CN110927396A - Duplex test strip for detecting methamphetamine and morphine as well as preparation method and application method thereof - Google Patents
Duplex test strip for detecting methamphetamine and morphine as well as preparation method and application method thereof Download PDFInfo
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Abstract
The invention relates to a duplex test strip for detecting methamphetamine and morphine, a preparation method and an application method thereof, wherein the test strip sequentially comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; the conjugate release pad is sprayed with methamphetamine antibody and morphine antibody marked by time-resolved fluorescent microspheres; the reaction membrane is coated with a detection line T1 of methamphetamine hapten-carrier protein conjugate, a detection line T2 of morphine hapten-carrier protein conjugate and a quality control line C of goat anti-mouse secondary antibody (or Biotin-BSA); the invention uses time-resolved fluorescent microspheres to mark antibodies, and adopts an immunochromatography technology to realize the simultaneous and rapid immunoassay of methamphetamine and morphine. The invention also provides a method for simultaneously detecting methamphetamine and morphine in a sample by applying the test strip. The invention has the advantages of high sensitivity, accurate quantification, quick detection and convenient operation, and can realize quick detection and field detection of a large batch of samples.
Description
Technical Field
The invention relates to detection of methamphetamine and morphine, in particular to a duplex test strip for simultaneously and quantitatively detecting methamphetamine and morphine by utilizing a time-resolved fluorescence microsphere immunochromatography technology, and a preparation method and an application method thereof.
Background
Methamphetamine, commonly known as methamphetamine, belongs to stimulants psychotropic drugs, has strong central nervous stimulation effect, and is a psychotropic drug and anesthetic strictly controlled by the nation. Currently, the abuse of amphetamine substances is on the rise internationally. Experts predict that amphetamine-type stimulants will become one of the most widely abused drugs in the 21 st century.
With the increasing year by year of criminal cases related to methamphetamine and the stricter state towards the management of methamphetamine, the field puts higher requirements on the detection of methamphetamine in biological specimens of methamphetamine and drug abusers.
Morphine, which is a main component of opium, heroin, etc., is extracted from opium, is a pure opioid receptor agonist, has analgesic and hypnotic effects, and is medically an narcotic analgesic. Morphine has the function of inhibiting the central nervous system, and is easy to be abused by addiction, because a consumer easily has tolerance and dependence.
At present, detection and monitoring methods for methamphetamine and morphine mainly comprise high performance liquid chromatography, gas chromatography, liquid chromatography or gas chromatography. These chromatographic methods have good sensitivity and specificity, but are complex to operate, low in throughput, long in time consumption and expensive in instrumentation. Enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography are currently internationally recognized mainstream techniques, and the two methods have the advantages of high detection speed, low price, simple operation and the like. However, the ELISA detection still requires professional personnel and needs a long time to display the result; the colloidal gold method can be used for qualitatively analyzing the methamphetamine and the morphine, is simple to operate, has short detection time, low sensitivity and large interference of human factors; at present, a single detection card is arranged on the market, and under the condition that the type of drugs in a sample is not determined, a plurality of repeated tests are required to be carried out, so that the waste of manpower, material resources and time is caused.
In summary, the detection technology of methamphetamine and morphine is not mature at present, and the development of a product and a method which have high sensitivity and simple operation and can realize the simultaneous rapid detection of a plurality of projects on a large batch of samples becomes a problem to be solved urgently.
Disclosure of Invention
The invention aims to overcome the defects that the existing method for detecting methamphetamine and morphine is complex in operation, long in time consumption, incapable of realizing rapid detection of a large batch of samples and incapable of simultaneously detecting methamphetamine and morphine, and provides a test strip for detecting methamphetamine and morphine, which is simple in operation, high in sensitivity, high in detection speed and low in cost, a preparation method and an application method thereof, so as to realize rapid detection and field monitoring of methamphetamine and morphine on a large batch of samples.
In order to realize the purpose of the invention, the duplex test strip for simultaneously detecting methamphetamine and morphine adopts the following technical scheme: a duplex test strip for simultaneously detecting methamphetamine and morphine comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; the method is characterized in that: the conjugate release pad is sprayed with methamphetamine antibody and morphine marked by time-resolved fluorescent microspheres; the reaction membrane is coated with a detection line T1 of methamphetamine hapten-carrier protein conjugate, a detection line T2 of morphine hapten-carrier protein conjugate and a quality control line C of goat anti-mouse secondary antibody (or Biotin-BSA).
The sample absorbing pad, the conjugate releasing pad, the reaction membrane and the water absorbing pad are all adhered to the bottom plate, and any two adjacent pads are partially overlapped in the up-down direction.
1/3-1/2 of the conjugate release pad is covered under the sample absorbing pad.
The time-resolved fluorescent microsphere takes rare earth ions with the diameter of 100nm-500nm as a marker, and the surface of the time-resolved fluorescent microsphere is modified with functional groups and used for covalent coupling of protein, antibody and nucleic acid.
The excitation wavelength of the time-resolved fluorescent microsphere is 300-400nm, and the emission wavelength is 500-700 nm.
The sample absorbing pad is a glass cellulose membrane.
The conjugate release pad is a fiberglass or polyester material.
The reaction membrane is a nitrocellulose membrane or a cellulose acetate membrane.
The bottom plate is a PVC bottom plate or other hard non-absorbent materials.
The absorbent pad is absorbent paper.
The method for preparing the duplex test strip for simultaneously detecting the methamphetamine and the morphine comprises the following steps:
1) marking methamphetamine antibodies and morphine antibodies by using fluorescent microspheres, and spraying the fluorescent microspheres on a conjugate release pad to prepare the conjugate release pad sprayed with methamphetamine monoclonal antibody-fluorescent microsphere markers and morphine monoclonal antibody-fluorescent microsphere markers;
2) respectively spraying methamphetamine hapten-carrier protein conjugate, morphine hapten-carrier protein conjugate and goat anti-mouse secondary antibody (or Biotin-BSA) on a reaction membrane as detection lines T1, T2 and a quality control line (C);
3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.
In the step 1), the method for labeling the methamphetamine antibody and the morphine antibody by using the fluorescent microspheres comprises the following steps: and (3) taking out the fluorescent microspheres for activation, then adding a methamphetamine antibody or a morphine antibody for covalent coupling, adding a sealing buffer solution for sealing after the covalent coupling is finished, centrifugally washing, and then placing at 4 ℃ for storage for later use.
The conjugate release pad in the step 1) is soaked in buffer solution for 2 hours, dried at 37 ℃ for 2 hours, stored in a dry environment and then sprayed with the labeled antibody. The buffer solution is 0.02-0.05mol/L Tris-HCl buffer solution with the pH value of 7.4, wherein the buffer solution contains 0.2-1% of bovine serum albumin, 0.1-5% of trehalose and 0.02-0.1% of Tween-20.
The invention relates to a method for detecting methamphetamine and morphine in a sample by applying the duplex test strip for simultaneously detecting the methamphetamine and the morphine, which comprises the following steps:
(1) and (5) restoring the test strip and the sample to be tested to room temperature.
(2) And starting the immunofluorescence analyzer, inserting the corresponding ID card, and reading the standard curve.
(3) And adding 60-120 mu L of sample to be detected into a sample hole of the test strip, reacting for 5-10min, and inserting the detection card into an immunofluorescence analyzer.
(4) The fluorescent microspheres trapped in the detection line and the quality control line emit strong fluorescent strips under the optimal excitation light;
(5) and reading the fluorescence intensity of the detection line and the quality control line by adopting an immunochromatography analyzer, giving out T1/C and a value T2/C, calculating the concentration of methamphetamine and morphine in the sample by using a built-in standard curve by using the analyzer, and judging whether the sample is positive or negative.
Compared with the prior art, the invention has the following advantages:
(1) the method can accurately quantify the contents of the methamphetamine and the morphine in the sample to be tested.
(2) The invention can simultaneously detect the methamphetamine and the morphine in the sample, and the methamphetamine and the morphine are complementary to interfere with each other.
(3) The invention adopts the reaction systems of the independent quality control line and the detection line without mutual interference and influence, and adopts the T/C value mode for calibration, thereby ensuring the accuracy of the test result.
(4) The invention adopts the time-resolved fluorescent microspheres, and the Stokes displacement is large (more than 150nm) and the fluorescence lifetime is 5-6 orders of magnitude higher than that of a background substance, so that the interference of various non-specific fluorescence can be effectively eliminated, and the detection sensitivity is improved.
(5) The invention provides a rapid pretreatment method for biological samples such as hair, blood, saliva, urine and the like, and ensures the rapid test.
The test strip provided by the invention has the advantages of high sensitivity, strong specificity, low cost, simplicity in operation, short detection time, no limitation of detection equipment, simplicity in storage and long quality guarantee period. The method for simultaneously detecting the methamphetamine and the morphine by using the test strip is simple, convenient, rapid, accurate, wide in application range, low in cost and easy to popularize and use.
Further, covering 1/3-1/2 of the conjugate release pad with the sample absorbent pad can extend the observation time of the test result, and allow the sample absorbent pad to sufficiently absorb the test liquid and sufficiently react with the antibody, thereby reducing errors.
Drawings
Fig. 1 is a schematic diagram of a duplex test strip for detecting methamphetamine and ketamine of the present invention. Wherein 1 is a sample absorption pad, 2 is a conjugate release pad, 3 is a reaction membrane, 4 is a water absorption pad, 5 is a bottom plate, 6-7 is a detection line, and 8 is a quality control line.
Fig. 2 is a schematic diagram of the structure of the test card. Where 9 is the detection window and 10 is the sample well.
FIG. 3 is a schematic diagram of the test result of the test strip sample.
FIG. 4 is a standard curve of methamphetamine for the test strip of the present invention.
FIG. 5 is a standard curve of ketamine for the test strips of the present invention.
Fig. 6 is a schematic structural diagram of a saliva collecting device. Where 11 is the collection well lid, 12 is the collection well, 13 is the collection tube, 14 is the collection tube lid, and 15 is the lowest collection line.
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. In addition, various changes or modifications of the present invention may be made by those skilled in the art within the scope defined by the appended claims, and these changes or modifications should also fall within the scope of the present invention.
The duplex test strip for simultaneously detecting methamphetamine and morphine disclosed by the invention comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the sample absorption pad is arranged on the bottom plate; the conjugate release pad is sprayed with an antibody marked by a time-resolved fluorescent microsphere; the reaction membrane is coated with a detection line T1 of methamphetamine hapten-carrier protein conjugate, a detection line T2 of morphine hapten-carrier protein conjugate and a quality control line C of goat anti-mouse secondary antibody (or Biotin-BSA). The sample absorbing pad, the conjugate releasing pad, the reaction membrane and the water absorbing pad are all adhered to the bottom plate, and any two adjacent pads are partially overlapped along the up-down direction. Covering 1/3 of the conjugate release pad with the sample absorbent pad can prolong the observation time of the test result, and allow the sample absorbent pad to absorb the test liquid and react with the antibody sufficiently, thereby reducing errors, and in other embodiments, 1/2 of the conjugate release pad can be covered under the sample absorbent pad. The time-resolved fluorescent microsphere takes rare earth ions with the diameter of 100nm-500nm as a marker, and the surface of the time-resolved fluorescent microsphere is modified with functional groups for covalent coupling of protein, antibody and nucleic acid. The excitation wavelength of the time-resolved fluorescent microsphere is 300-400nm, and the emission wavelength is 500-700 nm. The sample absorbing pad is a glass cellulose membrane. The conjugate release pad is a fiberglass or polyester material. The reaction membrane is a nitrocellulose membrane or a cellulose acetate membrane. The bottom plate is a PVC bottom plate or other hard non-absorbent materials. The absorbent pad is absorbent paper.
The preparation method of the test strip for detecting methamphetamine and morphine mainly comprises the following steps:
1) marking methamphetamine antibodies and morphine antibodies by fluorescent microspheres, and spraying the fluorescent microspheres on a conjugate release pad to prepare the conjugate release pad sprayed with the methamphetamine antibodies and morphine antibodies marked by the fluorescent microspheres;
2) respectively spraying the methamphetamine hapten-carrier protein conjugate, the morphine hapten-carrier protein conjugate and the goat anti-mouse secondary antibody (or Biotin-BSA) on reaction membranes as detection lines T1, T2 and a quality control line C to prepare the reaction membranes coated with the detection line T1 of the methamphetamine hapten-carrier protein conjugate, the detection line T2 of the morphine hapten-carrier protein conjugate and the quality control line C coated with the goat anti-mouse secondary antibody (or Biotin-BSA);
3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.
The following steps are detailed:
1. preparation of fluorescent microsphere marker:
labeling a methamphetamine antibody and a morphine antibody by fluorescent microspheres: 1mg of fluorescent microspheres were centrifuged at 15000rpm for 10min, and the pellet was collected and resuspended in 1mL of coupling buffer. Then adding EDC and NHS according to the molar ratio of 1:2-1:20, incubating for 20-30min at room temperature after vortex oscillation, centrifuging for 10min at 15000rpm, and collecting the precipitate. Adding coupling buffer solution to resuspend the microspheres, adding 40-150 mu g of antibody into the solution, fully mixing uniformly, stirring at room temperature to react for 2-4h, centrifuging at 10000rpm for 10min to remove supernatant, adding 1mL of blocking buffer solution, reacting at room temperature for 1-2h after mixing uniformly, centrifuging and washing for 3 times by using the blocking buffer solution, then resuspending and precipitating by using 0.02M PBS (containing 0.2-1% BSA and 0.1-5% trehalose) with pH7.4 to obtain the prepared fluorescent microsphere labeled antibody, and placing at 4 ℃ for later use.
2. Preparation of fluorescent microsphere pad:
the conjugate release pad was soaked in 0.02-0.05M Tris-HCl buffer (containing 0.1-5% trehalose and 0.02-1% Tween-20) containing 0.2-1% bovine serum albumin (mass fraction), pH7.4 for 2h, and dried at 37 ℃ for 2 h. Mixing the antibody of the methamphetamine marked by the fluorescent microspheres and the morphine antibody by using a film spraying instrument, uniformly spraying the mixture on a conjugate release pad, spraying 1-9 mu L of the antibody marked by the fluorescent microspheres on every 1cm of the conjugate release pad, drying the mixture for 1-2h at 37 ℃, and placing the mixture in a dry environment for later use.
3. Preparation of NC film:
separately coating methamphetamine hapten-carrier protein conjugate, morphine hapten-carrier protein conjugate and goat anti-mouse secondary antibody (or Biotin-BSA) on NC membrane: adjusting the methamphetamine hapten-carrier protein conjugate and the morphine hapten-carrier protein conjugate to 0.5-2mg/mL by using 0.02M PBS (pH7.4), coating on an NC film to form a detection line (T1, T2), wherein the coating amount is 5-10 muL/cm; a goat anti-mouse secondary antibody (or Biotin-BSA) was adjusted to 0.1-0.5mg/mL with 0.01M PBS (pH7.4), and coated on an NC membrane to form a quality control line C in an amount of 5-10. mu.L/cm. And (3) drying the coated reaction membrane at 37 ℃ for 1-2h, and placing the reaction membrane in a drying environment for later use.
4. Preparation of time-resolved fluorescent microsphere immunoassay card
Sequentially sticking a sample absorption pad, a conjugate release pad sprayed with an antibody marked by fluorescent microspheres, a reaction membrane sprayed with methamphetamine hapten-carrier protein and morphine hapten-carrier protein conjugates as detection lines and goat-anti-mouse secondary antibodies (or Biotin-BSA) as quality control lines, and a water absorption pad on a PVC (polyvinyl chloride) base plate; wherein, the binder release pad is covered by the sample absorption pad from the 1/3 area of the starting end, the tail end of the binder release pad is connected with the starting end of the reaction film, the tail end of the reaction film is connected with the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the PVC soleplate, and the tail end of the water absorption pad is aligned with the tail end of the PVC soleplate; the detection line and the quality control line on the reaction membrane are strip-shaped strips which are vertical to the long phase of the test strip; the detection line is located on the side near the end of the conjugate release pad; the quality control line is positioned on the side away from the end of the conjugate release pad; and cutting the test paper into strips with the width of 4mm after the assembly is finished, thus obtaining the immunochromatographic test paper strip.
The immunochromatographic test strip is fixed on a plastic bottom card, the surface of the test strip is tightly pressed by a surface card, and a sample hole 10 and a detection window 9 are respectively reserved on the parts of the surface card corresponding to the test strip sample absorption pad and the NC membrane, as shown in figure 2. The detection card is assembled and then put into an aluminum foil bag, a drying agent is added for sealing, and the detection card can be stored for 12 months in a room-temperature drying environment.
A method for detecting methamphetamine and morphine in a sample by using the test strip comprises the following steps:
1) restoring the test strip and the sample to be tested to room temperature;
2) starting an immunofluorescence analyzer, inserting a corresponding ID card, and reading a standard curve;
3) adding 60-120 mu L of sample to be detected into a sample hole 9 of the test strip, reacting for 5-10min, and inserting the detection card into an immunofluorescence analyzer;
4) the fluorescent microspheres trapped in the detection line and the quality control line emit strong fluorescent strips under the optimal excitation light;
5) the immunochromatography analyzer is adopted to read the fluorescence intensity of the detection line and the quality control line, and give T1/C and T2/C values, and the analyzer can calculate the concentration of methamphetamine and morphine in the sample through a built-in standard curve and judge the negative and positive, as shown in figure 3.
Test example 1 detection of methamphetamine and morphine content in Hair samples
1. Establishment of methamphetamine standard curve
Blank hair samples are taken, cut into pieces and subjected to ultrasonic treatment or vortex grinding or grinding, methamphetamine is added into the treated solution respectively until the final concentration is 0.5ppb, 1ppb, 2.5ppb, 5ppb, 10ppb, 25ppb, 50ppb and 100ppb, test strips are taken for detection, and each sample is tested for three times. And averaging the three repeated results of the determination, and calibrating on an immunofluorescence analyzer.
2. Establishment of morphine detection standard curve
Blank hair samples are taken, cut into pieces and ultrasonically treated or vortexed or ground, morphine is added into the treated solution to the final concentration of 0.5ppb, 1ppb, 2.5ppb, 5ppb, 10ppb, 25ppb, 50ppb and 100ppb respectively, test strips are taken for detection, and each sample is repeatedly tested for three times. And averaging the three repeated results of the determination, and calibrating on an immunofluorescence analyzer.
3. Detection of methamphetamine and morphine in hair samples
(1) Pretreatment of samples
① restoring the sample to room temperature of 20-25 deg.C before detection;
② weighing 10-20 mg of hair sample, and cutting into polystyrene centrifuge tubes;
③ adding 1mL of sample extract, and performing ultrasonic treatment in water bath for 5 min;
(2) detection with test strip
① the immunofluorescent analyzer was turned on, the corresponding ID card was inserted, and the standard curve was read.
② the test card is placed flat with its front side facing upwards, 60-120 μ L of sample to be tested is added into the sample hole of the test paper, and after reaction at room temperature for 5-10min, the test card is placed into an immunofluorescence analyzer for detection
③ the fluorescent microspheres trapped on the detection line and the quality control line emit strong fluorescent bands under the best excitation light;
④ the immunochromatography analyzer is used to read the fluorescence intensity of the detection line and the quality control line and give out T1/C, T2/C value, the analyzer can calculate the concentration of methamphetamine and morphine in the sample through the built-in standard curve and judge the negative and positive.
Test example 2 detection of Methamphetamine and morphine content in blood samples
In this example, the test strip was prepared in the same manner as in example 1 except that the sample absorbing pad was a blood filtration membrane.
1. Haemolytic blood sample
(1) Establishment of methamphetamine standard curve
Taking a blank blood sample, diluting the blank blood sample by using a sample diluent according to the proportion of 1:5-1:20, adding methamphetamine into a hemolyzed blood sample solution after dilution to the final concentration of 0.1ppb, 0.25ppb, 0.5ppb, 1ppb, 2.5ppb, 5ppb, 10ppb, 25ppb, 50ppb and 100ppb respectively, taking a test strip for detection, and repeatedly measuring each sample for three times. And averaging the three repeated results of the determination, and calibrating on an immunofluorescence analyzer.
(2) Establishment of morphine Standard Curve
Taking a blank blood sample, diluting the blank blood sample by using a sample diluent according to the proportion of 1:5-1:20, adding morphine into a hemolyzed blood sample solution after dilution to the final concentration of 0.5ppb, 1ppb, 2.5ppb, 5ppb, 10ppb, 25ppb, 50ppb and 100ppb respectively, taking a test strip for detection, and repeatedly measuring each sample for three times. And averaging the three repeated results of the determination, and calibrating on an immunofluorescence analyzer.
(2) Detection of methamphetamine and morphine in hemolyzed blood samples
① pretreatment of sample
a, restoring the sample to room temperature of 20-25 ℃ before detection;
b diluting the hemolyzed blood sample with a sample diluent in a ratio of 1:5 to 1: 20.
② the test paper strip is used for detection
a, starting the immunofluorescence analyzer, inserting the corresponding ID card, and reading the standard curve.
b, horizontally placing the detection card with the right side facing upwards, adding 60-120 mu L of sample to be detected into a sample hole of the test strip, reacting for 5-10min at room temperature, and then placing the detection card into an immunofluorescence analyzer for detection
c, the fluorescent microspheres trapped on the detection line and the quality control line emit strong fluorescent strips under the optimal excitation light;
and d, reading the fluorescence intensity of the detection line and the quality control line by using an immunochromatography analyzer, giving a T1/C, T2/C value, and simultaneously calculating the concentrations of the methamphetamine and the morphine in the sample by using a built-in standard curve by using the analyzer, and judging whether the sample is positive or negative.
2. Fresh blood sample
This example is the same as the hemolyzed blood sample example described above, but the fresh blood sample can be directly applied for detection without dilution before testing.
Test example 3 detection of Methamphetamine and morphine content in urine sample
In this example, all preparation methods and sample detection methods of the test strip were the same as those of examples 1-2.
Test example 4 measurement of Methamphetamine and morphine content in saliva sample
In this embodiment, all preparation methods and sample detection methods of the test strip are the same as those in embodiments 1-2, and a saliva collecting device is used for collecting saliva, as shown in fig. 6, and includes a collecting groove cover 11, a collecting groove 12, a collecting tube 13, and a collecting tube cover 14, and a lowest collecting line 15 is disposed on the collecting tube 13.
The saliva sample was collected as follows:
1. spitting saliva into collection trough until saliva amount reaches minimum collection line.
2. The collection well lid is closed until a click is heard and the sample diluent in the lid flows into the collection tube and mixes with the saliva sample.
3. And (4) unscrewing the collection groove, covering the collection tube cover, and turning upside down to mix evenly.
4. And sucking the diluted saliva sample for detection.
Establishment of basic parameters
Detection limit: the blank samples are used for repeated measurement for 20 times, the mean M and the standard deviation SD of 20 results are calculated, the detection limit of the method is reported by adding two times of the standard deviation (M +2SD) to the blank mean, and the detection limit of methamphetamine and morphine is 1 ppb.
Linear range: taking concentration values of 0.5ppb, 1ppb, 2.5ppb, 5ppb, 10ppb, 25ppb, 50ppb, 100ppb and the like of methamphetamine and morphine for detection, repeatedly measuring each concentration for three times, and carrying out linear analysis on the average value of the measured concentration and a theoretical value to obtain a linear equation y of-1.4247 x +0.7197 of the methamphetamine, wherein R is20.9916. (the experimental results and analysis are shown in table 1 and fig. 4); the linear equation for morphine was found to be y-1.7339 x +0.9425, R20.9886. (see Table 2, FIG. 5 for results and analysis)
TABLE 1 Methylamphetamine Standard test results
TABLE 2 morphine standards test results
Accuracy: the sample diluent is used for preparing a methamphetamine standard substance with the concentration of 2.5ppb, a morphine standard substance with the concentration of 5ppb and a mixed solution of methamphetamine and morphine with the concentration of 5ppb respectively, the test paper strip prepared in the embodiment 1 is used for detection, the detection is repeated for three times, and the detection result is calculated by taking the average value. The recovery rate is the detection concentration/actual addition concentration multiplied by 100 percent, and the calculated recovery rate of the methamphetamine is 98.13 percent; the recovery rate of morphine is 109%; the recovery rate of methamphetamine in the mixed solution is 110 percent, and the recovery rate of morphine is 108.33 percent.
Precision: taking three batches of the test strips for detecting the time-resolved fluorescence quantitative methamphetamine prepared in the example 1, detecting the mixed solution of the 5ppb methamphetamine and the morphine, and carrying out parallel detection on the standard substance of each batch of the test strips for 10 times, wherein the results show that the CV values of the methyl benzene medical records in the three batches are 3.392%, 4.005% and 3.858% respectively, and the CV value among the three batches is 3.683%; CV values for morphine 3.476%, 3.896%, 3.291%, respectively; the CV value was 3.498% between three batches.
From the data, compared with the existing method for detecting methamphetamine and morphine, the test strip disclosed by the invention has the characteristics of sensitivity, rapidness and accurate quantification, is not limited by an operation place, is small and portable in a matched detection instrument, has lower requirements on personnel, and is suitable for large-scale popularization and use.
Claims (13)
1. A duplex test strip for detecting methamphetamine and morphine comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; the method is characterized in that: the conjugate release pad is sprayed with methamphetamine antibody and morphine antibody marked by time-resolved fluorescent microspheres; the reaction membrane is coated with a detection line T1 of methamphetamine hapten-carrier protein conjugate, a detection line T2 of morphine hapten-carrier protein conjugate and a quality control line C of goat anti-mouse secondary antibody or Biotin-BSA.
2. The test strip of claim 1, wherein: the sample absorbing pad, the conjugate releasing pad, the reaction membrane and the water absorbing pad are all adhered to the bottom plate, and any two adjacent pads are partially overlapped in the up-down direction.
3. The test strip of claim 2, wherein: 1/3-1/2 of the conjugate release pad is covered under the sample absorbing pad.
4. The test strip of any one of claims 1-3, wherein: the excitation wavelength of the time-resolved fluorescent microsphere is 300-400nm, and the emission wavelength is 500-700 nm.
5. The test strip of any one of claims 1-4, wherein: the time-resolved fluorescent microsphere takes rare earth ions with the diameter of 100nm-500nm as a marker, and the surface of the time-resolved fluorescent microsphere is modified with functional groups for covalent coupling of protein, antibody and nucleic acid.
6. The test strip of any one of claims 1-5, wherein: the conjugate release pad is a fiberglass or polyester material.
7. The test strip of any one of claims 1-6, wherein: the reaction membrane is a nitrocellulose membrane or a cellulose acetate membrane.
8. The test strip of any one of claims 1-7, wherein: the samples tested included: hair, blood, saliva, urine, or other biological samples.
9. The test strip of claim 8, wherein: where the sample to be tested is hair, the sample is subjected to a pre-treatment comprising grinding, sonication or vortexing of the hair.
10. A method of preparing the test strip of any one of claims 1-9, comprising the steps of:
1) respectively marking methamphetamine antibody and morphine antibody by fluorescent microspheres, and spraying the mixed solution of the methamphetamine antibody and the morphine antibody on a conjugate release pad to prepare the conjugate release pad sprayed with a methamphetamine monoclonal antibody-fluorescent microsphere marker and a morphine monoclonal antibody-fluorescent microsphere marker;
2) respectively spraying methamphetamine hapten-carrier protein conjugate, morphine hapten-carrier protein conjugate and goat anti-mouse secondary antibody (or Biotin-BSA) on a reaction membrane as a detection line T1, a detection line T2 and a quality control line C;
3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.
11. The method for preparing the test strip of claim 10, wherein in the step 1), the method for labeling the methamphetamine antibody and the morphine antibody with the fluorescent microspheres comprises the following steps: and (3) taking out the fluorescent microspheres for activation, then adding a methamphetamine antibody or a morphine antibody for covalent coupling, adding a sealing buffer solution for sealing after the covalent coupling is finished, centrifugally washing, and then placing at 4 ℃ for storage for later use.
12. The method of claim 10 or 11, wherein the conjugate release pad of step 1) is first soaked in a buffer solution for 2 hours, dried at 37 ℃ for 2 hours, stored in a dry environment, and then sprayed with the labeled antibody. The buffer solution is 0.02-0.05mol/L Tris-HCl buffer solution with the pH value of 7.4, wherein the buffer solution contains 0.2-1% of bovine serum albumin, 0.1-5% of trehalose and 0.02-0.1% of Tween-20.
13. A method for detecting methamphetamine and morphine in a sample using the duplex test strip of any one of claims 1-9, the method comprising the steps of:
1) restoring the test strip and the sample to be tested to room temperature;
2) starting an immunofluorescence analyzer, inserting a corresponding ID card, and reading a standard curve;
3) adding 60-120 mu L of sample to be detected into a sample hole of the test strip, reacting for 5-10min, and inserting the detection card into an immunofluorescence analyzer;
4) the fluorescent microspheres trapped in the detection line and the quality control line emit strong fluorescent strips under the optimal excitation light;
5) and reading the fluorescence intensity of the detection line and the quality control line by adopting an immunochromatography analyzer, giving out T1/C and T2/C values, calculating the concentration of methamphetamine and morphine in the sample by using a built-in standard curve by using the analyzer, and judging whether the sample is positive or negative.
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| CN113391062A (en) * | 2021-05-25 | 2021-09-14 | 黄淮学院 | Morphine immunofluorescence chromatography rapid detection test paper strip, preparation method and detection method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113391062A (en) * | 2021-05-25 | 2021-09-14 | 黄淮学院 | Morphine immunofluorescence chromatography rapid detection test paper strip, preparation method and detection method thereof |
| CN113391062B (en) * | 2021-05-25 | 2024-06-07 | 黄淮学院 | Morphine immunofluorescence chromatography rapid detection test strip, preparation method and detection method thereof |
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