CN114113576B

CN114113576B - Colloidal gold test strip for detecting cocaine, preparation method and method for testing cocaine

Info

Publication number: CN114113576B
Application number: CN202111169242.XA
Authority: CN
Inventors: 李辉; 靖乐; 谢崇禹; 李芊芊; 李少光; 夏帆
Original assignee: China University of Geosciences
Current assignee: China University of Geosciences
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2023-05-12
Anticipated expiration: 2041-09-30
Also published as: CN114113576A

Abstract

The invention provides a colloidal gold test strip for detecting cocaine, a preparation method and a method for testing cocaine, which comprises the following steps: a sample pad, a gold mark pad, a reaction membrane and a water absorption pad which are sequentially overlapped; the gold label pad is fixed with a conjugate of a first nucleic acid aptamer segment and nano gold, the molecular structure of the first nucleic acid aptamer segment is 5' -X1-DNA1-Y1-3', the reaction membrane is provided with a detection line and a quality control line, the detection line is fixed with a second nucleic acid aptamer segment and a labeled conjugate, and the molecular structure of the second nucleic acid aptamer segment is 5' -X1-DNA2; DNA1 and DNA2 are nucleic acid fragments comprising specific sites capable of binding cocaine, and DNA1 and DNA2 are combined with cocaine to form a three grass leaf hairpin structure with cocaine molecules as the center. The invention has the beneficial effects that: the sandwich colloidal gold test strip is prepared by adopting the first nucleic acid aptamer fragments and the second nucleic acid aptamer fragments which respectively contain a part and are specifically combined with cocaine, and has the advantages of rapid detection, strong specificity and high sensitivity.

Description

Colloidal gold test strip for detecting cocaine, preparation method and method for testing cocaine

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a colloidal gold test strip for detecting cocaine, a preparation method and a method for testing cocaine.

Background

Cocaine (cocaine) is one of the most abused drugs in the world today, a diester alkaloid composed of carboxymethanolic ester of ecgonine and hydroxybenzoate, the chemical name benzoylmethyl ecgonine (Benzoyl Methyl Ecgonine, BME), of formula C ₁₇ H ₂₁ NO ₄ The molecular weight is 303. The cocaine is a white crystal with a melting point of 98 ℃, is insoluble in water and is easily dissolved in organic solvents. Cocaine has damaging effects on the digestive system, immune system, cardiovascular system and genitourinary system of the human body. Especially as a dose-dependent hepatotoxin, can lead to large hepatocytesQuantitative necrosis manifested by elevation of ALT, AST, bilirubin in serum, pathological changes including local necrosis, inflammatory infiltration, steatosis, etc]. The human can be poisoned and killed by taking 0.5-1g of pure cocaine orally (30 mg for allergic people), and 30mg can be poisoned and killed by intramuscular injection or mucous membrane administration (the mortem blood concentration is 0.9-21 mg/L).

Currently, cocaine detection methods mainly include High Performance Liquid Chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), solvent microextraction-on-line stripping-field amplification sample injection-capillary electrophoresis-ultraviolet detection (SME-OLBE-FASI-CE-UV), direct real-time ionization time-of-flight mass spectrometry (DART-TOF-MS), and the like. The above method, although having high sensitivity and precision, requires expensive instruments, equipment and specially trained technicians, and cannot realize on-site large-scale instant detection. Therefore, sample feeding delay, improper storage of the sample and the like can occur, so that the sample cannot be accurately detected at the first time.

A nucleic acid aptamer (aptamer) is a single stranded oligonucleotide molecule (ssDNA or RNA) with a low number of bases (10-80) that can be obtained by in vitro screening by exponential enrichment of ligand system evolution techniques (Systematic Evolution of Ligands by Exponential Enrichment, SELEX). The aptamer has a short sequence folded to form a unique three-dimensional configuration, such as hairpin, stem-loop, convex-loop, clover and pseudoknot structures. These highly structured nucleic acid aptamers are capable of tightly binding to target molecules based on van der waals forces, hydrogen bonding, and hydrophobic interactions, among others. Compared with antibodies, the aptamer has the advantages of wide target range, small molecular weight, no immunogenicity, high stability, short preparation period, easy modification and the like. Therefore, the aptamer has wide application in drug detection, environmental analysis, heavy metal detection, clinical diagnosis and identification, food safety rapid detection, disease treatment and other aspects. In the aspect of detection of nucleic acid aptamer aiming at cocaine, a colloidal gold test paper is designed in the development of a specific cocaine nucleic acid aptamer test strip, and the colloidal gold test strip adopts a screened nucleic acid aptamer in a detection line, and a mouse anti-BSA monoclonal antibody is still adopted in a quality control line, so that the defects of long antibody preparation period, harsh transportation conditions and the like still exist. Therefore, it is necessary to prepare a test strip for detecting cocaine colloidal gold more rapidly and conveniently.

Disclosure of Invention

In order to solve the technical problems, the invention provides a colloidal gold test strip for detecting cocaine, a preparation method and a method for testing cocaine.

The specific technical scheme is as follows:

a colloidal gold test strip for detecting cocaine, which is characterized by comprising:

a sample pad, a gold mark pad, a reaction membrane and a water absorption pad which are sequentially overlapped;

the gold-labeled pad is fixed with a conjugate of a first nucleic acid aptamer segment and nano gold, and the molecular structure of the first nucleic acid aptamer segment is 5'-X1-DNA1-Y1-3', wherein X1 is a marker, and Y1 is a group capable of being coupled with the nano gold;

the reaction membrane is provided with a detection line and a quality control line, the detection line is fixed with a second nucleic acid aptamer segment and a marked conjugate, the molecular structure of the second nucleic acid aptamer segment is 5' -X1-DNA2, X1 is a marker, and the quality control line is fixed with the marked conjugate;

wherein, DNA1 and DNA2 are nucleic acid fragments comprising specific sites capable of binding to cocaine, and DNA1 and DNA2 form a three grass leaf hairpin structure with cocaine molecules as the center after being combined with cocaine.

It should be emphasized that in the present invention, DNA1 and DNA2 are combined with cocaine to form a three-leaf hairpin specific structure, while DNA1 and DNA2 alone are combined with cocaine to form no specific structure. In the present invention, the labeled conjugate can specifically bind to the label.

Further, the sequence of the nucleic acid fragment of the DNA1 is shown as SEQ ID No.2, and the sequence of the nucleic acid fragment of the DNA2 is shown as SEQ ID No. 3.

Further, the label is selected from one of biotin, fluorescein, chemiluminescent or radioactive substances; the label conjugate is selected from one of avidin or streptavidin.

It should be noted that the above-mentioned label may be selected without affecting the gold-labeled signal or enhancing the gold-labeled signal.

Further, the particle size of the nano gold is not more than 20 nm-40 nm.

Further, the particle size of the nano gold is not more than 25nm.

Further, Y1 is mercapto.

The preparation method of the single nucleic acid aptamer sandwich type colloidal gold test strip for detecting cocaine is characterized by comprising the following steps:

step S1, designing a DNA1 fragment and a DNA2 fragment;

step S2, sequentially performing coupling and aging reaction on a colloidal gold solution and a first nucleic acid aptamer fragment, and then performing solid-liquid separation, adding colloid Jin Chong suspension into the solid, and re-suspending to obtain gold-labeled compound re-suspension;

step S3, treating the sample pad substrate and the gold-labeled pad substrate by using sample treatment fluid and heavy suspension respectively;

step S4: fixing the gold-labeled compound heavy suspension on the gold-labeled pad matrix, and respectively marking the detection line and the quality control line on the reaction film;

step S5: and sequentially overlapping the sample pad, the gold-labeled pad, the reaction membrane and the water-absorbing pad on a bottom plate to obtain the colloidal gold test strip.

Further, in the step S1, the selected aptamer is truncated to obtain the DNA1 and the DNA2; the nucleic acid aptamer comprises a specific site capable of being combined with cocaine, and after the specific site is combined with the cocaine, a three-grass-leaf hairpin structure taking a cocaine molecule as a core is formed; cutting off the nucleic acid aptamer at a position far away from the specific site of the aptamer to obtain DNA1 and DNA2 of which a part can be combined with the specific site of cocaine;

preferably, the sequence of the nucleic acid aptamer is shown in SEQ.ID.NO. 1.

Further, the sample pad substrate and the gold mark pad substrate are made of glass fibers, and the reaction film substrate is made of nitrocellulose.

Further, the colloidal gold solution comprises nano gold with the particle size of 20 nm-40 nm.

Further, the colloidal gold solution includes nano gold having a particle size of 20nm.

Further, the resuspension includes: 0.06 to 1.2 percent w/v bovine serum albumin, 1.5 to 2.2 percent w/v sucrose, 0.20 to 0.30 percent w/v tween-20, 0.015 to 0.025 percent sodium azide and 0.008 to 0.012M phosphate buffer salt solution; the sample treatment solution comprises 2.5 to 3.2 percent w/v bovine serum albumin, 0.03 to 0.06 percent w/v Tween-20 and 0.008 to 0.012M phosphate buffer salt solution.

Further, in the step S2, the preparation of the colloidal gold solution is further included, after deionized water and chloroauric acid solution are uniformly mixed, the solution is heated until the solution starts to slightly boil, the sodium citrate solution is added at one time, when the color is continuously heated until the color becomes red and is not changed, the solution is kept in a boiling state and is continuously heated for 5-15 min, and the solution is kept stand and cooled to room temperature.

Further, the mass ratio of chloroauric acid to sodium citrate is 1: (1-2).

Further, the mass ratio of chloroauric acid to sodium citrate is 1:1.

Further, in the step S4, the second aptamer segment is incubated with a labeled conjugate solution, and the reaction is performed to obtain a T-line streaking solution; mixing the marked conjugate solution to obtain a C line marking liquid; and scribing the reaction film by adopting the T line scribing liquid and the C line scribing liquid to obtain the detection line and the quality control line.

A method for detecting cocaine, which is characterized by comprising:

and (3) dropwise adding a test sample into the cocaine detection colloidal gold test strip for reaction, and observing the signal change condition of the detection line and the quality control line.

Further, the method comprises the steps of:

if the detection line and the quality control line are both color-changed, cocaine exists in the sample; if the detection line does not change in color and the quality control line changes in color, no cocaine exists in the sample; if the quality control lines are not changed in color, the detection result is invalid.

Preferably, the detection method further comprises: preparing standard solutions of cocaine with different concentrations, and then respectively dripping the standard solutions of cocaine into the colloidal gold test strip for reaction to obtain a standard curve of the concentration and the signal gray value at the detection line; and then, dripping a sample on the colloidal gold test paper for reaction, and bringing the gray value measured by the detection line into the standard curve to quantitatively test the cocaine.

Compared with the prior art, the invention has the beneficial effects that:

(1) The invention adopts the first nucleic acid aptamer fragments and the second nucleic acid aptamer fragments which respectively contain a part and are specifically combined with cocaine to prepare the sandwich colloidal gold test strip, thereby completely replacing the traditional antigen-antibody reaction, and having the advantages of rapid detection, strong specificity and high sensitivity.

(2) According to the invention, the single nucleic acid aptamer is cut off on the premise of keeping forming a specific secondary structure site, the screening process is simplified, and the sandwich colloidal gold test strip can be prepared by using the single nucleic acid aptamer, so that the generated specific reaction fragment is combined with the sandwich of cocaine in the later period, and the matching performance is high.

(3) According to the invention, gold nanoparticles are generated by reduction reaction of chloroauric acid and sodium citrate, and the mass ratio of chloroauric acid to sodium citrate is controlled to be 1: (1-2) to make the grain diameter within the range of 20-40 nm, wherein when the grain diameter is 20nm, the outlet color is more beautiful and the grain diameter distribution is more uniform.

(4) The re-suspension can not re-suspend the gold-labeled compound after the aging step, but also pre-activate the gold-labeled pad, thereby being beneficial to release and interception of the gold-labeled compound after the later sample addition; and the sample pad after the pre-activation of the sample treatment liquid avoids reducing the nonspecific influence of the detection sample liquid on the test strip.

(5) The heavy suspension is compounded by adopting bovine serum albumin, sucrose, tween-20 and sodium azide, and the proper amount of the bovine serum albumin can balance the charge on the surface of the colloidal gold under the condition of not inducing bacterial proliferation, so that the proportion of various components is reasonable, the viscosity of the whole system is increased, and a milder and stable solution environment is provided for the colloidal gold.

Drawings

FIG. 1 is a schematic diagram showing the design of a DNA1 nucleic acid fragment and a DNA2 nucleic acid fragment according to the embodiment 1 of the present invention;

FIG. 2 is a visual illustration of colloidal gold solutions prepared according to various methods provided in examples 2-4 of the present invention;

FIG. 3 is an ultraviolet-visible absorption spectrum of colloidal gold solutions prepared by different methods according to examples 2-4 of the present invention

FIG. 4 is a transmission electron microscope image of the colloidal gold of example 2;

FIG. 5 is a block diagram and a flow chart of the colloidal gold test strip of the present invention;

FIG. 6 is a chart showing the specificity of the colloidal gold test strip of example 13;

FIG. 7 is a chart showing the test line results of the sensitivity test of the colloidal gold test strip of example 14;

FIG. 8 is a graph showing the sensitivity test standard of the colloidal gold test strip of example 14;

in FIG. 5, 1-nanogold, 2-first nucleic acid aptamer fragment, 201-coupling group, 202-DNA1, 203-label of first nucleic acid aptamer fragment, 3-second nucleic acid aptamer fragment, 301-DNA2, 302-label of second nucleic acid aptamer fragment, 4-sample pad, 5-gold-labeled pad, 6-reaction membrane, 601-detection line, 602-quality control line, 7-absorbent pad, 8-target.

Detailed Description

Example 1

The present embodiment provides a first nucleic acid aptamer segment and a second nucleic acid aptamer segment for performing a sandwich colloidal gold reaction.

The molecular structure of the first nucleic acid aptamer fragment is 5'-X1-DNA1-Y1-3', X1 is biotin, Y1 is sulfhydryl, and the sequence of the DNA1 is shown as SEQ ID No. 2.

The molecular structure of the second nucleic acid aptamer fragment is 5' -X1-DNA2, wherein X1 is biotin, and the sequence of the DNA2 is shown as SEQ ID No. 3.

The embodiment also provides a design method of the DNA1 and the DNA2, and the specific flow principle is shown in figure 1, comprising the following steps:

(1) Screening out a nucleic acid aptamer (1 a) capable of specifically binding with cocaine, wherein the nucleic acid aptamer comprises a specific site, and the nucleic acid aptamer can form a specific secondary structure after being bound with cocaine molecules through the specific site, specifically a three grass leaf hairpin structure (1 b) taking the cocaine molecules as cores. The sequence of the aptamer is shown as SEQ.ID.NO.1, and the aptamer comprises a ring structure fragment at a part which is not combined with the cocaine molecule.

(2) The part of the aptamer, which is far away from the specific site, is selected to be cut off, and particularly a ring structure fragment is adopted to obtain a part of DNA1 fragment and a part of DNA2 fragment which respectively contain the specific site, so that the target molecule cocaine becomes a connector, and the DNA1 fragment and the DNA2 fragment can form a specific three-grass leaf hairpin structure through reaction (1 c) with the cocaine.

Examples 2 to 4

Examples 2 to 4 provide a method for preparing colloidal gold, comprising the following steps:

99mL of deionized water and 1mL of 1% w/v chloroauric acid solution were added sequentially to a clean 250mL Erlenmeyer flask, and mixed by shaking. Heating by a constant temperature electric heating stirrer, regulating the rotation speed and the voltage of the electric heating stirrer, keeping the rotation speed and the voltage unchanged, heating until the solution starts to slightly boil, and rapidly adding 1% w/v sodium citrate solution with different volumes after filtration into a conical flask at one time. The heating is continued, the color of the solution gradually changes from colorless to gray after a few minutes of reaction, then changes from gray to black, then changes from black to purple, and finally gradually changes from red to red. And when the color of the solution keeps red and does not change, keeping the solution in a boiling state and heating for 10min. Then the electric heater is turned off, and the mixture is kept stand and cooled to room temperature. And (3) storing the prepared colloidal gold solution in a clean sterile silk mouth bottle, sealing, and preserving in a dark place at 4 ℃. The amount of 1% w/v sodium citrate added and the color of the colloidal gold solution required for preparing colloidal gold of different particle sizes in examples 2 to 4 are shown in Table 1 and FIG. 2.

Table 1 sodium citrate addition

Examples	1% sodium citrate addition (mL)	Color of colloidal gold solution
			Example 2	1	Red color
Example 3	1.5	Deep red color
			Example 4	2	Mauve color

The colloidal gold is further identified by adopting an ultraviolet spectrophotometry and a transmission electron microscope scanning.

According to the judgment of the color of the colloidal gold solution, the red corresponds to about 20nm of nano gold, the deep red corresponds to about 30nm of nano gold, and the purple red corresponds to about 40nm of nano gold.

Ultraviolet spectrophotometry:

and (3) taking a proper amount of three colloidal gold solutions, carrying out ultraviolet-visible spectrophotometry scanning at the wavelength of 450-650nm, measuring the maximum absorption wavelength of the colloidal gold solutions with different particle diameters, and simultaneously observing the peak shape and the peak width of the maximum absorption peak. As shown in fig. 3, three colloidal gold solutions having different particle diameters have a single absorption peak in the wavelength range of 450-600 nm. The maximum absorption peak wavelength of the colloidal gold of example 2 was 520nm, the maximum absorption peak wavelength of the colloidal gold of example 3 was 522nm, the maximum absorption peak wavelength of the colloidal gold of example 4 was 530nm, and the maximum absorption peak wavelengths of the colloidal gold of three particle sizes were all within the standard range. In addition, the maximum absorption peak width of the colloidal gold solutions with three particle sizes is smaller, which indicates that the colloidal gold solutions with three particle sizes have uniform gold particle sizes and qualified quality.

Transmission electron microscope scanning identification method

A clean piece of filter paper was laid on the petri dish, and then a copper mesh for transmission electron microscopy was placed right side up on the filter paper. 25 μl of the prepared colloidal gold solution of example 2 was dropped onto the copper mesh, and the dish was placed in a ventilated place, so that the copper mesh was dried slowly. And then placing the prepared copper mesh under a transmission electron microscope to observe the size, morphology and distribution uniformity of the colloidal gold particles. As can be seen from fig. 4, the colloidal gold particles in the prepared colloidal gold solution were uniform in size, well dispersed, and free from aggregation, and the particle size of the colloidal gold particles in the colloidal gold solution was confirmed to be about 20nm by an electron microscope spectrum scale.

In summary, the colloidal gold prepared in examples 2 to 4 can be used to prepare colloidal gold test strips, and further, the colloidal gold prepared in example 2 is most excellent in terms of its color and particle size uniformity and dispersibility.

Example 5

The embodiment provides a preparation method of a sample pad, and the specific operation method is as follows:

glass fiber is selected as sample pad material, which is put into 0.01M phosphate buffer salt solution (pH 7.4) containing 3% w/v bovine serum albumin and 0.05% w/v Tween-20 to be soaked for more than 1 hour, dried for more than 1 hour at 37 ℃ and stored at 4 ℃ for standby.

Examples 6 to 8

Examples 6 to 8 provide a method for preparing a gold-labeled pad, comprising the following steps:

1. coupling DNA1 (first nucleic acid aptamer fragment) modified with sulfhydryl and biotin with colloidal gold, adding 2 mu L of 100 mu M (first nucleic acid aptamer fragment) reduced by TECP per milliliter of colloidal gold solution, and reacting for more than 16 hours in a dark place; then adding 1M NaCl for aging reaction to make the final concentration of NaCl be 35mM, and making the NaCl react for 12 hours in a dark place; the solution was centrifuged at 12000g for 20 min at 4℃to remove the supernatant, and the pellet was resuspended in a 0.01M phosphate buffer solution (pH 7.4) containing 1% w/v bovine serum albumin, 2% w/v sucrose, 0.25% w/v Tween-20, 0.02% w/v sodium azide to give a gold-labeled conjugate complex suspension. Wherein the selected colloidal gold is shown in Table 2.

2. Glass fiber is selected as a gold-labeled pad material, the glass fiber is soaked in the heavy suspension for more than 1 hour in advance, and is dried for 2 hours at 37 ℃ and stored at 4 ℃ for standby.

3. And uniformly dripping the gold-labeled conjugate heavy suspension on the treated gold-labeled pad, drying at 37 ℃ for 2 hours, and preserving at 4 ℃ for later use.

TABLE 2 gold-labeled pads of examples 6-8 colloidal gold

Examples	Colloidal gold
		Example 6	Example 2
Example 7	Example 3
		Example 8	Example 4

Example 9

The embodiment provides a preparation method of a reaction film, which specifically comprises the following steps:

scribing on nitrocellulose membrane: diluting the synthesized DNA2 (second aptamer fragment) modified with the biotin label into a solution with the concentration of 250 mu M, mixing the solution with a streptavidin solution with the concentration of 5mg/mL according to the volume ratio of 1.1:1, and reacting for 1 hour at room temperature to obtain a T line streaking solution; 2mg/mL of streptavidin solution is used as a C line streaking solution; the two prepared solutions are respectively marked on the positions of a detection line and a quality control line on a nitrocellulose membrane at a flow rate of 2 mu L/cm by a film marking instrument, the two lines are separated by 3 mm, and then the nitrocellulose membrane is dried for half an hour at 37 ℃ for standby.

Examples 10 to 12

Examples 10 to 12 provide colloidal gold test strips for detecting cocaine, which are shown in fig. 5a and 5c, and include a sample pad, a gold-labeled pad, a reaction membrane, and a water-absorbent paper, which are sequentially overlapped.

Examples 10 to 12 also provide a colloidal gold test strip assembly method:

and respectively overlapping and adhering the sample pad, the gold mark pad, the reaction film and the absorbent paper on the PVC bottom plate in sequence, and then cutting into detection strips with the width of 4 mm by using a strip cutting machine, and loading the detection strips into a plastic card to form the colloidal gold detection test strip. The gold label pad selected for the colloidal gold test strips selected in examples 10 to 12 is shown in table 3.

TABLE 3 gold-labeled pad for colloidal gold test strip for detecting cocaine in examples 10-12

The application method of the colloidal gold test strip of the embodiment 10 to the embodiment 12 specifically comprises the following steps:

qualitative detection:

dripping the sample on a sample pad, reacting for 15min, and observing, as shown in fig. 5d, if the quality control line and the detection line both show red lines, the sample is proved to contain cocaine positively; if the quality control line is red and the detection line is not red as shown in fig. 5b, the sample is proved to be negative without cocaine; if the quality control line is not out, the result is invalid.

Quantitative detection:

and (3) preparing cocaine standard solutions with different concentrations, respectively dripping the standard solutions on sample pads of the colloidal gold test strips in the same batch, reacting for 15min, and then carrying out gray value analysis on the test result by using Image J software to establish a standard curve of the concentration and gray value of the test strips.

And (3) dripping the sample on a sample pad, reacting for 15min, and bringing the gray value into a standard curve to obtain the accurate concentration.

Example 13

The specific analysis of the colloidal gold test strip of example 10 is provided in this example, and comprises the following specific steps: respectively sucking 100 mu L of water and 1mM of cocaine standard substance solution, dripping the solution on a sample pad of the prepared colloidal gold test strip, and observing after reaction time is 15 minutes; as can be seen from fig. 6, a red line appears on the quality control line of the 6a test strip (water), and the detection line is not out. The quality control line and the detection line of the test strip (cocaine) of 6b are red, which indicates that the sample liquid contains cocaine with the concentration higher than the lowest detection concentration.

Example 14

The present embodiment provides a sensitivity analysis of the colloidal gold test strip of embodiment 10, which comprises the following specific steps: cocaine standard substances are respectively prepared into standard solutions of 10 mu M (7 b test strip is used for carrying out subsequent test), 50 mu M (7 c test strip is used for carrying out subsequent test), 250 mu M (7 d test strip is used for carrying out subsequent test), 500 mu M (7 e test strip is used for carrying out subsequent test), 1mM (7 f test strip is used for carrying out subsequent test) and 5mM (7 g test strip is used for carrying out subsequent test). 100. Mu.L of each cocaine standard solution of the gradient concentration was tested with a test strip prepared in the same batch and with water as a blank (NC) (7 a strip for subsequent testing). The test results of test strips to which cocaine solutions of different concentrations were added dropwise were observed and compared, and the analysis of fig. 7 gave a minimum detection line of 10 μm for the test strip. Finally, gray value analysis can be performed on the test result by using Image J software, a standard curve (figure 8) of the test strip is established, the ordinate is the ratio of the peak area of the T line of the sample to be tested to the peak area of the C line of the blank sample, and the abscissa is the logarithmic value of the concentration of the cocaine solution.

It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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Claims

1. A colloidal gold test strip for detecting cocaine, comprising:

the reaction membrane is provided with a detection line and a quality control line, the detection line is fixed with a second nucleic acid aptamer segment and a marked conjugate, the molecular structure of the second nucleic acid aptamer segment is 5' -X1-DNA2, X1 is a marker, and the quality control line is fixed with the marked conjugate; the label and the label conjugate can be specifically combined;

wherein, the DNA1 and the DNA2 are nucleic acid fragments comprising specific sites capable of being combined with cocaine, the sequence of the nucleic acid fragment of the DNA1 is shown as SEQ ID NO.2, the sequence of the nucleic acid fragment of the DNA2 is shown as SEQ ID NO.3, and both the DNA1 and the DNA2 are combined with cocaine to form a three grass leaf hairpin structure taking cocaine molecules as the center;

the X1 is selected from one of biotin, fluorescein, chemiluminescent substance or radioactive substance; y1 is mercapto; the label conjugate is selected from one of avidin or streptavidin.

2. The cocaine detection colloidal gold test strip of claim 1, wherein the particle size of the nano gold is 20 nm-40 nm.

3. The method for preparing the colloidal gold test strip for detecting cocaine according to any one of claims 1 to 2, which is characterized by comprising the following steps:

step S1, designing a DNA1 fragment and a DNA2 fragment;

step S2, sequentially performing coupling and aging reaction on a colloidal gold solution and the first nucleic acid aptamer fragment, and then performing solid-liquid separation, and adding colloid Jin Chong suspension into the solid to re-suspend the solid to obtain gold-labeled compound re-suspension;

4. The method according to claim 3, wherein in the step S1, the selected aptamer is truncated to obtain the DNA1 and the DNA2; the nucleic acid aptamer comprises a specific site capable of being combined with cocaine, and after the specific site is combined with the cocaine, a three-grass-leaf hairpin structure taking a cocaine molecule as a core is formed; and cutting off the nucleic acid aptamer at a position far away from the specific site of the aptamer to obtain DNA1 and DNA2 with a part of the specific site capable of being combined with cocaine.

5. The method according to claim 4, wherein the sequence of the aptamer is shown in SEQ ID No. 1.

6. A method of preparation according to claim 3, wherein the resuspension comprises: 0.06-1.2% w/v bovine serum albumin, 1.5-2.2% w/v sucrose, 0.20-0.30% w/v tween-20, 0.015-0.025% sodium azide and 0.008-0.012M phosphate buffer salt solution; the sample treatment fluid comprises 2.5-3.2% w/v bovine serum albumin, 0.03-0.06% w/v Tween-20 and 0.008-0.012M phosphate buffer salt solution.

7. The preparation method of claim 3, further comprising the preparation of colloidal gold solution in step S2, wherein deionized water and chloroauric acid solution are uniformly mixed, then heated until the solution starts to slightly boil, sodium citrate solution is added at one time, and when the solution is continuously heated until the color becomes red and does not change any more, the solution is kept in a boiling state and is continuously heated for 5-15 min, and then the solution is left to stand and cooled to room temperature.

8. The method according to claim 3, wherein in step S4, the second nucleic acid aptamer fragment is incubated with a labeled conjugate solution, and reacted to form a T-line streaking solution; mixing the marked conjugate solution to obtain a C line marking liquid; and scribing the reaction film by adopting the T line scribing liquid and the C line scribing liquid to obtain the detection line and the quality control line.

9. A method for detecting cocaine, comprising:

dripping a test sample into the cocaine detection colloidal gold test strip according to any one of claims 1-2 for reaction, and observing the signal change condition of the detection line and the quality control line.

10. The method of detecting according to claim 9, comprising:

if the detection line and the quality control line are red lines, cocaine exists in the sample; if the detection line does not have a red line and the quality control line has a red line, no cocaine exists in the sample; if the quality control lines are not red lines, the detection result is invalid.