CN107656083B - Self-sampling immune detection paper chip and preparation method thereof - Google Patents
Self-sampling immune detection paper chip and preparation method thereof Download PDFInfo
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Abstract
A self-injection immune detection paper chip and a preparation method thereof, wherein the self-injection immune detection paper chip is used for detecting the concentration of an antigen and comprises the following components from top to bottom along the lamination direction: the microfluidic layer is provided with a microfluidic channel and a sample inlet hole which are connected through a communication channel, the microfluidic channel is used for self-sampling, and the sample inlet hole is used for introducing a tissue body to be detected obtained by sampling into a detection area; an auxiliary electrode layer; the detection layer and the working electrode layer are modified with electroactive composite materials on the surfaces, and the surfaces of the electroactive composite materials are used for fixing antibodies; the auxiliary electrode layer, the detection layer and the working electrode layer form a detection area which comprises an electrochemical detection circuit, and the concentration of the antigen in the liquid to be detected is detected and obtained in an electrochemical mode based on the specific binding principle of the antigen-antibody and the characteristics of the electroactive composite substance. Because the advantages of the microfluidic technology and the paper are combined, automatic sample injection is realized through the arranged microfluidic channel and the sample injection hole, the operation is easy, and professional operators are not needed.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a self-injection immune detection paper chip and a preparation method thereof.
Background
The antigen is a substance capable of inducing an immune response, and most antigens have specificity and are capable of specifically binding to an antibody. There are many kinds of antigens, including proteins and polypeptides. The content of certain antigens, such as disease markers and the like, has important significance for human health. The detection of the substances can be used for mechanism research, early diagnosis, early intervention and treatment of some serious diseases.
Currently, there are a variety of immunoassay methods available for high-sensitivity detection of antigens, including radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay, and the like. However, the above methods all require pretreatment of the antigen or antibody to be detected, and thus have limitations in terms of instant detection.
Disclosure of Invention
Based on the above problems, the main objective of the present invention is to provide a trace electrochemical immunoassay paper chip and a method for preparing the same, which are used to solve at least one of the above technical problems.
In order to achieve the above object, according to one aspect of the present invention, there is provided a self-sampling immunoassay paper chip for detecting antigen concentration, comprising, from top to bottom in a stacking direction: the microfluidic layer is provided with a microfluidic channel and a sample inlet hole which are connected through a communication channel, the microfluidic channel is used for self-sampling, and the sample inlet hole is used for introducing the tissue fluid to be detected obtained by sampling into the detection area; an auxiliary electrode layer; the detection layer and the working electrode layer are modified with electroactive composite materials on the surfaces, and the surfaces of the electroactive composite materials are used for fixing antibodies; the auxiliary electrode layer, the detection layer and the working electrode layer form a detection area which comprises an electrochemical detection circuit, and the concentration of the antigen in the liquid to be detected is detected and obtained in an electrochemical mode based on the specific binding principle of the antigen-antibody and the characteristics of the electroactive composite material.
In some embodiments of the present invention, the electroactive composite material includes a composite material of a carbon nanomaterial, an electroactive material, and nanogold; the carbon nano material comprises a multi-walled carbon nano tube or aminated graphene; the electroactive substance comprises thionine, Prussian blue or potassium ferricyanide; preferably, in the composite material, the ratio of the carbon nano material to the electroactive substance to the nanogold is 1: 5-1: 2: 10.
In some embodiments of the present invention, the working electrode layer comprises at least two circumferentially arranged circular working electrodes; the auxiliary electrode layer comprises a first through hole arranged opposite to the sample inlet hole, at least two second through holes arranged opposite to the round working electrode, and at least two pairs of combined counter electrodes and reference electrodes; the combination of the counter electrode and the reference electrode is respectively matched with partial edges of at least two second through holes; the circular working electrode, the counter electrode and the reference electrode are all connected with electrode leads so as to be connected with an external device.
In some embodiments of the present invention, the detection layer comprises a third through hole facing the first through hole, and symmetrical communication holes extending along the length direction of the detection layer and facing the circular working electrode, thereby forming a detection region.
In some embodiments of the present invention, the microfluidic layer and the detection layer each have at least two sets of edge through holes arranged along the circumferential direction, and the at least two sets of edge through holes are opposite to the electrode leads of the working electrode, the counter electrode and the reference electrode.
In some embodiments of the invention, the upper surface of the microfluidic layer is provided with a first filter layer; the first filter layer is provided with a first filter hole which is arranged opposite to the microfluidic channel and is used for filtering the tissue fluid to be detected when the microfluidic channel performs self-sampling; preferably, the porosity of the first filtering pores is less than 3 μm.
In some embodiments of the present invention, a second filtering layer is disposed on a lower surface of the microfluidic layer, and has a second filtering hole disposed opposite to the sample inlet, for further filtering the tissue fluid to be measured obtained by sampling.
In some embodiments of the present invention, the width of the communication channel connecting the microfluidic channel and the sample inlet is inversely proportional to the sampling rate.
In some embodiments of the present invention, the size of the above-mentioned self-injection immunoassay paper chip is 10.5mm × 35 mm.
In some embodiments of the present invention, the microfluidic channels, the communication channels, and the sampling holes of the microfluidic layer are located in a hydrophobic region, and other regions are hydrophilic.
In order to achieve the above object, the present invention further provides a method for preparing a self-injection immunoassay paper chip, which is used for preparing the self-injection immunoassay paper chip, and comprises the following steps: printing a preset microfluidic channel on the surface of the microfluidic layer through a wax-spraying printer, and heating the microfluidic layer to enable the area of the microfluidic channel of the microfluidic layer to have hydrophobicity; respectively printing a working electrode, a counter electrode and a reference electrode on the working electrode layer and the auxiliary electrode layer by a screen printer; modifying the surface of the working electrode with an electroactive composite material; integrally packaging the microfluidic layer, the auxiliary electrode layer, the detection layer and the working electrode layer; when the self-sampling immunoassay paper chip is used, the antibody is dripped on the surface of the working electrode modified with the electroactive composite material.
In some embodiments of the present invention, the electroactive composite material is prepared by: step 1, dissolving a multi-walled carbon nanotube/aminated graphene into a polyethyleneimine aqueous solution to enable the multi-walled carbon nanotube/aminated graphene to be non-covalently functionalized; step 2, adding an electroactive substance into the solution, and stirring vigorously for a certain time to combine the non-covalent functionalized multi-walled carbon nanotube/aminated graphene with the electroactive substance; and 3, mixing the solution obtained in the step 2 with the nano gold particle solution, adding the chitosan solution after stirring vigorously, and carrying out ultrasonic mixing to obtain the electroactive composite material.
The self-injection immune detection paper chip and the preparation method thereof provided by the invention have the following beneficial effects:
1. because the advantages of the microfluidic technology and the paper are combined, the liquid flows on the paper by utilizing the capillary action, and the automatic sample injection is realized through the arranged microfluidic channel and the sample injection hole, the operation is easy, and professional operators are not needed; the surface of the working electrode layer is modified with the electroactive composite material, so that an antibody can be fixed, and when an antigen is combined with the antibody fixed on the surface of the electroactive composite material, the activity of the electroactive composite material is influenced, so that the current generated by the interaction of the electroactive composite material and the surface of the working electrode is influenced, and the concentration of the antigen can be indirectly measured, so that a detected sample does not need to be pretreated, the specific combination principle of the antigen and the antibody and the characteristics of the electroactive composite material can be directly detected, the detection time is short, and the rapid trace detection can be realized;
2. the first filtering layer with the first filtering holes is arranged, so that the serum sample and the whole blood sample can be detected by selecting the filtering holes with different pores; and the amount of sample required for the detection process is small, about 10 μm;
3. the microfluidic channel is printed on the surface of the microfluidic layer by a wax-spraying printer and then heated, so that the area where the microfluidic channel is located has hydrophobicity, the paper chip and the capillary action of liquid are combined, the sampling can be rapidly carried out, the flowing direction of the liquid can be well guided, and the rapid and efficient detection can be realized;
4. the combination of at least two groups of working electrodes, reference electrodes and counter electrodes is provided, so that different antibodies are dripped on the surfaces of the at least two groups of combined working electrodes, the detection of the concentration of at least two antigens can be realized by one-time sampling, and the sample amount required in the detection process is further reduced;
5. the manufacturing cost is low, and the disposable portable use is convenient for detection.
Drawings
FIG. 1 is a schematic diagram of a layered structure of a self-sampling immunoassay paper chip according to an embodiment of the present invention;
FIG. 2 is a flow chart of the preparation of the electroactive composite material in the method for preparing the self-injection immunoassay paper chip according to an embodiment of the present invention;
fig. 3 is a schematic diagram of the use and detection of the self-injection immunoassay paper chip according to an embodiment of the present invention.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
The label-free electrochemical rapid detection technology can carry out rapid and trace detection on the analyzed antigen (protein and polypeptide) by directly measuring the current change formed when the antigen and the antibody are combined, so that the related research also becomes a hot spot direction of the advanced scientific research of the international biosensor, and has important scientific significance and application prospect.
In recent years, nanomaterials gradually enter the frontier of analytical chemistry, play a key role in various analytical methods, are increasingly researched in terms of application of nanomaterials in electroanalytical chemistry and electrochemical sensors, and show a trend of converting traditional electrochemical electrodes into nanomaterial-modified electrodes. The nanometer material has many unique properties different from the traditional material, compared with the conventional electrode, the nanometer material modified electrode has larger effective surface area, high substance transmission speed and high catalytic activity, and in addition, the nanometer material modified electrode also has the capability of regulating and controlling local environment on the surface of the electrode. Among them, carbon nanotubes and graphene are widely used for modification of biosensors due to their excellent conductivity and certain electrocatalysis function.
The microfluidic paper chip is called a paper chip for short, and combines the advantages of the microfluidic technology and paper. Compared with the traditional electrode, the paper chip has the advantages that: 1. the paper has rich sources and can be produced in batches; 2. an external pump is not needed, the main component of the paper is cellulose, and the fluid flows on the paper through the capillary action, so that automatic sample injection detection can be realized; 3. the consumption of the sample is lower, the biocompatibility is good, and the property of the paper can be changed through chemical modification; 4. disposable portable analysis, easy and simple to handle, do not even need professional operating personnel. The paper chip provides a wide platform for portable detection and field real-time monitoring required in clinical diagnosis, environmental monitoring and food safety analysis, and is a powerful tool for low-cost and rapid detection instant diagnosis in less developed areas with shortage of medical care personnel and medical equipment.
The invention aims at the rapid and portable detection requirement of antigens (proteins and polypeptides), prepares a novel automatic-feeding type trace electrochemical immunodetection paper chip by synthesizing a nano composite material based on a label-free electrochemical rapid detection technology and an antigen-antibody specific combination principle, and carries out trace and rapid detection tests by taking tumor markers (carcinoembryonic antigen CEA, neuron specific enolase NSE and the like), neurotrophic factors (brain-derived neurotrophic factor BDNF and the like) and hormones and the like as entry points.
Specifically, the invention provides a self-sample-injection immune detection paper chip for detecting the concentration of an antigen, which comprises the following components from top to bottom along the lamination direction: the microfluidic layer is provided with a microfluidic channel and a sample inlet hole which are connected through a communication channel, the microfluidic channel is used for self-sampling, and the sample inlet hole is used for introducing the tissue fluid to be detected obtained by sampling into the detection area; an auxiliary electrode layer; the detection layer and the working electrode layer are modified with electroactive composite materials on the surfaces, and the surfaces of the electroactive composite materials are used for fixing antibodies; the auxiliary electrode layer, the detection layer and the working electrode layer form a detection area which comprises an electrochemical detection circuit, and the concentration of the antigen in the liquid to be detected is detected and obtained in an electrochemical mode based on the specific binding principle of the antigen-antibody and the characteristics of the electroactive composite material.
Therefore, the invention combines the advantages of the microfluidic technology and the paper, utilizes the capillary action flow of the liquid on the paper, realizes automatic sample injection through the arranged microfluidic channel and the sample injection hole, is easy to operate and does not need professional operators; the surface of the working electrode layer is modified with the electroactive composite material, so that an antibody can be fixed, the activity of the electroactive composite material is influenced when an antigen is combined with the antibody fixed on the surface of the electroactive composite material, the current generated by the interaction of the electroactive composite material and the surface of the working electrode is influenced, and the concentration of the antigen can be indirectly measured.
In some embodiments of the present invention, the electroactive composite material includes a composite material of a carbon nanomaterial, an electroactive material, and nanogold; the carbon nano material can be a multi-walled carbon nanotube or aminated graphene, but not limited to this, and any carbon nano material capable of forming the composite material required by the invention can be used; the electroactive substance includes thionine, prussian blue or potassium ferricyanide, but not limited thereto, and any electroactive substance that can affect the activity of an antigen when the antigen is bound to an antibody, thereby affecting the magnitude of a current generated by the interaction between the electroactive substance and the surface of the working electrode can be used.
In some embodiments of the present invention, in the composite material, a ratio of the carbon nanomaterial, the electroactive material, and the nanogold is 1: 5 to 1: 2: 10.
In some embodiments of the present invention, the working electrode layer comprises at least two circumferentially arranged circular working electrodes; the auxiliary electrode layer comprises a first through hole arranged opposite to the sample inlet hole, at least two second through holes arranged opposite to the round working electrode, and at least two pairs of combined counter electrodes and reference electrodes; the combination of the counter electrode and the reference electrode is respectively matched with partial edges of at least two second through holes; the circular working electrode, the counter electrode and the reference electrode are all connected with electrode leads so as to be connected with an external device. The working electrode, the counter electrode and the reference electrode form a three-electrode system, the three electrodes are all connected with an electrochemical workstation through electrode leads to form an electrochemical detection circuit, and therefore the concentration of the antigen in the liquid to be detected is detected and obtained in an electrochemical mode based on the specific binding principle of the antigen-antibody and the characteristics of the electroactive composite material. And because the combination of at least two groups of working electrodes, reference electrodes and counter electrodes is provided, different antibodies are dripped on the surfaces of the at least two groups of combined working electrodes, so that the detection of the concentrations of at least two antigens can be realized by one-time sampling, and the sample amount required in the detection process is further reduced.
In some embodiments of the present invention, the detection layer comprises a third through hole facing the first through hole, and symmetrical communication holes extending along the length direction of the detection layer and facing the circular working electrode, thereby forming a detection region.
In some embodiments of the present invention, the microfluidic layer and the detection layer each have at least two sets of edge through holes arranged along the circumferential direction, and the at least two sets of edge through holes face the electrode leads of the working electrode, the counter electrode and the reference electrode so as to be connected to the electrochemical workstation.
In some embodiments of the invention, the upper surface of the microfluidic layer is provided with a first filter layer; the first filter layer is provided with a first filter hole which is arranged opposite to the microfluidic channel and is used for filtering liquid to be detected when the microfluidic channel performs self-sampling; preferably, the porosity of the first filtering pores is less than 3 μm. Specifically, the pore diameter of the first filtering hole can be specifically set, so that cells and molecules with large diameters such as white blood cells and red blood cells in the whole blood can be effectively filtered by adopting a small filtering pore, and the detection of a sample of the whole blood can be realized.
In some embodiments of the present invention, a second filtering layer is disposed on a lower surface of the microfluidic layer, and has a second filtering hole disposed opposite to the sample inlet, for further filtering the tissue fluid to be measured obtained by sampling.
In some embodiments of the present invention, the width of the communication channel connecting the microfluidic channel and the sample inlet is inversely proportional to the sampling rate. Therefore, the communication channel can be set to a larger width according to the actual sampling rate, so as to achieve the purpose of quick detection.
In some embodiments of the present invention, the size of the self-feeding immunoassay paper chip is 10.5mm × 35mm, and the size is adopted to fully consider the convenience of the connection operation of the self-feeding immunoassay paper chip and the back-end instrument.
In some embodiments of the present invention, the areas of the microfluidic channels, the communication channels, and the sampling holes of the microfluidic layer have hydrophobicity, and other areas have hydrophilicity, so that the flow direction of the liquid can be well guided by the microfluidic channels, and rapid and efficient detection can be achieved.
In order to achieve the above object, the present invention further provides a method for preparing a self-injection immunoassay paper chip, which is used for preparing the self-injection immunoassay paper chip, and comprises the following steps: printing a preset microfluidic channel on the surface of the microfluidic layer through a wax-spraying printer, and heating the microfluidic layer to enable the area of the microfluidic channel of the microfluidic layer to have hydrophobicity; respectively printing a working electrode, a counter electrode and a reference electrode on the working electrode layer and the auxiliary electrode layer by a screen printer; modifying the surface of the working electrode with an electroactive composite material; integrally packaging the microfluidic layer, the auxiliary electrode layer, the detection layer and the working electrode layer; when the self-sampling immunoassay paper chip is used, the antibody is dripped on the surface of the working electrode modified with the electroactive composite material.
The electroactive composite material is prepared by the following steps: step 1, dissolving a multi-walled carbon nanotube/aminated graphene into a polyethyleneimine aqueous solution to enable the multi-walled carbon nanotube/aminated graphene to be non-covalently functionalized; step 2, adding an electroactive substance into the solution, and stirring vigorously for a certain time to combine the non-covalent functionalized multi-walled carbon nanotube/aminated graphene with the electroactive substance; and 3, mixing the solution obtained in the step 2 with the nano gold particle solution, adding the chitosan solution after stirring vigorously, and carrying out ultrasonic mixing to obtain the electroactive composite material.
In summary, the self-sample-injection immune whole blood detection paper chip designed by the invention can perform label-free, high-sensitivity, trace, instant and rapid detection on protein (disease markers and neurotrophic factors) and polypeptide molecules based on the combined application of a microfluidic paper chip, a carbon nano composite material preparation and modification technology, a label-free electrochemical detection technology and the like, has low detection requirement, can detect serum and whole blood samples, breaks through the traditional detection technology, and provides a novel detection device and method for detecting protein and polypeptide molecules.
In some embodiments of the present invention, as shown in fig. 1, a novel self-sampling immunoassay paper chip is provided, which comprises 6 layers, namely, a first filter layer 1, a microfluidic layer 2, a second filter layer 3, an auxiliary electrode layer 4, a detection layer 5 and a working electrode layer 6, specifically, the first filter layer 1 is provided with a first filter hole 11, the microfluidic layer 2 is provided with a microfluidic channel 22 and a sample inlet hole 23 connected through a communication channel 21, wherein the microfluidic channel 22 is disposed at a position opposite to the first filter hole 11; the second filter layer 3 is provided with a second filter hole 31 opposite to the sample inlet hole 23; the auxiliary electrode layer 4 is provided with a first through hole 41 opposite to the second filtering hole 31 and two second through holes 42 symmetrically arranged at two sides of the first through hole 41; the edges of the two second through holes 42 are respectively provided with a counter electrode 43 and a reference electrode 44 which are matched with part of the edges, the counter electrode 43 and the reference electrode 44 are separately arranged and are respectively connected with external equipment through a counter electrode lead 45 and a reference electrode lead 46; the detection layer 5 is provided with a third through hole 51 opposite to the first through hole 41, the third through hole 51 is provided with symmetrical through holes 52 extending in the length direction of the detection layer 5, the end part 520 of the through hole 52 is a detection area of the detection layer 5, and the position of the detection area is opposite to the position of the second through hole 42; the working electrode layer 6 is provided with a circular working electrode 61 facing the detection area, the circular working electrode is connected with external equipment through a working electrode lead 62, and the size of the circular working electrode is matched with that of the second through hole.
The paper chip designed in this embodiment is a 3D structure paper chip, and is a 6-layer structure formed by integrated packaging, and since the fluid flows on the paper through capillary action, the paper chip realizes self-sample injection of the tissue fluid to be tested by the microfluidic channel 22, and the tissue fluid to be tested after self-sample injection is filtered by the first filtering hole 11 at the same time, so as to filter out the cells and molecules with large diameters such as white blood cells and red blood cells in the whole blood; the tissue fluid to be measured reaches the sample inlet hole 23 through the connecting channel 21 and then permeates through the second filtering hole 31, so that the residual large-diameter cells and molecules are further filtered; after further filtration, the liquid to be measured reaches the detection region 520 at the end of the communication hole 52 via the first through hole 41 and the third through hole 51, so that the counter electrode 43, the reference electrode 44, and the working electrode 61 are in the same liquid environment.
Wherein, the first layer 1, the third layer 3 and the fifth layer 5 of the paper chip adopt filter paper with the porosity of less than 3 μm, the particle retention degree is less than 3 μm, and cells and molecules with large diameters such as white blood cells, red blood cells and the like can be effectively filtered; the second layer 2, the fourth layer 4 and the sixth layer 6 adopt filter paper with the porosity smaller than the porosity of the first layer, the third layer and the fifth layer. The detection of serum and whole blood samples can be realized by arranging the first, third and fifth layers of porosity.
The surface of the working electrode is modified with an electroactive composite material, and the electroactive composite material is prepared by the following preparation method:
as shown in FIG. 2, first, 1mg of multi-walled carbon nanotubes was dissolved in 10mL of an aqueous Polyethyleneimine (PEI) solution, and 2mL of the solution was taken at a concentration of 1mg/mL-1And 2mL of the dispersion of the multi-walled carbon nanotube (2 mg/mL)-1Mixing the thionine solution or Prussian blue solution, stirring vigorously for 24h, centrifuging to remove unbound thionine or Prussian blue molecules, mixing 100 μ L of thionine/Prussian blue-multi-walled carbon nanotube mixed solution with 250 μ L of gold nanoparticle solution, stirring vigorously for 12h, adding 100 μ L of chitosan solution, and ultrasonic mixing to obtain multi-walled carbonnanotube-thionine/Prussian blue-nano gold-chitosan mixed liquor, namely the electroactive composite material.
Because in the preparation of the electroactive composite material, the multi-walled carbon nanotube is modified by utilizing the non-covalent function of the polyethyleneimine, compared with common graphene, the catalytic action is stronger, so that the finally generated signal current is larger, and the detection range of the self-sampling immunoassay paper chip is wider.
In some embodiments of the present invention, a method for preparing a self-injection immunoassay paper chip is provided, which comprises the following specific steps:
1. the structure of each layer of the self-sampling immune detection paper chip is designed by using computer software, and the main structure comprises a microfluidic channel structure of the paper chip and a structure of an electrode. The paper chip adopts a three-electrode system, the working electrode is circular, the diameter of the working electrode is 3mm, the counter electrode and the reference electrode are both arc-shaped, when the working electrode and the reference electrode are combined together, the middle graph of the working electrode and the reference electrode is circular, the diameter of the working electrode is also 3mm, and the working electrode and the counter electrode are matched with each other in area;
2. because the printing breadth of the wax-spraying printer is limited, the filter paper with the A1 breadth is cut into the A4 breadth by a paper cutter and is suitable for printing, the first layer, the third layer and the fifth layer of the paper chip adopt the filter paper with the porosity of 1 mu, the particle retention degree is 1 mu m, and cells and molecules with large diameters such as white blood cells, red blood cells and the like can be effectively filtered; the second, fourth and sixth layers were made of filter paper with a porosity of 11 and a particle retention of 11 μm. (ii) a
3. Printing the designed paper chip channel pattern on the surface of the second layer of filter paper by using a digital wax-spraying printer, and simultaneously printing corresponding patterns on the filter paper of other layers;
4. and putting the wax-sprayed filter paper into an oven, wherein the wax on the surface of the paper is melted and infiltrated into the paper due to heating, so that the wax-sprayed area is changed into a hydrophobic area, and the wax-not-sprayed area is changed into a hydrophilic area. The wax can diffuse to the periphery while penetrating into the interior, so that the areas of the hydrophilic and hydrophobic regions can be accurately controlled by accurately controlling the heating temperature and time;
5. and respectively printing a three-electrode system (comprising a working electrode, a counter electrode and a reference electrode) on the surface of the paper heated by the wax spraying by using a screen printer. The working electrode and the counter electrode are made of conductive carbon paste, and the reference electrode is made of conductive Ag/AgCl paste.
6. Designing a scribing graph, and manufacturing a double-sided adhesive tape with a corresponding structure by using a scribing mechanism;
7. preparing a carbon nano material-electroactive material composite nano material based on the carbon nano material and the electroactive material;
8. adding nanogold to prepare a carbon nanomaterial-electroactive substance-nanogold composite nanomaterial, and modifying a working electrode of the paper chip; dripping 10 μ L of the mixed solution of multi-walled carbon nanotube-thionine/Prussian blue-nanogold on the surface of the working electrode, putting the working electrode into an oven, and baking for 20min at 50 ℃.
9. Packaging the modified working electrode and the structures of other paper chips together by using the carved double-sided adhesive tape so as to be used for detection;
10. and (3) carrying out actual test on the sample by utilizing the antigen-antibody specific adsorption principle.
The working principle and the using method of the self-sampling immune detection paper chip prepared by the embodiment are shown in figure 3, and 10 mul of the immune detection paper chip with the concentration of 200mg/mL is taken-1The antibody corresponding to the antigen to be detected in the tissue fluid to be detected is dripped on the surface of the electrode, and the electrode is placed in a refrigerator and placed for 6 hours at 4 ℃ for combining the antigen. The remaining active sites on the electrode surface were then blocked with 10 μ L of 1% bovine serum albumin at room temperature to reduce specific repair.
The self-sampling immune detection paper chip automatically samples tissue fluid to be detected, the tissue fluid flows to the detection layer, wherein the antigen is combined with the antibody fixed on the surface of the electroactive composite material, although the antigen is specifically combined with the antibody and does not generate electron transfer, because the electroactive material is fixed on the surface of the electrode, the detected current is the current generated by the interaction of the electroactive material and the surface of the electrode, after the antigen is combined with the antibody fixed on the surface of the electrode, the activity of the fixed electroactive material can be influenced, so that the detected current is influenced, the more the antigen is combined, the smaller the detected current is, the inverse relation between the antigen concentration and the current magnitude is formed, and the concentration of the antigen in the tissue fluid to be detected can be indirectly measured by detecting the current magnitude.
The following describes the self-injection immunoassay paper chip and the preparation method and the application method thereof in detail by specific embodiments.
Example 1
The embodiment provides an automatic sample introduction type trace rapid electrochemical immune whole blood detection paper chip and a preparation method thereof, which are used for trace rapid electrochemical detection of CEA and NSE with two parameters automatically fed simultaneously, and specifically adopt a structure shown in figure 1.
The preparation and use processes of the automatic feeding type trace rapid electrochemical whole blood detection paper chip for detecting the tumor marker are as follows:
(1) selecting two parameters of carcinoembryonic antigen (CEA) and neuron specific alkene purification enzyme (NSE) for combined determination;
(2) preparing an automatic feeding type trace rapid electrochemical whole blood detection paper chip: printing the designed channel pattern on the surface of the corresponding filter paper by a wax-spraying printer; the paper is placed in an oven, and the wax on the surface of the paper is melted and permeates into the paper due to heating, so that the wax spraying area is changed into a hydrophobic area, and the non-wax spraying area is changed into a hydrophilic area, therefore, the areas of the hydrophilic area and the hydrophobic area can be accurately controlled by accurately controlling the heating temperature and time; printing a counter electrode and a reference electrode on the surface of the fourth layer of paper and printing a working electrode on the surface of the sixth layer of paper by using a screen printer;
(3) preparing a multi-walled carbon nanotube-electroactive substance-nanogold composite nanomaterial: dissolving 1mg of multi-wall carbon nano tube into 10mL of PEI aqueous solution, and taking 2mL of PEI aqueous solution with the concentration of 1mg/mL-1And 2mL of the dispersion of the multi-walled carbon nanotube (2 mg/mL)-1Mixing the thionine solution or Prussian blue solution, stirring vigorously for 24h, centrifuging to remove unbound thionine or Prussian blue molecules, and collecting 100 μ L of thionine/Prussian blue-multi-walled carbon nanotube mixed solution and 250 μ LAnd mixing the L nano gold particle solution, stirring vigorously for 12h, adding 100 mu L of chitosan solution, and carrying out ultrasonic mixing to obtain the multi-wall carbon nano tube-thionine/Prussian blue-nano gold-chitosan mixed solution.
(4) Modification of the working electrode: transferring 10 μ L of the multi-walled carbon nanotube-electroactive substance-nanogold composite nanomaterial onto the surface of the working electrode by using a pipette, and baking at 50 ℃ for 20 min. Then, 10. mu.L of 200mg/mL was taken-1The CEA and NSE antibodies are respectively coated on the surface of the electrode in a dripping way, and are placed in a refrigerator and are placed for 6h at the temperature of 4 ℃ for binding the antibodies. Finally, 10 μ L of 1% BSA solution was used at room temperature to cover the remaining active sites.
(5) And manufacturing a double-sided adhesive tape by using a scribing machine, and carrying out integrated packaging on the paper chip.
The automatic sample introduction type trace rapid electrochemical immune whole blood detection paper chip prepared by the embodiment can automatically introduce samples, the required sample amount is about 10 mu L, and the samples are automatically introduced through a microfluidic channel and a sample introduction hole; and (3) after sample introduction, standing for 10 minutes at room temperature to combine the antigen and the antibody, directly connecting the paper chip with an electrochemical workstation through a rear-end interface, detecting the current response of a working electrode by adopting a differential pulse voltammetry method, and inversely calculating the concentration of CEA and NSE in the sample to be detected according to the change value of a current signal.
Example 2
The embodiment provides an automatic sample introduction type trace rapid electrochemical immune whole blood detection paper chip and a preparation method thereof, the automatic sample introduction type trace rapid electrochemical immune whole blood detection paper chip is used for detecting neurotrophic factors (BDNF), and the preparation and use processes are as follows:
(1) selecting BDNF parameters for determination;
(2) preparing an automatic feeding type trace rapid electrochemical whole blood detection paper chip: printing the designed channel pattern on the surface of the corresponding filter paper by a wax-spraying printer; the paper is placed in an oven, and the wax on the surface of the paper is melted and permeates into the paper due to heating, so that the wax spraying area is changed into a hydrophobic area, and the non-wax spraying area is changed into a hydrophilic area, therefore, the areas of the hydrophilic area and the hydrophobic area can be accurately controlled by accurately controlling the heating temperature and time; printing a counter electrode and a reference electrode on the surface of the fourth layer of paper and printing a working electrode on the surface of the sixth layer of paper by using a screen printer;
(3) preparing an aminated graphene-electroactive substance-nanogold composite nanomaterial: taking 2mL of the solution with the concentration of 1mg/mL-1And 2mL of the dispersion of aminated graphene (2 mg/mL)-1Mixing the thionine solution or the Prussian blue solution, stirring vigorously for 24h, removing unbound thionine or Prussian blue molecules through centrifugal separation, mixing 100 mu L of thionine/Prussian blue-multi-walled carbon nanotube mixed solution with 250 mu L of nano gold particle solution, stirring vigorously for 12h, adding 100 mu L of chitosan solution, and mixing ultrasonically to obtain the aminated graphene-thionine/Prussian blue-nano gold-chitosan mixed solution.
(4) Modification of working electrode 8: and transferring 10 mu L of the aminated graphene-electroactive substance-nanogold composite nanomaterial onto the surface of the working electrode by using a liquid transfer machine, and baking for 20min at 50 ℃. Then, 10. mu.L of 200mg/mL was taken-1The BDNF antibody is dripped on the surface of an electrode and placed in a refrigerator for 6 hours at 4 ℃ for binding the antibody. Finally, 10 μ L of 1% BSA solution was used at room temperature to cover the remaining active sites.
(5) And manufacturing a double-sided adhesive tape by using a scribing machine, and carrying out integrated packaging on the paper chip.
The automatic sample introduction type trace rapid electrochemical immune whole blood detection paper chip prepared by the embodiment can automatically introduce samples, the required sample amount is about 10 mu L, and the samples are automatically introduced through the microfluidic channel and the sample introduction hole. And (3) after sample introduction, standing for 10 minutes at room temperature to combine the antigen and the antibody, directly connecting the paper chip with an electrochemical workstation through a rear-end interface, detecting the current response of a working electrode by adopting a differential pulse voltammetry method, and inversely calculating the BDNF concentration in the sample to be detected according to the change value of a current signal.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A self-sampling immune detection paper chip is used for detecting the concentration of an antigen and comprises the following components from top to bottom along the lamination direction:
the microfluidic layer is provided with a microfluidic channel and a sample inlet hole which are connected through a communication channel, the microfluidic channel is used for self-sampling, and the sample inlet hole is used for introducing the tissue fluid to be detected obtained by sampling into the detection area;
the auxiliary electrode layer comprises a first through hole arranged opposite to the sample inlet hole, and at least two pairs of combined counter electrodes and reference electrodes;
a detection layer, wherein the detection layer comprises a third through hole which is opposite to the first through hole and is provided with symmetrical communicating holes in the extension direction of the length direction of the detection layer,
the surface of the working electrode layer is modified with an electroactive composite material, and the surface of the electroactive composite material is used for fixing an antibody; wherein the working electrode layer comprises at least two circular working electrodes arranged along the circumferential direction,
the communicating hole is opposite to the round working electrode; the circular working electrode, the counter electrode and the reference electrode are all connected with electrode leads so as to be connected with an external device;
the auxiliary electrode layer also comprises at least two second through holes which are arranged opposite to the round working electrode, and the combination of the counter electrode and the reference electrode is respectively matched with the partial edges of the at least two second through holes;
the auxiliary electrode layer, the detection layer and the working electrode layer form the detection area, the detection area comprises an electrochemical detection circuit, and the concentration of the antigen in the tissue fluid to be detected is detected and obtained in an electrochemical mode based on the specific binding principle of the antigen-antibody and the characteristics of the electroactive composite material.
2. The self-feeding immunoassay paper chip of claim 1, wherein:
the electroactive composite material comprises a composite material containing a carbon nanomaterial, an electroactive material and nanogold;
the carbon nanomaterial comprises a multi-walled carbon nanotube or aminated graphene;
the electroactive substance comprises thionine, Prussian blue or potassium ferricyanide;
in the composite material, the ratio of the carbon nano material to the electroactive substance to the nanogold is 1: 5-1: 2: 10.
3. The self-feeding immune detection paper chip according to claim 1,
the microfluidic layer and the detection layer are respectively provided with at least two groups of edge through holes arranged along the circumferential direction, and the at least two groups of edge through holes are over against the electrode leads of the working electrode, the counter electrode and the reference electrode.
4. The self-feeding immune detection paper chip according to claim 1,
the upper surface of the microfluidic layer is provided with a first filter layer;
the first filter layer is provided with a first filter hole which is arranged opposite to the microfluidic channel and is used for filtering the tissue fluid to be detected when the microfluidic channel performs self-sampling; the porosity of the first filtering pores is less than 3 μm;
and the lower surface of the micro-fluidic layer is provided with a second filter layer which is provided with a second filter hole opposite to the sample inlet hole and is used for further filtering the tissue fluid to be detected obtained by sampling.
5. The self-feeding immune detection paper chip according to claim 1,
the width of a communication channel connecting the microfluidic channel and the sample inlet hole is inversely proportional to the sampling rate;
the size of the self-injection immune detection paper chip is 10.5mm multiplied by 35 mm.
6. The self-feeding immunoassay paper chip of claim 1, wherein the microfluidic channel, the communication channel and the feeding hole of the microfluidic layer are hydrophobic in the region, and hydrophilic in other regions.
7. A method for preparing a self-feeding immunoassay paper chip, which is used for preparing the self-feeding immunoassay paper chip of any one of claims 1 to 6, comprising the following steps:
printing a preset microfluidic channel on the surface of a microfluidic layer through a wax-spraying printer, and heating the microfluidic layer to make the microfluidic channel, a communication channel and a sample inlet hole of the microfluidic layer have hydrophobic properties;
respectively printing a working electrode, a counter electrode and a reference electrode on the working electrode layer and the auxiliary electrode layer by a screen printer;
modifying the surface of the working electrode with an electroactive composite material;
integrally packaging the microfluidic layer, the auxiliary electrode layer, the detection layer and the working electrode layer;
when the self-injection immunoassay paper chip is used, the antibody is dripped on the surface of the working electrode modified with the electroactive composite material.
8. The method for preparing the self-feeding immunoassay paper chip of claim 7, wherein the electroactive composite material is prepared by the following steps:
step 1, dissolving a multi-walled carbon nanotube or aminated graphene into a polyethyleneimine aqueous solution to enable the multi-walled carbon nanotube or aminated graphene to be non-covalently functionalized;
step 2, adding an electroactive substance into the solution, and stirring vigorously for a certain time to combine the non-covalent functionalized multi-walled carbon nanotube or the aminated graphene with the electroactive substance;
and 3, mixing the solution obtained in the step 2 with the nano gold particle solution, adding a chitosan solution after stirring vigorously, and carrying out ultrasonic mixing to obtain the electroactive composite material.
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| CN109060916A (en) * | 2018-07-31 | 2018-12-21 | 山东师范大学 | Electrochemical sensor electrodes nano surface material method of modifying and sensor |
| CN109060923B (en) * | 2018-08-20 | 2023-06-09 | 浙江大学 | Body surface sweat electrochemical sensor with paper folding structure and monitoring method |
| CN109061190B (en) * | 2018-08-22 | 2021-12-07 | 苏州微湃医疗科技有限公司 | Preparation of multi-channel biosensor array based on paper chip and application of multi-channel biosensor array in immunodetection |
| CN109298061B (en) * | 2018-10-22 | 2021-01-08 | 山东师范大学 | Portable micro cancer antigen multi-parameter quantitative sensing detection system and method |
| CN109254043B (en) * | 2018-10-29 | 2020-10-02 | 济南大学 | Preparation of automatic cleaning paper-based sensing device and application of automatic cleaning paper-based sensing device in ion analysis |
| CN109604629B (en) * | 2018-11-27 | 2022-06-28 | 湖南科技大学 | Composite material and preparation method and application thereof |
| CN109781811A (en) * | 2019-02-28 | 2019-05-21 | 中国科学院电子学研究所 | A kind of paper chip aptamer sensor and preparation method thereof |
| CN111172025B (en) * | 2020-01-14 | 2022-11-15 | 中国科学院烟台海岸带研究所 | Compound micro-fluidic chip device of hydrogen sulfide activity detection based on cell |
| CN111781263B (en) * | 2020-07-15 | 2023-03-28 | 桂林电子科技大学 | Preparation method of electrochemical immunosensor based on rGO/PB @ AuPtNPs nanocomposite |
| CN113125717B (en) * | 2021-04-07 | 2024-02-13 | 江苏大学 | Method and device for detecting concentration of salmonella in food based on microfluidic chip |
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