CN110261605B - Method for improving detection sensitivity of test paper by modifying chitosan - Google Patents

Method for improving detection sensitivity of test paper by modifying chitosan Download PDF

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CN110261605B
CN110261605B CN201910552425.6A CN201910552425A CN110261605B CN 110261605 B CN110261605 B CN 110261605B CN 201910552425 A CN201910552425 A CN 201910552425A CN 110261605 B CN110261605 B CN 110261605B
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唐蕊华
刘丽娜
张素风
钱立伟
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Shaanxi University of Science and Technology
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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
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
    • G01N2400/24—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar beta-D-Glucans, i.e. having beta 1,n (n=3,4,6) linkages between saccharide units, e.g. xanthan
    • G01N2400/28—Chitin, chitosan

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Abstract

The invention discloses a method for improving detection sensitivity of test paper by chitosan modification, which specifically comprises the following steps: step 1, preparing a chitosan solution; step 2, manufacturing a blank test strip: step 3, preparing a chitosan modified test strip according to the blank test strip prepared in the step 2; step 4, preparing chitosan modified lateral flow chromatography test paper; and 5, detecting the test paper manufactured in the step 4. The invention solves the problems of complex operation, high cost, long time consumption and poor uniformity of the conventional method for improving the sensitivity of the lateral flow test paper.

Description

Method for improving detection sensitivity of test paper by modifying chitosan
Technical Field
The invention belongs to the technical field of test paper sensitivity test, and relates to a method for improving test paper detection sensitivity by chitosan modification.
Background
In resource-limited environments, lateral flow test strips have been widely used for disease diagnosis, food safety testing, and environmental testing. Compared with the traditional detection technology which is expensive, time-consuming and can be completed by professional technicians in experimental operation. For example: enzyme linked immunosorbent assay and polymerase chain reaction; the lateral flow chromatography test paper has the advantages of low cost, simple operation, rapidness and portability. Currently, lateral flow test strips have been used to detect various targets, such as: nucleic acid, protein, virus, bacteria, heavy metal and other markers. However, the disadvantage of low sensitivity limits the wide application of lateral flow test strips.
Currently, researchers have used different methods to enhance lateral flow strip detection sensitivity. For example: enzyme-based signal enhancement techniques, probe-based signal enhancement techniques, techniques for slowing down the flow rate to enhance the signal, and techniques for concentrating the sample to enhance the signal, among others. However, these techniques have drawbacks such as requiring expensive chemical reagents or complicated knitting processes. Many researchers try to improve the sensitivity of the paper-based detection device by changing the characteristics of the material, but the existing methods have the defects of high background noise, long material synthesis time, complex operation and the like, and cannot be used for mass production.
Disclosure of Invention
The invention aims to provide a method for improving the detection sensitivity of test paper by chitosan modification, which solves the problems of complex operation, high cost, long time consumption and poor uniformity of the conventional method for improving the sensitivity of lateral flow test paper.
The invention adopts the technical scheme that a method for improving the detection sensitivity of test paper by modifying chitosan specifically comprises the following steps:
step 1, preparing a chitosan solution;
step 2, manufacturing a blank test strip:
step 3, preparing a chitosan modified test strip according to the blank test strip prepared in the step 2;
step 4, preparing chitosan modified lateral flow chromatography test paper;
and 5, detecting the test paper manufactured in the step 4.
The present invention is also characterized in that,
the specific process of the step 1 is as follows: weighing 15-25 mg of chitosan, dissolving in 100ml of 1% glacial acetic acid solution, and diluting to 0.005-25mg/ml of chitosan solution.
The specific process of the step 2 is as follows: and sequentially overlapping the nitrocellulose membrane, the combination pad, the sample pad and the absorbent paper in pairs and pasting the nitrocellulose membrane, the combination pad, the sample pad and the absorbent paper on the back rubber plate to prepare blank test paper, and cutting the blank test paper into test paper strips with the width of 3-7 mm.
The specific process of the step 3 is as follows: and (2) directly dripping 0.5-2 mu L of the chitosan solution prepared in the step (1) on the surface quality control line and the detection line of the nitrocellulose membrane of the blank test strip prepared in the step (2), and placing the blank test strip in an oven for drying at 25-37 ℃ for 30-60 min.
The specific process of the step 4 is as follows: dripping 8-15 mu L of nano gold solution modified with a detection probe on a bonding pad, respectively dripping 0.5-2 mu L of quality control line probe solution with the concentration of 2.5nM-100nM on the position of a quality control line modified by chitosan on the surface of a nitrocellulose membrane, dripping 0.5-2 mu L of detection probe with the concentration of 2.5nM-100nM on the position of a detection line modified by chitosan on the surface of the nitrocellulose membrane to prepare chitosan modified lateral flow chromatography test paper, and then placing the lateral flow chromatography test paper in an oven at 25-37 ℃ for drying for 60-120 min.
The specific process of the step 5 is as follows: taking the lateral flow chromatography test paper dried in the step 4, dropwise adding 60-120 mu L of a sample to be detected on a sample pad of the lateral flow chromatography test paper by using a pipettor until the absorbent paper is completely soaked, observing the color development conditions of the control line and the detection line, and if the control line and the detection line are both developed, indicating that the detection result is positive; if the contrast line is colored, the detection line is not colored, which indicates that the detection result is negative; if the contrast line and the detection line do not develop color, the test paper is invalid.
The test paper has the beneficial effects that the test paper enhances the detection signal by changing the characteristics of the paper base material (such as the nitrocellulose membrane). Compared with the traditional lateral flow chromatography test paper detection method, the method has the greatest improvement that natural degradable biomolecule chitosan is introduced to the surface of the paper base material, so that the characteristics of the material such as porosity, pore size, surface functional groups and the like are changed, the adsorption capacity of biomolecules on the surface of the material is improved, the number of chromogenic particles is further increased, and the test signal of the test paper is improved. The method is low in cost, environment-friendly, simple and convenient, can realize batch production, is wide in application, and can be used for detecting target objects such as nucleic acid, protein, metal ions and chemical molecules on different paper-based detection platforms.
Drawings
FIG. 1 is a schematic diagram showing the detection result of normal test paper in example 1 of the method for improving test sensitivity of test paper by chitosan modification of the present invention;
fig. 2 is a schematic diagram of a sensitivity detection result of chitosan modified test paper in example 1 of the method for enhancing detection sensitivity of test paper by chitosan modification of the present invention.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
The invention relates to a method for improving detection sensitivity of test paper by chitosan modification, which specifically comprises the following steps:
step 1, preparing a chitosan solution;
weighing 15-25 mg of chitosan, dissolving the chitosan in 100ml of 1% glacial acetic acid solution, and diluting the chitosan solution into 0.005-25mg/ml of chitosan solution for later use;
step 2, manufacturing a blank test strip:
sequentially overlapping and adhering the nitrocellulose membrane, the combination pad, the sample pad and the absorbent paper on a back rubber plate in pairs in sequence to prepare blank test paper, and cutting the blank test paper into test paper strips with the width of 3-7 mm;
step 3, preparing a chitosan modified test strip according to the blank test strip prepared in the step 2;
and (3) directly dripping 0.5-2 mu L of the chitosan solution prepared in the step (1) on the surface quality control line and the detection line of the nitrocellulose membrane of the blank test strip prepared in the step (2), and placing the blank test strip in an oven for drying for 30-60min at the temperature of 25-37 ℃.
Step 4, manufacturing lateral flow chromatography test paper;
preparation of normal test paper: dripping 8-15 mu L of nano gold solution modified with a detection probe on a combination pad, and respectively dripping 0.5-2 mu L of quality control line probe solution with the concentration of 2.5nM-100nM on the position of a quality control line on the surface of the nitrocellulose membrane; 0.5-2 mu L of detection probe solution with the concentration of 2.5nM-100nM is dripped at the position of the detection line on the surface of the nitrocellulose membrane to prepare normal lateral flow chromatography test paper, and then the lateral flow chromatography test paper is placed in an oven for drying for 60-120min at the temperature of 25-37 ℃;
preparing chitosan modified lateral flow chromatography test paper: dripping 8-15 mu L of nano gold solution modified with a detection probe on a bonding pad, dripping 0.5-2 mu L of quality control line probe solution with the concentration of 2.5nM-100nM on the position of a quality control line modified by chitosan on the surface of a nitrocellulose membrane, dripping 0.5-2 mu L of detection probe with the concentration of 2.5nM-100nM on the position of a detection line modified by chitosan on the surface of the nitrocellulose membrane to prepare chitosan modified lateral flow chromatography test paper, and then placing the lateral flow chromatography test paper in an oven at 25-37 ℃ for drying for 60-120 min;
step 5, taking the lateral flow chromatography test paper dried in the step 4, dropwise adding 60-120 mu L of a sample to be detected on a sample pad of the lateral flow chromatography test paper by using a pipettor until the absorbent paper is completely soaked, observing the color development conditions of the control line and the detection line, and if the control line and the detection line are both developed, indicating that the detection result is positive; if the contrast line is colored, the detection line is not colored, which indicates that the detection result is negative; if the contrast line and the detection line do not develop color, the test paper is invalid.
Example 1
A method for improving detection sensitivity of test paper by chitosan modification comprises the following steps:
step 1, preparing a chitosan solution;
weighing 15mg of chitosan, dissolving in 100ml of 1% glacial acetic acid solution, and diluting to 0.005mg/ml of chitosan solution for later use;
step 2, manufacturing a blank lateral flow chromatography test strip:
sticking the nitrocellulose membrane, the combination pad, the sample pad and the absorbent paper on a back rubber plate in a manner of overlapping 2mm in sequence, and then cutting the nitrocellulose membrane, the combination pad, the sample pad and the absorbent paper into test strips with the width of 3 mm;
step 3, directly dripping 1.5 mu L of chitosan solution with the concentration of 0.005mg/ml on the surface quality control line and the detection line of the nitrocellulose membrane of the blank test strip, and placing the test strip in a drying oven at 37 ℃ for drying for 30 min;
step 4, preparing normal test paper: respectively adding 10 mu L of nano gold drops modified with detection probes on a normal test strip combination pad, and dropping 0.5 mu L of quality control line with the concentration of 5nM on the surface quality control line position of a normal nitrocellulose membrane; 0.5 mu L of detection line with the concentration of 5nM is dripped to the position of the detection line on the surface of the normal nitrocellulose membrane, and the nitrocellulose membrane is placed in an oven at 37 ℃ to be dried for 120 min.
Preparing chitosan modified lateral flow chromatography test paper: dripping 10 mu L of nano gold solution modified with a detection probe on a bonding pad, and respectively dripping 0.5 mu L of quality control line probe solution with the concentration of 2.5nM on the position of a quality control line of the nitrocellulose membrane surface modified by chitosan; 0.5 mu L of detection probe with the concentration of 2.5nM is taken and dripped on the detection line position of the nitrocellulose membrane surface after chitosan modification to prepare chitosan modified lateral flow chromatography test paper, and then the lateral flow chromatography test paper is placed in an oven at 25 ℃ and dried for 60 min;
and 5, taking 100 mu L of targets to be detected with the concentrations of 50nM, 25nM, 10nM, 5nM, 2.5nM, 1nM, 0.5nM, 0.25nM, 0.1nM, 0.05nM and 0nM respectively, dropwise adding the targets to be detected to sample pads of the normal test paper and the chitosan modified test paper respectively, enabling the samples to flow along the lateral flow test paper direction by capillary force in the horizontal direction, and observing a detection result after 15min, wherein the detection result is shown in figure 1. The results in fig. 1 show that the detection sensitivity of the normal test paper is 0.5nM, and the results in fig. 2 show that the detection sensitivity of the chitosan modified test paper is 0.05nM, and compared with the test paper with chitosan modified test paper, the detection sensitivity of the test paper is improved by 10 times. In addition, the color development intensity of the chitosan modified test paper is obviously improved compared with that of normal test paper, and the result shows that the detection signal of the test paper is obviously enhanced.
Example 2
A method for improving detection sensitivity of test paper by chitosan modification comprises the following steps:
step 1, preparing a chitosan solution;
weighing 20mg of chitosan, dissolving in 100ml of 1% glacial acetic acid solution, and diluting to 0.08mg/ml of chitosan solution for later use;
step 2, manufacturing a blank lateral flow chromatography test strip:
sticking the nitrocellulose membrane, the combination pad, the sample pad and the absorbent paper on a back rubber plate in a mode of overlapping 2mm in sequence, and then cutting the nitrocellulose membrane, the combination pad, the sample pad and the absorbent paper into test strips with the width of 5 mm;
step 3, directly dripping 0.5 mu L of chitosan solution with the concentration of 0.08mg/ml on the surface quality control line and the detection line of the nitrocellulose membrane of the blank test strip, and placing the test strip in a 30 ℃ oven for drying for 45 min;
step 4, preparing normal test paper: respectively adding 8 mu L of nano gold drops modified with detection probes on the normal test strip combination pads; dripping 1 mu L of quality control line with the concentration of 2.5nM on the surface of the normal nitrocellulose membrane; and dropwise adding 1 mu L of detection line with the concentration of 2.5nM to the surface detection line position of the normal nitrocellulose membrane, and drying in an oven at 30 ℃ for 100 min.
Preparing chitosan modified lateral flow chromatography test paper: dripping 8 mu L of nano gold solution modified with a detection probe on a bonding pad, and respectively dripping 1 mu L of quality control line probe solution with the concentration of 10nM on the position of a quality control line modified by chitosan on the surface of a nitrocellulose membrane; dripping 1 mu L of detection probe with the concentration of 10nM on the detection line position modified by chitosan on the surface of the nitrocellulose membrane to prepare chitosan modified lateral flow chromatography test paper, and then placing the lateral flow chromatography test paper in an oven at 30 ℃ to dry for 100 min;
and 5, taking 60 mu L of targets to be detected with the concentrations of 50nM, 25nM, 10nM, 5nM, 2.5nM, 1nM, 0.5nM, 0.25nM, 0.1nM, 0.05nM and 0nM respectively, dropwise adding the targets to be detected to sample pads of the normal test paper and the chitosan modified test paper respectively, enabling the samples to flow along the direction of the lateral flow test paper by virtue of capillary force in the horizontal direction, observing a detection result after 15min, wherein the detection result is similar to that of example 1, and compared with the normal test paper, the detection limit and the color development strength of the chitosan modified test paper are obviously enhanced.
Example 3
A method for improving detection sensitivity of test paper by chitosan modification comprises the following steps:
step 1, preparing a chitosan solution;
weighing 25mg of chitosan, dissolving the chitosan in 100ml of 1% glacial acetic acid solution, and diluting the chitosan solution into 0.1mg/ml of chitosan solution for later use;
step 2, manufacturing a blank lateral flow chromatography test strip:
sticking the nitrocellulose membrane, the combination pad, the sample pad and the absorbent paper on a back rubber plate in a mode of overlapping 2mm in sequence, and then cutting the nitrocellulose membrane, the combination pad, the sample pad and the absorbent paper into test strips with the width of 3 mm;
step 3, directly dripping 2 mu L of chitosan solution with the concentration of 0.1mg/ml on the surface quality control line and the detection line of the nitrocellulose membrane of the blank test strip, and placing the test strip in an oven for drying at 25 ℃ for 60 min;
step 4, preparing normal test paper: respectively adding 15 mu L of nano gold drops modified with detection probes on the combination pads of the normal test paper strips; dripping 2 mu L of a quality control line with the concentration of 100nM on the surface of the normal nitrocellulose membrane at the position of the quality control line; and (3) dropwise adding 2 mu L of a detection line with the concentration of 100nM to the position of the detection line on the surface of the normal nitrocellulose membrane, and drying in an oven at 25 ℃ for 60 min.
Preparing chitosan modified lateral flow chromatography test paper: 15 mu L of nano gold solution modified with a detection probe is dripped on the bonding pad, and 2 mu L of quality control line probe solution with the concentration of 100nM is dripped on the position of the quality control line of the nitrocellulose membrane surface after chitosan modification; dripping 2 mu L of detection probe with the concentration of 100nM on the detection line position modified by chitosan on the surface of the nitrocellulose membrane to prepare chitosan modified lateral flow chromatography test paper, and then placing the lateral flow chromatography test paper in an oven at 37 ℃ for drying for 120 min;
and 5, taking 120 mu L of targets to be detected with the concentrations of 50nM, 25nM, 10nM, 5nM, 2.5nM, 1nM, 0.5nM, 0.25nM, 0.1nM, 0.05nM and 0nM respectively, dropwise adding the targets to be detected to sample pads of normal test paper and chitosan modified test paper respectively, enabling the samples to flow along the direction of lateral flow test paper by virtue of capillary force in the horizontal direction, observing a detection result after 15min, wherein the detection result is similar to that of example 1, and compared with the normal test paper, the detection limit and the color development strength of the chitosan modified test paper are obviously enhanced.

Claims (1)

1. A method for improving test paper detection sensitivity by chitosan modification is characterized by comprising the following steps: the method specifically comprises the following steps:
step 1, preparing a chitosan solution;
the specific process of the step 1 is as follows: weighing 15-25 mg of chitosan, dissolving the chitosan in 100ml of 1% glacial acetic acid solution, and diluting the chitosan solution into 0.005-25mg/ml of chitosan solution;
step 2, manufacturing a blank test strip:
the specific process of the step 2 comprises the following steps: sequentially overlapping and sticking the nitrocellulose membrane, the combination pad, the sample pad and the absorbent paper on the back rubber plate in pairs to prepare blank test paper, and cutting the blank test paper into test paper strips with the width of 3-7 mm;
step 3, preparing a chitosan modified test strip according to the blank test strip prepared in the step 2;
the specific process of the step 3 is as follows: taking 0.5-2 mu L of the chitosan solution prepared in the step 1, directly dripping the chitosan solution on the surface quality control line and the detection line of the nitrocellulose membrane of the blank test strip prepared in the step 2, and placing the blank test strip on an oven for drying for 30-60min at 25-37 ℃;
step 4, preparing chitosan modified lateral flow chromatography test paper;
the specific process of the step 4 is as follows: dripping 8-15 mu L of nano gold solution modified with a detection probe on a bonding pad, dripping 0.5-2 mu L of quality control line probe solution with the concentration of 2.5nM-100nM on the position of a quality control line modified by chitosan on the surface of a nitrocellulose membrane, dripping 0.5-2 mu L of detection probe with the concentration of 2.5nM-100nM on the position of a detection line modified by chitosan on the surface of the nitrocellulose membrane to prepare chitosan modified lateral flow chromatography test paper, and then placing the lateral flow chromatography test paper in an oven at 25-37 ℃ for drying for 60-120 min;
step 5, detecting the test paper manufactured in the step 4;
the specific process of the step 5 comprises the following steps: taking the lateral flow chromatography test paper dried in the step 4, dropwise adding 60-120 mu L of a sample to be detected on a sample pad of the lateral flow chromatography test paper by using a pipettor until the absorbent paper is completely soaked, observing the color development conditions of the control line and the detection line, and if the control line and the detection line are both developed, indicating that the detection result is positive; if the contrast line is colored, the detection line is not colored, which indicates that the detection result is negative; if the contrast line and the detection line do not develop color, the test paper is invalid.
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