CN111505298B - A copper ion-labeled colloidal gold test strip for detecting endotoxin and its preparation and detection method - Google Patents
A copper ion-labeled colloidal gold test strip for detecting endotoxin and its preparation and detection method Download PDFInfo
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Abstract
本发明涉及一种铜离子标记的用于检测内毒素的胶体金试纸条及其制备和检测方法。一种铜离子标记的用于检测内毒素的胶体金试纸条，所述的试纸条包括底板以及依次搭接固定于底板上的样品垫、连接垫、硝酸纤维膜和吸收垫；所述的连接垫固定有铜离子标记的胶体金探针；所述的硝酸纤维膜上设置有检测线和质控线；所述的检测线固定有适配体捕获探针；所述的质控线固定有链霉亲和素；本发明同时公开了一种采用上述铜离子标记的胶体金试纸条检测内毒素的方法。本发明通过铜离子介导的金纳米颗粒聚集体和亲和性高的适配体作为检测探针和捕获探针，与传统的胶体金试纸条相对比特异性强、检测限低、灵敏度高，显色效果好。
The invention relates to a copper ion-labeled colloidal gold test strip for detecting endotoxin and a preparation and detection method thereof. A copper ion-labeled colloidal gold test strip for detecting endotoxin, the test strip comprises a bottom plate, a sample pad, a connection pad, a nitrocellulose membrane and an absorption pad which are sequentially overlapped and fixed on the bottom plate; the A copper ion-labeled colloidal gold probe is fixed on the connection pad; a detection line and a quality control line are arranged on the nitrocellulose membrane; the detection line is fixed with an aptamer capture probe; the quality control line is Streptavidin is immobilized; the invention also discloses a method for detecting endotoxin by using the copper ion-labeled colloidal gold test strip. The invention uses copper ion-mediated gold nanoparticle aggregates and aptamers with high affinity as detection probes and capture probes. Compared with traditional colloidal gold test strips, the invention has stronger specificity, lower detection limit and higher sensitivity. Good color rendering.
Description
Technical Field
The invention relates to a copper ion labeled colloidal gold test strip for detecting endotoxin, and a preparation method and a detection method thereof, and belongs to the technical field of biochemical detection.
Background
Endotoxin is one of the components of gram-negative bacterial outer membrane, and trace amount of endotoxin can cause body temperature rise and a series of harmful symptoms, such as diarrhea, asthma, intravascular coagulation, infectious shock and the like. Therefore, rapid and sensitive detection of endotoxin is of great importance for food safety and medical supplies.
The traditional endotoxin detection method is mainly based on a limulus test method and a rabbit heat source inspection method, but the two methods mainly adopt enzyme catalysis reaction, so that the measurement result is easily influenced by enzyme, the measurement result is inaccurate, and meanwhile, the sensitivity is low, the detection limit is high, and the detection time is long. In recent years, biosensors have attracted much attention. For example, polypeptide biosensors, protein biosensors, antibody biosensors, and aptamer biosensors can specifically recognize endotoxin molecules, and although these sensors improve sensitivity to a certain extent and reduce the detection limit, they have disadvantages of complex detection, weak specificity, long time consumption, high cost, and the like, and have high professional requirements, are difficult to operate for non-professional people, are limited to laboratory studies, and have not been applied to rapid field detection.
Chinese patent document CN104698159A discloses a method for detecting endotoxin content, comprising the following steps: s1, preparing at least five standard solutions containing endotoxin with different concentrations; s2, mixing the limulus reagent diluent with each endotoxin standard solution in proportion; s3, respectively detecting the time required when the elasticity reaches 0.1 amplitude by adopting a thromboelastogram instrument for each mixed solution, and drawing a time-endotoxin concentration double-logarithm diagram to obtain a standard curve chart; s4, mixing the limulus reagent diluent with the sample liquid to be detected according to a specific ratio, detecting the time required when the elasticity reaches 0.1 amplitude by using a thromboelastogram instrument, and comparing with a standard curve graph, thereby obtaining the endotoxin content in the sample liquid to be detected. However, the method still has the defects of complex operation and long detection time.
Chinese patent document CN105116142A discloses a novel bacterial endotoxin test paper and a test method, wherein Phos-tag is combined with the phosphate group of endotoxin, polymyxin B is combined with the lipid A of endotoxin, and the test can be carried out quickly, conveniently and timely, but the test paper has the problem of weak specificity.
At present, no report is found on a test strip which takes a copper ion labeled colloidal gold probe and an aptamer as capture probes.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a copper ion labeled colloidal gold test strip for detecting endotoxin and a preparation and detection method thereof.
The invention is realized by the following technical scheme:
a copper ion labeled colloidal gold test strip for detecting endotoxin comprises a base plate, and a sample pad, a connecting pad, a nitrocellulose membrane and an absorbing pad which are sequentially fixed on the base plate in an overlapping manner;
a colloidal gold probe marked by copper ions is fixed on the connecting pad; the nitrocellulose membrane is provided with a detection line and a quality control line; an aptamer capture probe is fixed on the detection line; streptavidin is fixed on the quality control line.
According to the invention, preferably, the aptamer capture probe is an aptamer modified by 5' end biotin, and is immobilized by streptavidin to form an aptamer-biotin-streptavidin structure together.
According to the invention, the sample pad, the connecting pad, the nitrocellulose membrane and the absorption pad are preferably overlapped by 2-3 mm, the interval between the detection line and the quality control line is 3-6 mm, and the width of the bottom plate is 4 mm.
Preferably, according to the invention, the sequence of the aptamer is 5 '-CTTCTGCCCGCCTCCTTCC-TAGCCGGATCGCGCTGGCCAGATGATATAAAGGGTCAGCCCCCCA-GGAGACGAGATAGGCGGACACT-3'.
The invention also discloses a preparation method of the copper ion labeled colloidal gold test strip for detecting endotoxin, which comprises the following steps:
s1: preparing a sample pad;
s2: preparing a connecting pad, fixing a copper ion labeled colloidal gold probe on the connecting pad, and drying at room temperature;
s3: treatment of nitrocellulose membrane: fixing an aptamer capture probe and streptavidin on a nitrocellulose membrane by a three-dimensional membrane scribing and gold spraying instrument to obtain a detection line and a quality control line;
s4: assembling the test strip: and sequentially fixing the sample pad, the connecting pad, the nitrocellulose membrane and the absorbing pad on the base plate to obtain the copper ion labeled colloidal gold test strip for detecting endotoxin.
According to a preferred embodiment of the present invention, the step S1 is specifically: the glass fiber was soaked in a buffer solution (pH 8.0) containing 0.25% of polyethylene glycol octyl phenyl ether (triton X-100), 0.05mol/L of tris (hydroxymethyl) aminomethane, 5% of tween-20, and 0.15mol/L of sodium chloride for 1h, then dried at room temperature and stored in a desiccator. The processing method can effectively promote the flow of the sample to be detected and reduce the retention on the sample pad.
Preferably, the copper ion-labeled colloidal gold probe in S2 is prepared as follows:
firstly preparing gold nanoparticles by a sodium citrate reduction method, then modifying cysteamine molecules on the surfaces of the gold nanoparticles, centrifuging the composite solution at the rotating speed of 8000rpm for 10min to remove free cysteamine molecules, finally adding a copper ion standard solution to promote the gold nanoparticles to agglomerate, centrifuging at the rotating speed of 8000rpm for 10min to remove unadsorbed copper ions, discarding supernatant fluid to obtain a copper ion-labeled colloidal gold probe, and re-dispersing the obtained red particles in 50 mu L of colloidal gold probe containing 20mmol/L Na3PO45% BSA, 0.25% Tween-20 and 10% sucrose in elution buffer, for immobilizing and facilitating release of the probe from the conjugate pad, reducing non-specific adsorption onto the nitrocellulose membrane.
Further preferably, the molar ratio of the gold nanoparticles to the cysteamine molecules to the copper ions is 150:1: 1.5.
According to the invention, the method for detecting endotoxin by using the copper ion labeled colloidal gold test strip for detecting endotoxin comprises the following steps:
(1) dispersing a sample to be detected in a phosphate buffer solution to obtain a liquid to be detected;
(2) dropwise adding the solution to be detected on the sample pad, and obtaining a test paper color development result after 0.5-2 min; the detection line and the quality control line are both red, and the sample to be detected contains endotoxin; the detection line is colorless, the quality control line is red, and the sample to be detected has no endotoxin.
According to the invention, the concentration of the phosphate buffer solution is preferably 0.005-0.015 mol/L.
The detection principle and the technical characteristics of the invention are as follows:
the colloidal gold test strip is prepared based on the nucleic acid aptamer combined colloidal gold color development principle, target molecule endotoxin can be specifically adsorbed with a colloidal gold probe marked by copper ions, and an aptamer capture probe modified by biotin is specifically combined on a detection line to form a sandwich structure, and a red color band is generated. In the preparation process of the copper ion labeled colloidal gold probe, the gold nanoparticles are prepared by reducing sodium citrate, and the method can ensure that the copper ion labeled colloidal gold probe prepared from the gold nanoparticles can provide a strong surface plasma band for colorimetric determination; the molar ratio of the gold nanoparticles to the cysteamine to the copper ions is 150:1:1.5, so that the situation that when the amount of the copper ions is too small, fewer colloidal gold probes marked by the copper ions captured by the target molecules and endotoxin are caused is avoided, the color of a color band is lightened, and the detection effect is poor; when the amount of copper ions is too large, the agglomeration degree of the cysteamine functionalized gold nanoparticles is increased, and the color development is influenced. In the method for detecting endotoxin by using the endotoxin colloidal gold test paper based on the copper ion label, the sample pad is soaked for 1 hour in a buffer solution (pH 8.0) containing 0.25 percent of polyethylene glycol octyl phenyl ether (Triton X-100), 0.05mol/L of tris (hydroxymethyl) aminomethane, 5 percent of Tween-20 and 0.15mol/L of sodium chloride, so that the flow of a sample to be detected can be effectively promoted, the retention on the sample pad is reduced, and the color band display of a detection line and a quality control line is clearer.
Compared with the prior art, the invention has the following advantages:
(1) the colloidal gold test strip prepared by the invention has the advantages that the copper ion mediated gold nanoparticle aggregate and the aptamer with high affinity are used as the detection probe and the capture probe, compared with the traditional colloidal gold test strip, the colloidal gold test strip has strong specificity, low detection limit, high sensitivity and good color development effect, and the detection result can be directly obtained without further calculation or analysis according to the color development conditions of the detection line and the quality control line.
(2) According to the method for detecting endotoxin by using the colloidal gold test strip, the sample pad is soaked in a buffer solution (pH 8.0) containing 0.25% of polyethylene glycol octyl phenyl ether (Triton X-100), 0.05mol/L of tris (hydroxymethyl) aminomethane, 5% of Tween-20 and 0.15mol/L of sodium chloride for 1h, so that the flow of a sample to be detected can be effectively promoted, the retention on the sample pad is reduced, and the color development effect is more visual and vivid.
(3) In the preparation process of the copper ion labeled colloidal gold probe, the gold nanoparticles are prepared by reducing sodium citrate, and the method can ensure that the copper ion labeled colloidal gold probe prepared from the gold nanoparticles can provide a strong surface plasma band for colorimetric determination; the molar ratio of the gold nanoparticles to the cysteamine to the copper ions is 150:1:1.5, so that the situation that when the amount of the copper ions is too small, fewer colloidal gold probes marked by the copper ions captured by the target molecules and endotoxin are caused is avoided, the color of a color band is lightened, and the detection effect is poor; when the amount of copper ions is too large, the agglomeration degree of the cysteamine functionalized gold nanoparticles is increased, and the color development is influenced.
(4) The method for detecting endotoxin by using the colloidal gold test strip is simple and convenient to operate and short in detection time, the color development can be seen only in 0.5-2 min after the sample is dripped, the result is judged by the color development, and a non-professional person can use the method without professional training, so that the method is suitable for field real-time detection; but also can be produced in batches, is convenient to carry, is stable and is easy to store.
Drawings
FIG. 1 is a schematic structural diagram of a copper ion labeled colloidal gold test strip for detecting endotoxin in the present invention;
FIG. 2 is a graph showing the results of comparative experiments of the test strips of example 1, comparative example 1 and comparative example 2.
Detailed Description
The present invention will be described in further detail below, but is not limited thereto.
Example 1
As shown in fig. 1, a copper ion labeled colloidal gold test strip for detecting endotoxin comprises a base plate, and a sample pad, a connection pad, a nitrocellulose membrane and an absorption pad which are sequentially fixed on the base plate in an overlapping manner;
a colloidal gold probe marked by copper ions is fixed on the connecting pad; the nitrocellulose membrane is provided with a detection line and a quality control line; an aptamer capture probe is fixed on the detection line; streptavidin is fixed on the quality control line.
The aptamer capture probe is an aptamer modified by 5' -end biotin, and is fixed by streptavidin to form an aptamer-biotin-streptavidin structure together;
the sample pad, the connecting pad, the nitrocellulose membrane and the absorption pad are overlapped by 2mm, the interval between the detection line and the quality control line is 4mm, and the width of the bottom plate is 4 mm.
Wherein the sequence of the aptamer is 5 '-CTTCTGCCCGCCTCCTTCC-TAGCCGGATCGCGCTGGCCAGATGATATAAAGGGTCAGCCCCCCA-GGAGACGAGATAGGCGGACAC T-3'.
The preparation method of the copper ion labeled colloidal gold test strip for detecting endotoxin comprises the following steps:
s1: preparing a sample pad: a sample made of glass fiber was padded in a buffer solution (pH 8.0) containing 0.25% of polyethylene glycol octyl phenyl ether (triton X-100), 0.05mol/L of tris, 5% tween-20, and 0.15mol/L of sodium chloride for 1h, and then dried at room temperature and stored in a desiccator;
s2: preparing a connecting pad, fixing a copper ion labeled colloidal gold probe on the connecting pad, and drying at room temperature;
s3: treatment of nitrocellulose membrane: fixing an aptamer capture probe and streptavidin on a nitrocellulose membrane through a three-dimensional membrane scribing and gold spraying instrument to obtain a detection line and a quality control line, wherein the interval between the detection line and the quality control line is 5 mm;
s4: assembling the test strip: and sequentially fixing the sample pad, the connecting pad, the nitrocellulose membrane and the absorbing pad on the base plate to obtain the copper ion labeled colloidal gold test strip for detecting endotoxin.
The step of S1 is specifically: : the glass fiber was soaked in a buffer solution (pH 8.0) containing 0.25% of polyethylene glycol octyl phenyl ether (triton X-100), 0.05mol/L of tris (hydroxymethyl) aminomethane, 5% of tween-20, and 0.15mol/L of sodium chloride for 1h, then dried at room temperature and stored in a desiccator. The processing method can effectively promote the flow of the sample to be detected and reduce the retention on the sample pad.
The copper ion-labeled colloidal gold probe in S2 is prepared as follows:
firstly preparing gold nanoparticles by a sodium citrate reduction method, then modifying cysteamine molecules on the surfaces of the gold nanoparticles, centrifuging the composite solution at the rotating speed of 8000rpm for 10min to remove free cysteamine molecules, finally adding a copper ion standard solution to promote the gold nanoparticles to agglomerate, centrifuging at the rotating speed of 8000rpm for 10min to remove unadsorbed copper ions, discarding the supernatant to obtain a copper ion-labeled colloidal gold probe, and re-dispersing the obtained red particles in 50 mu L of colloidal gold probe containing 20mM Na3PO45% BSA, 0.25% Tween-20 and 10% sucrose in elution buffer, for immobilizing and facilitating release of the probe from the conjugate pad, reducing non-specific adsorption onto the nitrocellulose membrane.
Wherein the molar ratio of the gold nanoparticles to the cysteamine molecules to the copper ions is 150:1: 1.5.
The method for detecting endotoxin by using the copper ion labeled colloidal gold test strip for detecting endotoxin comprises the following steps:
(1) dispersing 50 mu L of endotoxin standard solution with the concentration of 500pg/mL into 150 mu L of phosphate buffer solution with the concentration of 0.01mol/L to obtain endotoxin solution to be detected;
(2) dripping the solution to be detected on the sample pad, and obtaining a test paper color development result after 0.5 min; the detection line and the quality control line are both red, and the sample to be detected contains endotoxin; the detection line is colorless, and the quality control line shows red color without endotoxin in the sample to be detected.
In the embodiment, the detection line color development color band is clear and obvious, the quality control line color development color band is clear and obvious, and the sample to be detected contains endotoxin.
Example 2
A copper ion labeled colloidal gold test strip for detecting endotoxin, the structure and the preparation method are the same as those of the embodiment 1.
The method for detecting endotoxin by using the copper ion labeled colloidal gold test strip for detecting endotoxin comprises the following steps:
(1) dispersing 40 mu L of endotoxin standard solution with the concentration of 600pg/mL into 150 mu L of phosphate buffer solution with the concentration of 0.012mol/L to obtain endotoxin solution to be detected;
(2) dripping the solution to be detected on the sample pad, and obtaining a test paper color development result after 1 min; the detection line and the quality control line are both red, and the sample to be detected contains endotoxin; the detection line is colorless, and the quality control line shows red color without endotoxin in the sample to be detected.
In the embodiment, the detection line color development color band is clear and obvious, the quality control line color development color band is clear and obvious, and the sample to be detected contains endotoxin.
Comparative example 1
The structure and the preparation method of the copper ion labeled colloidal gold test strip for detecting endotoxin are the same as those in example 1, and the difference is that the molar ratio of gold nanoparticles to cysteamine molecules to copper ions is 150:1:1.
The method for detecting endotoxin by using the copper ion labeled colloidal gold test strip for detecting endotoxin comprises the following steps:
(1) dispersing 50 mu L of endotoxin standard solution with the concentration of 500pg/mL into 150 mu L of phosphate buffer solution with the concentration of 0.01mol/L to obtain a solution to be detected;
(2) dripping the solution to be detected on the sample pad, and obtaining a test paper color development result after 0.5 min; the detection line and the quality control line are both red, and the sample to be detected contains endotoxin; the detection line is colorless, and the quality control line shows red color without endotoxin in the sample to be detected.
In this example, the color development band of the detection line is less obvious, the color development band of the quality control line is less obvious, and the sample to be detected contains endotoxin.
Comparative example 2
The structure and the preparation method of the copper ion labeled colloidal gold test strip for detecting endotoxin are the same as those in example 1, and the difference is that the molar ratio of gold nanoparticles to cysteamine molecules to copper ions is 150:1: 2.
The method for detecting endotoxin by using the copper ion labeled colloidal gold test strip for detecting endotoxin comprises the following steps:
(1) dispersing 50 mu L of endotoxin standard solution with the concentration of 500pg/mL into 150 mu L of phosphate buffer solution with the concentration of 0.01mol/L to obtain a solution to be detected;
(2) dripping the solution to be detected on the sample pad, and obtaining a test paper color development result after 0.5 min; the detection line and the quality control line are both red, and the sample to be detected contains endotoxin; the detection line is colorless, and the quality control line shows red color without endotoxin in the sample to be detected.
In this embodiment, the detection line is a color band with a light color and is not obvious, the color band of the quality control line is very light and cannot be identified, and whether endotoxin is contained in the sample to be detected cannot be determined.
Comparative example 3
Endotoxin standard solution with the concentration of 500pg/mL was detected according to the rapid endotoxin detection limulus kit described in Chinese patent document CN 202119777U.
The test results of example 1 and comparative examples 1 to 2 are shown in fig. 2, and it can be seen from fig. 2 that the color bands of the test line and the quality control line of comparative example 1 are lighter; the color bands of the detection line and the quality control line in the embodiment 1 are more clear and obvious than that of the comparison example 1; the color bands of the detection line and the quality control line of comparative example 2 were extremely light and could not be recognized. When the amount of the copper ions is too small, the colloidal gold probes marked by the copper ions and captured by the endotoxin of the target molecule are less, so that the color of the color band is lightened, and the detection effect is poor; when the amount of copper ions is too large, the aggregation degree of the cysteamine functionalized gold nanoparticles is increased, and the color development is influenced. Therefore, the optimal molar ratio of the gold nanoparticles to the cysteamine to the copper ions is 150:1: 1.5. In the embodiment 1, whether the sample to be detected contains endotoxin can be obtained only within 0.5min, while in the comparative example 3, the result can be obtained only within about 30min, meanwhile, the operation is complicated, and higher requirements are also provided for experimenters carrying out the operation.
Claims (8)
1. A copper ion labeled colloidal gold test strip for detecting endotoxin is characterized by comprising a base plate, and a sample pad, a connecting pad, a cellulose nitrate membrane and an absorption pad which are sequentially fixed on the base plate in a lap joint manner;
a colloidal gold probe marked by copper ions is fixed on the connecting pad; the cellulose nitrate membrane is provided with a detection line and a quality control line; an aptamer capture probe is fixed on the detection line; streptavidin is fixed on the quality control line;
the colloidal gold probe is prepared by the following method:
firstly, preparing gold nanoparticles by a sodium citrate reduction method, then modifying cysteamine molecules on the surfaces of the gold nanoparticles, centrifuging the composite solution at the rotating speed of 8000rpm for 10min to remove free cysteamine molecules, finally adding a copper ion standard solution to promote the gold nanoparticles to agglomerate, centrifuging at the rotating speed of 8000rpm for 10min, removing unadsorbed copper ions, and discarding supernatant to obtain a copper ion labeled colloidal gold probe;
the molar ratio of the gold nanoparticles to the cysteamine molecules to the copper ions is 150:1: 1.5.
2. The copper ion labeled colloidal gold test strip for detecting endotoxin as claimed in claim 1, wherein the aptamer capture probe is an aptamer modified by biotin at the 5' end, and then the aptamer-biotin-streptavidin structure is formed by immobilization through streptavidin.
3. The copper ion-labeled colloidal gold test strip for detecting endotoxin of claim 1, wherein the sample pad, the connecting pad, the nitrocellulose membrane and the absorbent pad are overlapped with each other by 2 to 3mm, the interval between the detection line and the quality control line is 3 to 6mm, and the width of the base plate is 4 mm.
4. The copper ion-labeled colloidal gold test strip for detecting endotoxin of claim 2, wherein the sequence of the aptamer is 5 '-CTTCTGCCCGCCTCCTTCC-TAGCCGGATCGCGCTGGCCAGATGATATAAAGGGTCAGCCCCCCA-GGAGACGAGATAGGCGGACACT-3'.
5. The method for preparing the copper ion labeled colloidal gold test strip for detecting endotoxin as claimed in claim 1, comprising the steps of:
s1: preparing a sample pad;
s2: preparing a connecting pad: fixing a copper ion-labeled colloidal gold probe on the connecting pad, and drying at room temperature;
s3: treatment of the nitrocellulose membrane: fixing an aptamer capture probe and streptavidin on a nitrocellulose membrane by a three-dimensional membrane scribing and gold spraying instrument to obtain a detection line and a quality control line;
s4: assembling the test strip: and sequentially fixing the sample pad, the connecting pad, the nitrocellulose membrane and the absorbing pad on the base plate to obtain the copper ion labeled colloidal gold test strip for detecting endotoxin.
6. The method for preparing the copper ion labeled colloidal gold test strip for detecting endotoxin as claimed in claim 5, wherein the step of S1 comprises: the glass fiber was soaked in a buffer solution containing 0.25% of polyethylene glycol octylphenyl ether, 0.05mol/L of tris (hydroxymethyl) aminomethane, 5% of tween-20, and 0.15mol/L of sodium chloride at pH 8.0 for 1h, then dried at room temperature and stored in a desiccator.
7. The method for detecting endotoxin by using the copper ion-labeled colloidal gold test strip for detecting endotoxin as claimed in any one of claims 1 to 4, which comprises the steps of:
(1) dispersing a sample to be detected in a phosphate buffer solution to obtain a liquid to be detected;
(2) dropwise adding the solution to be detected on the sample pad, and obtaining a test paper color development result after 0.5-2 min; the detection line and the quality control line are both red, and the sample to be detected contains endotoxin; the detection line is colorless, the quality control line is red, and the sample to be detected has no endotoxin.
8. The method for detecting endotoxin as claimed in claim 7, wherein the concentration of the phosphate buffer solution is 0.005 to 0.015 mol/L.
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