阅读说明：本技术 基于氧化锌纳米线荧光增强的纸基分析装置及方法 (Zinc oxide nanowire fluorescence enhancement-based paper base analysis device and method ) 是由 于海东 郭雪英 许智惠 于 2019-08-28 设计创作，主要内容包括：本发明公开了基于氧化锌纳米线荧光增强的纸基分析装置及纸基分析方法,包括检测层A、样品加入层B,所述检测层A包括三个检测区域,分别用于检测脂肪酸结合蛋白FABP、心肌肌钙蛋白cTnI和肌红蛋白myoglobin,样品被加入到所述样品加入层B的中心区域,然后将所述样品加入层B折叠到所述检测层A上让所述样品分流到所述三个检测区域实现对所述脂肪酸结合蛋白FABP、心肌肌钙蛋白cTnI和肌红蛋白myoglobin的同时检测。本发明的纸基分析装置及分析方法可以实现对三种心肌标志物的同时检测,并且对FABP、cTnI和myoglobin的检测都具有高灵敏度和良好的选择性,其检测限分别为1.36 ng/mL、1.00 ng/mL和2.38 ng/mL。(The invention discloses a paper-based analysis device based on zinc oxide nanowire fluorescence enhancement and a paper-based analysis method, wherein the paper-based analysis device comprises a detection layer A and a sample adding layer B, the detection layer A comprises three detection areas which are respectively used for detecting fatty acid binding protein FABP, cardiac troponin cTnI and myoglobin, a sample is added into the central area of the sample adding layer B, and then the sample adding layer B is folded onto the detection layer A to shunt the sample to the three detection areas so as to realize simultaneous detection of the fatty acid binding protein FABP, the cardiac troponin cTnI and the myoglobin. The paper-based analysis device and the analysis method can realize the simultaneous detection of three myocardial markers, have high sensitivity and good selectivity on the detection of FABP, cTnI and myoglobin, and have the detection limits of 1.36 ng/mL, 1.00 ng/mL and 2.38 ng/mL respectively.)

1. The paper base analysis device based on zinc oxide nanowire fluorescence enhancement is characterized by comprising a detection layer A and a sample adding layer B, wherein the detection layer A comprises three detection areas which are respectively used for detecting fatty acid binding protein FABP, cardiac troponin cTnI and myoglobin, a sample is added into the central area of the sample adding layer B, and then the sample adding layer B is folded on the detection layer A to shunt the sample to the three detection areas so as to realize the simultaneous detection of the fatty acid binding protein FABP, the cardiac troponin cTnI and the myoglobin.

2. the paper-based analysis method of the paper-based analysis device based on zinc oxide nanowire fluorescence enhancement is characterized by comprising the following steps:

Step SS 1: preparing a zinc oxide nanowire paper base;

step SS 2: preparing a paper-based fluorescence immune device;

step SS 3: the cardiac markers were determined by double antibody sandwich immunoassay.

3. The paper-based analysis method of the paper-based analysis device based on zinc oxide nanowire fluorescence enhancement as claimed in claim 2, wherein the step SS1 specifically comprises:

step SS 11: preparing a zinc oxide seed paper base;

step SS 12: preparing a zinc oxide nanowire growth solution;

Step SS 13: and (3) putting the zinc oxide seed paper base prepared in the step SS11 into the zinc oxide nanowire growth solution prepared in the step SS12, sealing the zinc oxide nanowire growth solution with tin foil paper, placing the zinc oxide seed paper base in an oven to react for 3 hours at 90 ℃, taking out the zinc oxide nanowire paper base, fully washing the zinc oxide seed paper base with ultrapure water, and drying the zinc oxide nanowire paper base at 90 ℃ to obtain the zinc oxide nanowire paper base.

4. the paper-based analysis method of the paper-based analysis device based on zinc oxide nanowire fluorescence enhancement of claim 3, wherein the step SS11 specifically comprises: and immersing the paper substrate used for reaction in a zinc oxide seed solution, namely 0.1 mol/L zinc acetate dihydrate for 60 s, taking out, and drying in an oven at 100 ℃ for 1 h to obtain a zinc oxide seed paper base for later use.

5. The paper-based analysis method of the paper-based analysis device based on zinc oxide nanowire fluorescence enhancement of claim 3, wherein the step SS12 specifically comprises: 3.72 g of zinc nitrate hexahydrate, 0.88g of hexamethylenetetramine and 1.52 g of polyethyleneimine are weighed according to the following proportion and dissolved in 500 mL of ultrapure water, 13.06mL of ammonia water is removed and added into the ultrapure water, and the solution with uniform phase, namely the zinc oxide nanowire growth solution, is obtained by ultrasonic treatment.

6. the paper-based analysis method of the paper-based analysis device based on zinc oxide nanowire fluorescence enhancement of claim 2, characterized in that the preparation of the paper-based fluorescence immunoassay device in the step SS2 specifically comprises:

Step SS 21: designing a pattern, and printing the pattern on the zinc oxide nanowire paper base by using a wax printer; after cooling, the zinc oxide nanowire paper base is placed in an oven to be heated, wax is melted and permeates into the whole zinc oxide nanowire paper base, and the final hole diameters of three detection areas of the paper base analysis device are 3.5 mm;

Step SS 22: treating the surface of the zinc oxide nanowire paper base with trimethoxy silane GPTMS to obtain an epoxy group capable of reacting with amino;

Step SS 23: respectively dripping the anti-FABP capture antibody solution, the anti-cTnI capture antibody solution and the anti-myoglobin capture antibody solution into corresponding detection areas, and incubating for 30 min at room temperature;

step SS 24: blocking unreacted epoxy groups on the surface of the zinc oxide nanowire paper base by using 1% methoxy polyethylene glycol thiol and 1% bovine serum albumin BSA (bovine serum albumin), and reducing non-specific adsorption to obtain a paper base fluorescence immune device;

Step SS 25: the paper-based fluorescent immunization device was stored at 4 ℃ for further use.

7. the paper-based analysis method of the paper-based analysis device based on zinc oxide nanowire fluorescence enhancement as claimed in claim 2, wherein the step SS3 specifically comprises:

step SS 31: adding a sample containing a cardiac marker to a central sample region of a paper-based fluorescence immunoassay device;

Step SS 32: the cardiac marker specifically binds to the corresponding capture antibody;

step SS 33: the myocardial marker is sandwiched between the capture antibody and the FITC-labeled antibody;

Step SS 34: the detection is carried out in a self-made ultraviolet box, an ultraviolet lamp irradiation device powered by a battery is used, and a fluorescence image excited by an ultraviolet lamp is captured by a smart phone.

8. The paper-based analysis method of the paper-based analysis device based on zinc oxide nanowire fluorescence enhancement of claim 7, wherein the step SS31 specifically comprises: adding the sample of the paper-based fluorescence immune device into the paper base of the layer B, and folding the paper base of the layer B to the top of the paper base of the detection layer A; adding sample solutions containing FABP, cTnI and myoglobin with different concentrations into a central sample area of the paper base of the sample adding layer B, and shunting the sample solutions to three detection areas of the paper base of the detection layer A by the capillary driving force of the sample adding layer B.

9. the paper-based analysis method of the paper-based analysis device based on zinc oxide nanowire fluorescence enhancement of claim 7, wherein the step SS32 specifically comprises: and after the target antigen in the sample solution reacts with the capture antibody pre-fixed on the detection area, adding the sample into the paper base of the layer B, and cutting off the paper base of the layer B, and only leaving the paper base of the detection layer A.

10. The paper-based analysis method of the paper-based analysis device based on zinc oxide nanowire fluorescence enhancement of claim 7, wherein the step SS33 specifically comprises: adding FITC-labeled anti-FABP, FITC-labeled anti-cTnI and FITC-labeled anti-myoglobin into corresponding detection areas respectively, and reacting for a period of time; each test area was washed separately by adding phosphate buffered saline containing 0.05% tween 20 to the test area, absorbing excess remaining solution with a piece of absorbent paper at the bottom of the paper-based assay device and washing off excess antibody.

Technical Field

The invention relates to a paper-based analysis device and method based on zinc oxide nanowire fluorescence enhancement, in particular to a paper-based analysis device and method for integrating zinc oxide nanowires on a paper base and simultaneously detecting various cardiac markers in a zinc oxide nanowire fluorescence enhancement signal mode, and belongs to the technical field of paper-based analysis and detection.

background

research on paper-based analysis and detection is started by the Whitesides subject group of Harvard university in 2007, and a paper-based analysis device is prepared by utilizing a photoetching method and used for detecting glucose and protein, so that the application of a paper-based analysis technology to medicine is promoted. The multilayer plane paper chips are also overlapped, and a first three-dimensional paper-based analysis device is manufactured by adopting double faced adhesive tape for fixation, so that the high-flux detection of various substances is realized, and the concept of using the paper base as a microfluid platform for multiple analyte detection is provided. Since then, paper-based analytical techniques have attracted increasing attention from researchers. The paper-based analysis device has the following advantages: the raw materials are rich, cheap and easy to obtain; the main component of the paper is cellulose, the internal structure of the paper is a three-dimensional net structure and has capillary force, and liquid flows on the paper through the capillary action of the paper without an external pump; the sample consumption is less; fourthly, the biocompatibility is good, and the paper base can be modified through physical or chemical modification; the paper base has good flexibility and can be folded, and high-flux detection can be realized; sixthly, the weight is light, the carrying is convenient, and the operation is simple and convenient. Based on these advantages, the paper-based analysis device has attracted much attention in recent years, provides great convenience for analysis and detection, and can be used for analysis and detection in clinical diagnosis, food quality control, environmental monitoring, and the like.

Acute Myocardial Infarction (AMI) is a serious life-threatening cardiovascular disease, and in order to alleviate the harm of acute myocardial infarction to human beings and improve the health level of people, a more sensitive detection method for the acute myocardial infarction disease needs to be developed. Currently, enzyme-linked immunosorbent assay, electrochemiluminescence, immunoturbidimetry and surface plasmon resonance are mainly used for monitoring AMI of patients, however, these detection methods can only be performed in hospitals or professional diagnostic centers, and require large-scale instruments and professional personnel for operation, thus many people cannot obtain timely diagnosis and treatment. And the traditional detection method has long time consumption, high price and low sensitivity, so that the practical application of the traditional detection method is limited, and the traditional detection method is particularly limited to be used in the areas with deficient medical resources. The paper-based analysis device is cheap, simple, portable and sensitive, and is expected to solve the problems, and the development of a new paper-based analysis and detection technology which is simple, convenient, cheap, high in sensitivity and high in detection speed is helpful for early prevention of acute myocardial infarction diseases.

Disclosure of Invention

the invention aims to overcome the defects in the prior art, solve the technical problems and provide a paper-based analysis device and method based on zinc oxide nanowire fluorescence enhancement.

The technical problems to be solved by the invention include: firstly, the price of a developed paper-based analysis device is low, and the problem of high cost for detecting AMI is solved; secondly, the developed paper-based analysis device has portability, and solves the problem that the detection of AMI depends on a detection center and a hospital excessively; thirdly, the developed paper-based analysis method can detect three cardiac markers simultaneously, and the accuracy of AMI diagnosis is improved.

the invention adopts the following technical scheme: the paper-based analysis device based on zinc oxide nanowire fluorescence enhancement is characterized by comprising a detection layer A and a sample adding layer B, wherein the detection layer A comprises three detection areas which are respectively used for detecting Fatty Acid Binding Protein (FABP), cardiac troponin I (cTnI) and myoglobin (myoglobin), a sample is added into the central area of the sample adding layer B, and then the sample adding layer B is folded onto the detection layer A to shunt the sample to the three detection areas so as to realize the simultaneous detection of the FABP, the cTnI and the myoglobin.

the invention also provides a paper base analysis method of the paper base analysis device based on zinc oxide nanowire fluorescence enhancement, which is characterized by comprising the following steps:

step SS 1: preparing a zinc oxide nanowire paper base;

Step SS 2: preparing a paper-based fluorescence immune device;

Step SS 3: the cardiac markers were determined by double antibody sandwich immunoassay.

as a preferred embodiment, step SS1 specifically includes:

step SS 11: preparing a zinc oxide seed paper base;

Step SS 12: preparing a zinc oxide nanowire growth solution;

step SS 13: and (3) putting the zinc oxide seed paper base prepared in the step SS11 into the zinc oxide nanowire growth solution prepared in the step SS12, sealing the zinc oxide nanowire growth solution with tin foil paper, placing the zinc oxide seed paper base in an oven to react for 3 hours at 90 ℃, taking out the zinc oxide nanowire paper base, fully washing the zinc oxide seed paper base with ultrapure water, and drying the zinc oxide nanowire paper base at 90 ℃ to obtain the zinc oxide nanowire paper base.

as a preferred embodiment, step SS11 specifically includes: and immersing the paper substrate used for reaction in a zinc oxide seed solution, namely 0.1 mol/L zinc acetate dihydrate for 60 s, taking out, and drying in an oven at 100 ℃ for 1 h to obtain a zinc oxide seed paper base for later use.

as a preferred embodiment, step SS12 specifically includes: 3.72 g of zinc nitrate hexahydrate, 0.88g of hexamethylenetetramine and 1.52 g of polyethyleneimine are weighed according to the following proportion and dissolved in 500 mL of ultrapure water, 13.06mL of ammonia water is removed and added into the ultrapure water, and the solution with uniform phase, namely the zinc oxide nanowire growth solution, is obtained by ultrasonic treatment.

as a preferred embodiment, the step of preparing the paper-based fluorescence immunoassay device in SS2 specifically comprises:

Step SS 21: designing a pattern, and printing the pattern on the zinc oxide nanowire paper base by using a wax printer; after cooling, the zinc oxide nanowire paper base is placed in an oven to be heated, wax is melted and permeates into the whole zinc oxide nanowire paper base, and the final hole diameters of three detection areas of the paper base analysis device are 3.5 mm;

Step SS 22: treating the surface of the zinc oxide nanowire paper base with trimethoxy silane GPTMS to obtain an epoxy group capable of reacting with amino;

step SS 23: respectively dripping the anti-FABP capture antibody solution, the anti-cTnI capture antibody solution and the anti-myoglobin capture antibody solution into corresponding detection areas, and incubating for 30 min at room temperature;

Step SS 24: blocking unreacted epoxy groups on the surface of the zinc oxide nanowire paper base by using 1% methoxy polyethylene glycol thiol and 1% bovine serum albumin BSA (bovine serum albumin), and reducing non-specific adsorption to obtain a paper base fluorescence immune device;

Step SS 25: the paper-based fluorescent immunization device was stored at 4 ℃ for further use.

as a preferred embodiment, step SS3 specifically includes:

Step SS 31: adding a sample containing a cardiac marker to the central sample region;

step SS 32: the cardiac marker specifically binds to the corresponding capture antibody;

Step SS 33: the myocardial marker is sandwiched between the capture antibody and the FITC-labeled antibody;

Step SS 34: the detection is carried out in a self-made ultraviolet box, an ultraviolet lamp irradiation device powered by a battery is used, and a fluorescence image excited by an ultraviolet lamp is captured by a smart phone.

as a preferred embodiment, step SS31 specifically includes: adding the sample of the paper-based fluorescence immune device into the paper base of the layer B, and folding the paper base of the layer B to the top of the paper base of the detection layer A; adding sample solutions containing FABP, cTnI and myoglobin with different concentrations into a central sample area of the paper base of the sample adding layer B, and shunting the sample solutions to three detection areas of the paper base of the detection layer A by the capillary driving force of the sample adding layer B.

as a preferred embodiment, step SS32 specifically includes: and after the target antigen in the sample solution reacts with the capture antibody pre-fixed on the detection area, adding the sample into the paper base of the layer B, and cutting off the paper base of the layer B, and only leaving the paper base of the detection layer A.

As a preferred embodiment, step SS33 specifically includes: FITC-labeled anti-FABP (FITC-labeled anti-FABP), FITC-labeled anti-cTnI (FITC-labeled anti-cTnI) and FITC-labeled anti-myoglobin (FITC-labeled anti-myoglobin) were added to the corresponding detection regions, respectively, and reacted for a certain period of time. Each test area was washed separately by adding phosphate buffered saline containing 0.05% tween 20 to the test area, absorbing excess remaining solution with a piece of absorbent paper at the bottom of the paper-based assay device and washing off excess antibody.

The invention achieves the following beneficial effects: firstly, the paper-based analysis device and the paper-based analysis method can realize the simultaneous detection of three cardiac markers, and have high sensitivity and good selectivity on the detection of FABP, cTnI and myoglobin, and the detection limits are 1.36 ng/mL, 1.00 ng/mL and 2.38 ng/mL respectively; secondly, the detection time is short, the detection can be completed quickly in 5 min, and the detection device is convenient to carry (only a smart phone and a portable ultraviolet lamp are needed in the detection process); thirdly, the paper adopted by the invention is a degradable ideal material, and the paper is used for replacing traditional materials such as plastics, glass and the like, so that the environmental pressure is reduced, the price is low, the environment is protected, and the post-treatment is simple.

Drawings

FIG. 1 is a flow chart of the present invention for preparing a zinc oxide nanowire paper substrate.

FIG. 2 is a flow chart for preparing a fluorescence immunoassay device according to the present invention.

FIG. 3 is a flow chart of the present invention for measuring cardiac markers using a double antibody sandwich immunoassay.

Detailed Description

The invention is further described below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.

The invention adopts the following technical scheme: the paper-based analysis device based on zinc oxide nanowire fluorescence enhancement is characterized by comprising a detection layer A and a sample adding layer B, wherein the detection layer A comprises three detection areas which are respectively used for detecting Fatty Acid Binding Protein (FABP), cardiac troponin I (cTnI) and myoglobin (myoglobin), a sample is added into the central area of the sample adding layer B, and then the sample adding layer B is folded onto the detection layer A to shunt the sample to the three detection areas so as to realize the simultaneous detection of the FABP, the cTnI and the myoglobin.

the invention also provides a paper base analysis method of the paper base analysis device based on zinc oxide nanowire fluorescence enhancement, which is characterized by comprising the following steps:

Step SS 1: preparing a zinc oxide nanowire paper base as shown in figure 1;

Step SS 2: preparing a paper-based fluorescence immunoassay device as shown in fig. 2;

step SS 3: the cardiac markers were determined using a double antibody sandwich immunoassay, as shown in FIG. 3.

as a preferred embodiment, step SS1 specifically includes:

Step SS 11: preparing a zinc oxide seed paper base;

Step SS 12: preparing a zinc oxide nanowire growth solution;

Step SS 13: and (3) putting the zinc oxide seed paper base prepared in the step SS11 into the zinc oxide nanowire growth solution prepared in the step SS12, sealing the zinc oxide nanowire growth solution with tin foil paper, placing the zinc oxide seed paper base in an oven to react for 3 hours at 90 ℃, taking out the zinc oxide nanowire paper base, fully washing the zinc oxide seed paper base with ultrapure water, and drying the zinc oxide nanowire paper base at 90 ℃ to obtain the zinc oxide nanowire paper base.

As a preferred embodiment, step SS11 specifically includes: immersing the whatman paper substrate used for reaction in a zinc oxide seed solution, namely 0.1 mol/L zinc acetate dihydrate for 60 s, taking out and drying in an oven at 100 ℃ for 1 h to obtain a zinc oxide seed paper base for later use.

as a preferred embodiment, step SS12 specifically includes: 3.72 g of zinc nitrate hexahydrate, 0.88g of hexamethylenetetramine and 1.52 g of polyethyleneimine are weighed according to the following proportion and dissolved in 500 mL of ultrapure water, 13.06mL of ammonia water is removed and added into the ultrapure water, and the solution with uniform phase, namely the zinc oxide nanowire growth solution, is obtained by ultrasonic treatment.

As a preferred embodiment, the step of preparing the paper-based fluorescence immunoassay device in SS2 specifically comprises:

Step SS 21: designing a pattern, and printing the pattern on the zinc oxide nanowire paper base by using a wax printer; after cooling, the zinc oxide nanowire paper base is placed in an oven to be heated, wax is melted and permeates into the whole zinc oxide nanowire paper base, and the final hole diameters of three detection areas of the paper base analysis device are 3.5 mm, as shown in fig. 2A and 2B;

Step SS 22: in order to enhance the antibody fixing capacity of the zinc oxide nanowire paper substrate and improve the detection performance of a paper-based analysis device, the surface of the zinc oxide nanowire paper substrate is treated by (3-glycidyloxypropyl) trimethoxysilane GPTMS to obtain an epoxy group capable of reacting with amino;

step SS 23: respectively dripping the anti-FABP capture antibody solution, the anti-cTnI capture antibody solution and the anti-myoglobin capture antibody solution into corresponding detection areas, and incubating for 30 min at room temperature;

step SS 24: blocking unreacted epoxy groups on the surface of the zinc oxide nanowire paper base by using 1% methoxy polyethylene glycol thiol and 1% bovine serum albumin BSA (bovine serum albumin), and reducing non-specific adsorption to obtain a paper base fluorescence immune device;

step SS 25: the paper-based fluorescent immunization device was stored at 4 ℃ for further use.

As a preferred embodiment, step SS3 specifically includes:

step SS 31: adding a sample containing a cardiac marker to the central sample region;

Step SS 32: the cardiac marker specifically binds to the corresponding capture antibody;

step SS 33: the myocardial marker is sandwiched between the capture antibody and the FITC-labeled antibody;

step SS 34: the detection is carried out in a self-made ultraviolet box, a battery-powered ultraviolet lamp irradiation device is used, a fluorescence image excited by an ultraviolet lamp is captured by a smart phone, and the detection is completed in about 5 min by the method. The results of the semi-quantitative test can also be read directly by the naked eye, conveniently and quickly, as shown in FIGS. 3A and 3B.

as a preferred embodiment, step SS31 specifically includes: adding the sample of the paper-based fluorescence immune device into the paper base of the layer B, and folding the paper base of the layer B to the top of the paper base of the detection layer A; adding sample solutions containing FABP, cTnI and myoglobin with different concentrations into a central sample area of the paper base of the sample adding layer B, and shunting the sample solutions to three detection areas of the paper base of the detection layer A by the capillary driving force of the sample adding layer B.

As a preferred embodiment, step SS32 specifically includes: and after the target antigen in the sample solution reacts with the capture antibody pre-fixed on the detection area, adding the sample into the paper base of the layer B, and cutting off the paper base of the layer B, and only leaving the paper base of the detection layer A.

As a preferred embodiment, step SS33 specifically includes: respectively adding FITC-labeled anti-FABP (FITC-labeled anti-FABP), FITC-labeled anti-cTnI (FITC-labeled anti-cTnI) and FITC-labeled anti-myoglobin (FITC-labeled anti-myoglobin) into corresponding detection areas, and reacting for a period of time; each test area was washed separately by adding phosphate buffered saline containing 0.05% tween 20 to the test area, absorbing excess remaining solution with a piece of absorbent paper at the bottom of the paper-based assay device and washing off excess antibody.

the noun explains: fatty acid-binding proteins (FABPs) are present in a variety of tissues, and the protein bound is albumin, most abundant in cardiac and skeletal muscle. FABPs are intracellular fatty acid carrier proteins that play an important role in intracellular utilization of fatty acids.

cardiac troponin (cTn) is a regulatory protein of myocardial muscle contraction.

cTn is composed of subunits of three different genes: cardiac troponin T (cTnT), cardiac troponin I (cTnI), and troponin C (TnC). Currently, cTnT and cTnI are used for ACS laboratory diagnostics.

myoglobin (Myoglobin) is a protein for storing and distributing oxygen in mammalian cells (mainly myocytes), and is composed of a polypeptide chain and a prosthetic heme, has a relative molecular mass of 16700, and contains 153 amino acid residues. Apomyoglobin from which heme is removed is called globin (globin), which has significant homology with the subunits of hemoglobin (a-globin chain and P-globin chain) in amino acid sequence, and their conformation and function are also very similar.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.