarray structure of the chromatographic pad is realized by a wax spraying printing mode, a required hydrophobic pattern is designed on a computer firstly, then the required hydrophobic pattern is printed on the chromatographic pad by a wax spraying printer, a thin layer of wax is adhered to the surface of the chromatographic pad, the thickness is about 10 mu m, and then the chromatographic pad printed with the hydrophobic pattern is heated for 2min at 150 ℃ on a heating table so as to ensure that the wax penetrates through the back of the chromatographic pad to form a closed hydrophilic-hydrophobic alternate region, thus obtaining the required microarray structure. The diameter of the 96/384 micropore formed after printing is 1-2mm, the same as the diameter of the bottom surface of the micropore of the porous PDMS resin chip, and the distance between the adjacent micropore arrays is 1-2 mm.
The absorption pad has hydrophilicity, and the absorption pad material is one of absorbent paper, cellulose membrane, filter paper, cellulose-glass fiber composite, non-woven fabric or sponge; the length of the absorption pad is 36-68mm, and the width of the absorption pad is 25-36 mm.
The microarray structure of the absorption pad is the same as the microarray structure of the chromatographic pad in realization method, the diameter of 96/384 micropores formed after printing is 1-2mm, the distance between adjacent micropore arrays is 1-2mm, and the diameter and the distance of the micropores are the same as those of the microarray of the chromatographic pad.
The PDMS resin chip is manufactured by a photoetching method, and has a length of 36-68mm, a width of 25-36mm and a thickness of 3-6 mm.
The specific implementation steps comprise three parts: firstly, fixing a capture antibody of a target on a hydrophilic area of a chromatography pad isolated by wax, secondly, carrying out a Raman label reaction of the target and metal nanoparticles in a test tube; thirdly, the reaction solution is subjected to immune infiltration reaction through a microarray on the test paper, and target detection is realized through signal acquisition.
The target in the second step is one of protein, nucleic acid, heavy metal ion, small organic molecule, fungus, bacteria or virus.
In the second specific implementation step, the surface of the metal nanoparticles is modified with a Raman label, and the surface of the metal nanoparticles is fixed with a detection antibody through electrostatic adsorption or covalent connection, so that the detection of the target is realized.
The metal nanoparticles comprise two types: (a) single structure nanomaterials: one of gold nanoparticles, silver nanoparticles or platinum nanoparticles, the particle size distribution range of which is 10-100 nm; (b) nanoparticles of core-shell structure: one of gold core silver shell nanoparticles, gold core gold shell nanoparticles, silver core silver shell nanoparticles, silica core gold shell nanoparticles, silica core silver shell nanoparticles, gold core silica shell nanoparticles or silver core silica shell nanoparticles, wherein the size range of the core is 10-100nm, and the thickness range of the shell is 1-100 nm.
The Raman label is one or more of Nile blue, 4-mercaptobenzoic acid, p-nitrobenzoic acid, crystal violet, methylene blue, rhodamine 6G, water-soluble 3H-indocyanine type bioluminescence labeling dye, malachite green isothiocyanate, 4' -bipyridine, 4-mercaptopyridine, p-mercaptoaniline, p-fluorophenylthiol, p-aminophenol and p-mercaptothiophenol.
The modification mode of the metal nano particles modified by the Raman label is that the Raman label is fixed on the surface of the metal nano particles or in the core-shell gaps of the metal nano particles with the core-shell structure through electrostatic adsorption or covalent connection.
The signal in the step III is a surface enhanced Raman scattering signal.
Theoretical model: as shown in fig. 2: a target in a sample firstly reacts with a Raman label based on metal nano particles in a solution, then reaction liquid carries out immune percolation reaction through a micropore channel in immune test paper to form a sandwich structure, and target detection is realized through Raman signal acquisition. A simplified model was used to analyze the reaction process. Approximating the porous structure in the chromatography pad to a plurality of cylindrical microtubes with diameter d, passing the sample through the chromatography pad at a flow rate of u, and sensing the length LsI.e., the detectable thickness of the chromatography pad. The sensing area of each micropore is S, and the radius is Rm. The inner surface of the porous structure is coated with a surface concentration of gamma capture antibody. The diffusion coefficient of the target is D, and the antigen-antibody binding constant is konMass transfer coefficient of kc。
There are two key dimensionless numbers in vertical flow immunoassays, the first being the dankel constant (Da), which describes the relationship between adsorption rate and transport rate:
da = adsorption/transport rate = kon·γ/kc（1a）；
The second is the Peclet constant (P)e) It can be used to compare the convection rate and diffusion rate:
Pe= convection velocity/diffusion velocity = (u/L)s)/(D/d2) (1b)；
To capture target molecules, antigens and capture antibodies efficiently at low concentrationsThe rate of body binding is faster than the rate of antigen molecule transport to the pore walls. High flow rate increases the delivery rate KcDa is reduced, reducing capture efficiency to some extent. However, this can be balanced by using an assay with fast binding kinetics: keep Pe<1 can ensure>Capture efficiency of 90%. The capture efficiency can be improved by reducing the pore size of the porous structure in the chromatography pad. When P is presenteAt < 1, the following constraints on the volumetric flow rate (Q) can be obtained:
Q＜ϕDLsS/d2(1c)；
in the formula (1c), ϕ represents the porosity of the membrane, and as can be seen from the formula (1c), the smaller the pore size of the membrane, the higher the maximum allowable flow rate, and the higher the flow rate can deliver more sample to the membrane within a fixed detection time, so that more target can be detected, and the influence of high flow rate on the capture efficiency is balanced. Therefore, on the basis of theoretical calculations, by appropriately increasing the flow rate: for example, the water absorption is enhanced by increasing the number of layers of the absorption pad, the flow velocity in the vertical direction is increased by reducing the diameter of the hydrophilic channel, and the flow velocity is adjusted by changing the circular truncated cone inclination angle of the PDMS inlet channel. Reduction of pore size of the membrane: such as a nitrocellulose membrane and the like, which replaces a micron-scale irregular channel by a nano-scale anodic alumina membrane with a vertical double-pass structure.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the microarray structure designed by the immune test paper can simultaneously detect a plurality of samples and a plurality of indexes, shortens the detection time and really realizes high-flux detection.
(2) The immune test paper with a microarray structure is used for replacing the traditional microfluidic chip, the operation is simple and convenient, the environment is friendly, the detection cost is greatly reduced, and the consumption of reagents is greatly reduced compared with the traditional test paper by the design of the microarray.
(3) The chromatographic pad provided by the invention can use the anodic alumina based on the nano-scale vertical double-channel structure to replace the traditional nitrocellulose membrane with the micron-scale irregular channel, thereby improving the capture efficiency of the target and further improving the signal-to-noise ratio, and further improving the detection sensitivity.
Description of the drawings:
FIG. 1 is a schematic diagram showing the structural composition of a high-throughput vertical flow immunoassay test paper assay microarray;
FIG. 2 is a schematic top view of a high throughput vertical flow immunoassay strip analysis microarray;
FIG. 3 is a schematic front view of a high throughput vertical flow immunoassay test strip analysis microarray;
FIG. 4 is a schematic left view of a high throughput vertical flow immunoassay strip analysis microarray;
FIG. 5 is a theoretical model of a vertical flow immunoassay performed with the immunoassay paper;
FIG. 6 is a flow chart of the specific steps of the high throughput vertical flow immunoassay test strip assay microarray;
in the figure: 1. a porous PDMS resin chip; 2. double-sided adhesive tape; 3. a chromatographic pad; 4. an absorbent pad; 5. a PDMS resin chip; 6. a target; 7. metal nanoparticles; 8. a signal.
The specific implementation mode is as follows:
the present invention is further illustrated by the following specific examples. It should be noted that these embodiments are provided to illustrate the realizability and superiority of the present invention, but the claims of the present invention are not limited to the following examples.
Example 1: multi-target 6 detection immune test paper analysis microarray for alpha fetoprotein, carcinoembryonic antigen and prostate specific antigen by taking nitrocellulose membrane as chromatography pad 3 material
1. Preparation of an immune test paper analysis microarray:
the high-throughput vertical flow immunoassay test paper analysis microarray takes a porous PDMS resin chip 1 of 96/384 as an inlet channel of a sample, and sequentially distributes the porous PDMS resin chip 1, a double-sided adhesive tape 2, a 96/384 microarray chromatography pad 3 isolated by wax, a 96/384 microarray absorption pad 4 isolated by wax, the double-sided adhesive tape 2 and a PDMS resin chip 5 as substrates from top to bottom. The absorption pad 4 and the PDMS resin chip 5 are overlapped up and down by the double-sided tape 2. The chromatography pad 3 and the absorption pad 4 have the same size of the micropore array and are overlapped up and down. The pore diameter of the micropore array on the chromatographic pad 3 is the same as that of the bottom of the micropore array of the porous PDMS resin chip 1, and the micropore array and the bottom of the micropore array are overlapped up and down through a double-sided adhesive tape 2.
Preparing a porous PDMS resin chip 1: preparing a truncated cone-shaped structure array on a silicon wafer through SU-8 photoresist, wherein the diameters of the upper bottom surface and the lower bottom surface are respectively 2.5mm and 1.5mm, and preparing the chip male die. And (3) pouring Polydimethylsiloxane (PDMS) on the chip male mold, wherein the pouring height is slightly lower than the height of the truncated cone column of the male mold to form a through hole array, and taking down the through hole array after molding to obtain the reverse mold with the truncated cone-shaped holes. And pouring the agarose which is melted by heating on a reverse mould, cooling and forming, and taking down to obtain the porous PDMS resin chip 1.
The prepared micropores of the porous PDMS resin chip 1 are truncated cone-shaped through holes, the diameters of the upper bottom surface and the lower bottom surface of each through hole are respectively 2.5mm and 1.5mm, the height of each through hole is 5mm, and the volume of each through hole is 16 mu l. The upper surface interval between adjacent through holes is 0.5mm, and the lower surface interval is 1.5 mm. The number of wells was 96, with a rectangular distribution (8 rows and 12 columns). The PDMS resin chip 5 has a length of 44mm, a width of 30mm and a height of 5 mm.
The chromatographic pad 3 was made of a nitrocellulose membrane of WhatmanBA85, and had a pore diameter of 0.45 μm and a thickness of 120. mu.m. Cutting the chromatographic pad 3 into a rectangular sheet with the length of 44mm and the width of 30mm, wherein the microarray structure of the chromatographic pad 3 is realized by a wax spraying printing mode, designing a required hydrophobic pattern on a computer, printing the hydrophobic pattern on the chromatographic pad 3 by a wax spraying printer, adhering a thin layer of wax on the surface of the chromatographic pad 3 with the thickness of about 10 mu m, and heating the chromatographic pad 3 printed with the hydrophobic pattern on a heating table at 150 ℃ for 2min to ensure that the wax penetrates through the back of the chromatographic pad 3 to form a closed hydrophilic-hydrophobic alternate region, thereby obtaining the required immunoassay paper microarray structure. The diameter of the 96 micro-holes formed after printing is 1.5mm, which is the same as the diameter of the lower bottom surface of the micro-holes of the porous PDMS resin chip 1, and the distance between the adjacent micro-hole arrays is 1.5 mm.
The material of the absorption pad 4 is absorbent paper, and the length of the absorption pad 4 is 44mm and the width is 30 mm. The microarray structure of the micro-porous array is the same as the realization method of the microarray structure of the chromatographic pad 3, the diameter of the 96 micro-pores formed after printing is 1.5mm, the distance between the adjacent micro-pores is 1.5mm, and the diameter and the distance of the micro-pores are the same as those of the micro-pores of the chromatographic pad 3. Mixing the alpha fetoprotein capture antibody, the carcinoembryonic antigen capture antibody and the prostate specific antigen capture antibody, sequentially fixing the antibodies on 96 hydrophilic micropores of the chromatographic pad 3, sealing the antibodies by bovine serum albumin, and finally assembling the immune test paper from bottom to top according to the sequence of a figure 1.
The PDMS resin chip 5 is 44mm in length, 30mm in width and 5mm in thickness.
2. Preparing an SERS nano label:
the method comprises the steps of selecting SERS nano-tags of gold nanoparticles (25 nm), and respectively marking an alpha-fetoprotein marked antibody, a carcinoembryonic antigen marked antibody and a prostate specific antigen marked antibody on the gold nanoparticles modified with Nile blue, 4-mercaptobenzoic acid and p-nitrobenzoic acid by an electrostatic adsorption method to form the SERS nano-tag with a plurality of targets 6.
3. And (3) detecting a target 6:
mixing the prepared three SERS nano-tags in each centrifugal tube, adding 96 cases of clinical serum into each centrifugal tube respectively, and reacting for about 5 min;
secondly, reaction liquid in 96 centrifuge tubes is added into the LVIII hole from the AI hole through the PDMS inlet channel in sequence, and after 5-10min, a portable Raman spectrometer is used for collecting signals 8 (surface enhanced Raman scattering signals 8) in sequence for each hydrophilic area on the chromatographic pad 3.
4. And (4) analyzing results:
if the characteristic peaks of nile blue, 4-mercaptobenzoic acid and p-nitrobenzoic acid all appear simultaneously in the Raman spectrum collected from the hydrophilic region of the chromatographic pad 3, the clinical serum sample detected has alpha-fetoprotein, carcinoembryonic antigen and prostate specific antigen; if the characteristic peak of the nile blue does not appear and the characteristic peaks of the 4-mercaptobenzoic acid and the p-nitrobenzoic acid appear, the carcinoembryonic antigen and the prostate specific antigen exist in the detection sample; if the characteristic peak of the 4-mercaptobenzoic acid does not appear and the characteristic peaks of the nile blue and the p-nitrobenzoic acid appear, the existence of alpha-fetoprotein and prostate specific antigen in the detection sample is indicated; if the characteristic peak of the p-nitrobenzoic acid does not appear and the characteristic peaks of the nile blue and the 4-mercaptobenzoic acid appear, the existence of alpha-fetoprotein and carcinoembryonic antigen in the detected sample is indicated; if the characteristic peaks of nile blue and 4-mercaptobenzoic acid do not appear and the characteristic peak of p-nitrobenzoic acid appears, the prostate specific antigen exists in the detection sample; if the characteristic peaks of nile blue and p-nitrobenzoic acid do not appear and the characteristic peak of 4-mercaptobenzoic acid appears, the carcinoembryonic antigen exists in the detection sample; if the characteristic peaks of the 4-mercaptobenzoic acid and the p-nitrobenzoic acid do not appear and the characteristic peak of the Nile blue appears, the existence of alpha-fetoprotein in the detection sample is indicated; if the characteristic peaks of the nile blue, the 4-mercaptobenzoic acid and the p-nitrobenzoic acid do not appear, the nile blue, the 4-mercaptobenzoic acid and the p-nitrobenzoic acid do not exist in the detection sample.
Example 2: the vertical double-pass anodic aluminum oxide film is used as the material of the chromatographic pad 3 for four inflammation items: c reaction protein, serum amyloid A, procalcitonin and interleukin 6 multi-target 6 detect the immune test paper microarray.
1. Preparation of an immune test paper analysis microarray:
the preparation method is the same as example 1, polyacrylic acid and protonated polyallylamine are used for modifying an anodic aluminum oxide film, a C-reactive protein capture antibody, a serum amyloid A capture antibody, a procalcitonin capture antibody and an interleukin 6 capture antibody are fully mixed and are sequentially fixed on 96 hydrophilic micropores of a chromatographic pad 3 and are sealed by bovine serum albumin, and finally the immune test paper is assembled from bottom to top according to the sequence of a figure 1.
2. Preparing an SERS nano label:
selecting SERS nano-tags of gold-core silver-shell nanoparticles (40 nm), and respectively marking a C-reactive protein labeled antibody, a serum amyloid A labeled antibody, a procalcitonin labeled antibody and an interleukin 6 labeled antibody on the gold-core silver-shell nanoparticles modified with Nile blue, 4-mercaptobenzoic acid, rhodamine 6G and crystal violet by an electrostatic adsorption method to form the multi-target 6 SERS nano-tags.
3. And (3) detecting a target 6:
mixing the prepared four SERS nano-tags in each centrifugal tube, adding 96 cases of clinical serum into each centrifugal tube in sequence, and reacting for about 5 min;
secondly, reaction liquid in 96 centrifuge tubes is added into the LVIII hole from the AI hole through the PDMS inlet channel in sequence, and after 5-10min, a portable Raman spectrometer is used for collecting signals 8 in sequence in each hydrophilic area on the chromatographic pad 3.
4. And (4) analyzing results:
if characteristic peaks of nile blue, 4-mercaptobenzoic acid, rhodamine 6G and crystal violet all appear simultaneously in a Raman spectrum collected from a hydrophilic region of the chromatographic pad 3, the clinical serum sample to be detected has C reactive protein, serum amyloid A, procalcitonin and interleukin 6; if the characteristic peak of the nile blue does not appear and the characteristic peaks of the 4-mercaptobenzoic acid, the rhodamine 6G and the crystal violet appear, indicating that serum amyloid A, procalcitonin and interleukin 6 exist in the detection sample; if the characteristic peak of the 4-mercaptobenzoic acid does not appear and the characteristic peaks of the nile blue, the rhodamine 6G and the crystal violet appear, indicating that C-reactive protein, procalcitonin and interleukin 6 exist in the detection sample; if the characteristic peak of rhodamine 6G does not appear and the characteristic peaks of nile blue, 4-mercaptobenzoic acid and crystal violet appear, indicating that C-reactive protein, serum amyloid A and interleukin 6 exist in the detection sample; if the characteristic peak of crystal violet does not appear and the characteristic peaks of nile blue, 4-mercaptobenzoic acid and rhodamine 6G appear, the C-reactive protein, serum amyloid A and procalcitonin exist in the detection sample. If the characteristic peaks of the nile blue and the 4-mercaptobenzoic acid do not appear and the characteristic peaks of the rhodamine 6G and the crystal violet appear, indicating that the procalcitonin and the interleukin 6 exist in the detection sample; if the characteristic peaks of the nile blue and the rhodamine 6G do not appear and the characteristic peaks of the 4-mercaptobenzoic acid and the crystal violet appear, indicating that serum amyloid A and interleukin 6 exist in the detection sample; if the characteristic peaks of the nile blue and the crystal violet do not appear and the characteristic peaks of the 4-mercaptobenzoic acid and the rhodamine 6G appear, the serum amyloid A and the procalcitonin exist in the detection sample.
If the characteristic peaks of the 4-mercaptobenzoic acid and the rhodamine 6G do not appear and the characteristic peaks of the nilla and the crystal violet appear, indicating that the C-reactive protein and the interleukin 6 exist in the detection sample; if the characteristic peaks of the 4-mercaptobenzoic acid and the crystal violet do not appear and the characteristic peaks of the nile blue and the rhodamine 6G appear, indicating that C-reactive protein and procalcitonin exist in the detection sample; if the characteristic peaks of rhodamine 6G and crystal violet do not appear and the characteristic peaks of nile blue and 4-mercaptobenzoic acid appear, indicating that C-reactive protein and serum amyloid A exist in the detection sample. If the characteristic peaks of nile blue, 4-mercaptobenzoic acid and rhodamine 6G do not appear and the characteristic peak of crystal violet appears, indicating that the interleukin 6 exists in the detection sample; if the characteristic peaks of nile blue, 4-mercaptobenzoic acid and crystal violet do not appear and the characteristic peak of rhodamine 6G appears, the procalcitonin exists in the detection sample; if the characteristic peaks of nile blue, rhodamine 6G and crystal violet do not appear and the characteristic peak of 4-mercaptobenzoic acid appears, indicating that serum amyloid A exists in the detection sample; if the characteristic peaks of the 4-mercaptobenzoic acid, the rhodamine 6G and the crystal violet do not appear and the characteristic peak of the nile blue appears, the C-reactive protein exists in the detection sample. If the characteristic peaks of nile blue, 4-mercaptobenzoic acid, rhodamine 6G and crystal violet do not appear, the clinical sample does not contain four inflammatory factors of C-reactive protein, serum amyloid A, procalcitonin and interleukin 6.
Claims (10)
1. A high-flux vertical flow immune test paper analysis microarray is characterized in that: the device comprises a porous PDMS resin chip, a chromatography pad, an absorption pad and a PDMS resin chip which are arranged in sequence from top to bottom; the pore diameter of the chromatographic pad is the same as that of the bottom of the porous PDMS resin chip, and the chromatographic pad and the bottom of the porous PDMS resin chip are overlapped up and down through a double-sided adhesive tape; the pore diameter of the chromatography pad is the same as that of the microarray of the absorption pad; the absorption pad and the PDMS resin chip are overlapped up and down through the double-sided adhesive tape.
2. The microarray for analyzing high-throughput vertical flow immunoassay test paper according to claim 1, wherein the method for preparing the porous PDMS resin chip comprises the following steps:
step 1: preparing a truncated cone-shaped structure array on a silicon wafer through photoresist to obtain a chip male die;
step 2: pouring polydimethylsiloxane on the male die of the chip, and forming a micropore array on the structure array to obtain a reverse die with micropores;
and step 3: pouring agarose on a reverse mould, cooling and forming to obtain the porous PDMS resin chip.
3. The high throughput vertical flow immunoassay strip of claim 2, wherein the microarray comprises: the micropore of the porous PDMS resin chip is a truncated cone-shaped through hole, the diameter of the upper bottom surface of the through hole is 2-4mm, the diameter of the lower bottom surface of the through hole is 1-2mm, and the depth of the through hole is 3-6 mm; the distance between the upper surfaces of the adjacent through holes is 0.4-1mm, and the distance between the lower bottom surfaces is 1-2 mm.
4. The high throughput vertical flow immunoassay test strip of claim 1, wherein: the chromatographic pad is a wax-isolated chromatographic pad; the material of the chromatographic pad is any one of alumina, polystyrene, silicon dioxide, polymethyl methacrylate, polyethylacrylate, polyethylene, cellulose nitrate, cellulose acetate, nylon and polyvinylidene fluoride; the length of the chromatographic pad is 36-68mm, and the width is 25-36 mm; the chromatography pad has vertically arranged holes with a diameter of 100-1000nm and a depth of 30-200 μm.
5. The high throughput vertical flow immunoassay test strip of claim 1, wherein: the absorption pad is isolated by wax; the material of the absorption pad is any one of absorbent paper, cellulose membrane, filter paper, cellulose-glass fiber composite, non-woven fabric and sponge; the absorbent pad has a length of 36-68mm and a width of 25-36 mm.
6. The high throughput vertical flow immunoassay test strip of claim 1, wherein: the PDMS resin chip is 36-68mm in length, 25-36mm in width and 3-6mm in thickness.
7. A high throughput vertical flow immunoassay strip microarray according to any of claims 1 to 6, wherein: the specific application steps of the immune test paper analysis microarray are as follows:
1) immobilizing a capture antibody of the target on a hydrophilic region of the chromatography pad;
2) reacting the target with a metal nanoparticle-based raman tag in a test tube;
3) the reaction solution is subjected to immunofiltration reaction by a microarray on the test paper, and target detection is realized by signal acquisition.
8. The high throughput vertical flow immunoassay test strip of claim 7, wherein: the target in the step 2) is any one of protein, nucleic acid, heavy metal ions, organic micromolecules, fungi, bacteria and viruses; the signal in the step 3) is a surface enhanced Raman scattering signal.
9. The high throughput vertical flow immunoassay test strip of claim 7, wherein:
the metal nanoparticles in the step 2) are any one of gold nanoparticles, silver nanoparticles, platinum nanoparticles, gold-core silver-shell nanoparticles, gold-core gold-shell nanoparticles, silver-core silver-shell nanoparticles, silica-core gold-shell nanoparticles, silica-core silver-shell nanoparticles, gold-core silica-shell nanoparticles and silver-core silica-shell nanoparticles;
the surface of the metal nano particle is modified with a Raman label, and the surface of the metal nano particle is fixed with a detection antibody through electrostatic adsorption or covalent connection;
the Raman label is any one or more of Nile blue, 4-mercaptobenzoic acid, p-nitrobenzoic acid, crystal violet, methylene blue, rhodamine 6G, water-soluble 3H-indocyanine type bioluminescence labeled dye, malachite green isothiocyanate, 4' -bipyridine, 4-mercaptopyridine, p-mercaptoaniline, p-fluorophenylthiol, p-aminophenol and p-mercaptothiophenol.
10. The high throughput vertical flow immunoassay strip of claim 9, wherein the microarray comprises: the modification method of the metal nano particles modified by the Raman label comprises the following steps: and fixing the Raman label on the surface of the metal nanoparticle or in the core-shell gap of the metal nanoparticle with the core-shell structure through electrostatic adsorption or covalent connection.
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