CN110927395A - Triple test strip for detecting methamphetamine, morphine and ketamine as well as preparation method and application method thereof - Google Patents
Triple test strip for detecting methamphetamine, morphine and ketamine as well as preparation method and application method thereof Download PDFInfo
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Abstract
The invention relates to a triple test strip for detecting methamphetamine, morphine and ketamine, 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 a methamphetamine antibody, a morphine antibody and a ketamine antibody marked by time-resolved fluorescent microspheres; the reaction membrane is coated with a detection line T1 of methamphetamine antigen, a detection line T2 of morphine antigen, a detection line T3 of ketamine antigen and a quality control line C of goat anti-mouse secondary antibody or Biotin-BSA; the immunochromatography technology of the invention realizes the simultaneous and rapid immunoassay of three drugs of methamphetamine, morphine and ketamine. The invention also provides a method for simultaneously detecting methamphetamine, morphine and ketamine 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 drugs such as methamphetamine, morphine, ketamine and the like, in particular to a triple test strip for simultaneously and quantitatively detecting methamphetamine, morphine and ketamine 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 of criminal cases related to methamphetamine and the stricter state of the country for the management of methamphetamine, the field puts higher requirements on the detection of the methamphetamine in biological specimens of the 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.
Ketamine, a separate anesthetic, is a derivative of phencyclidine (PCP), commonly known as "K powder," and is generally used clinically as an anesthetic. Ketamine can excite blood vessels, can kill when being sucked excessively, and has certain mental dependence. After the K powder is addicted, a smoker shakes his head violently under the action of drugs, so that the spine is easy to shake off; meanwhile, excessive rocking can cause heart and respiratory failure. Overdose or long-term intake of the medicine can cause fatal damage to heart, lung and nerves, and the damage to central nerves is worse than the ice toxicity.
At present, methods for detecting and monitoring drugs such as methamphetamine, morphine, ketamine and the like mainly comprise high performance liquid chromatography, gas chromatography, liquid chromatography or gas chromatography and the like. 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, the morphine and the ketamine, is simple to operate, has short detection time, but has 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, morphine and ketamine is not mature at present, and the problem to be solved urgently is to develop a product and a method which have high sensitivity and simple operation and can realize the rapid detection of a plurality of projects on a large batch of samples.
Disclosure of Invention
The invention aims to overcome the defects that the existing method for detecting methamphetamine, morphine and ketamine is complex in operation, long in time consumption, incapable of realizing rapid detection of a large batch of samples and capable of detecting multiple drugs simultaneously, and provides a triple test strip for detecting drugs, 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 that rapid detection and field monitoring of multiple drugs can be simultaneously performed on a large batch of samples.
In order to realize the purpose of the invention, the triple test strip for simultaneously detecting methamphetamine, morphine and ketamine adopts the following technical scheme: a triple test strip for simultaneously detecting methamphetamine, morphine and ketamine 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, morphine antibody and ketamine 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, a detection line T3 of ketamine 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 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 triple test strip for simultaneously detecting methamphetamine, morphine and ketamine comprises the following steps:
1) marking methamphetamine antibodies, morphine antibodies and ketamine antibodies by 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, morphine monoclonal antibody-fluorescent microsphere markers and ketamine monoclonal antibody-fluorescent microsphere markers;
2) respectively spraying methamphetamine hapten-carrier protein conjugate, morphine hapten-carrier protein conjugate, ketamine hapten-carrier protein conjugate and goat anti-mouse secondary antibody (or Biotin-BSA) on a reaction membrane as detection lines T1, T2, T3 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, the morphine antibody and the ketamine 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 or a ketamine 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.
Soaking the conjugate release pad in the step 1) with a buffer solution for 2h, drying the conjugate release pad at 37 ℃ for 2h, storing the conjugate release pad in a dry environment, and spraying a labeled antibody; the buffer solution is Tris-HCl buffer solution with the pH value of 7.4, wherein the buffer solution contains 0.02-0.05mol/L of Tris, 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, morphine and ketamine in a sample by using the triple test strip for simultaneously detecting methamphetamine, morphine and ketamine, 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, T2/C and T3/C, calculating the concentrations of methamphetamine, morphine and ketamine in the sample by using a built-in standard curve by using the analyzer, and judging whether the sample is negative or positive.
Compared with the prior art, the invention has the following advantages:
(1) the method can accurately quantify the contents of the methamphetamine, the morphine and the ketamine in the sample to be tested.
(2) The invention can simultaneously detect the methamphetamine, the morphine and the ketamine in the sample, and the three do not 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 methamphetamine, morphine and ketamine by using the test strip is simple, convenient, quick and accurate, has wide application range and low cost, and is 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 triple test strip for detecting methamphetamine, morphine and ketamine according to 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, 6-8 is a detection line, and 9 is a quality control line.
Fig. 2 is a schematic diagram of the structure of the test card. Where 10 is the detection window and 11 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.
Figure 5 is a standard curve of morphine for the test strip of the present invention.
FIG. 6 is a standard curve of ketamine for the test strips of the present invention.
Fig. 7 is a schematic structural diagram of a saliva collecting device. Where 12 is the collection well lid, 13 is the collection well, 14 is the collection tube, 15 is the collection tube lid, and 16 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 invention discloses a triple test strip for simultaneously detecting methamphetamine, morphine and ketamine, which is shown in figure 1 and 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 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, a detection line T3 of ketamine 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. The 1/3 of the conjugate release pad is covered by the sample absorption pad, so that the observation time of the detection result can be prolonged, the sample absorption pad can fully absorb the detection liquid and fully react with the antibody, and the error is reduced; in other embodiments, the conjugate release pad 1/2 may also be covered under the sample absorbing pad. The time-resolved fluorescent microsphere is a fluorescent microsphere taking rare earth ions with the diameter of 100nm-500nm as a marker, the surface of the fluorescent microsphere is modified with functional groups, and the fluorescent microsphere is 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 preparation method of the triple test strip for detecting methamphetamine, morphine and ketamine mainly comprises the following steps:
1) marking methamphetamine antibodies, morphine antibodies and ketamine antibodies by fluorescent microspheres, and spraying the fluorescent microspheres on the conjugate release pad to prepare the conjugate release pad sprayed with the methamphetamine antibodies, morphine antibodies and ketamine antibodies marked by the fluorescent microspheres;
2) respectively spraying the methamphetamine hapten-carrier protein conjugate, the morphine hapten-carrier protein conjugate, the ketamine hapten-carrier protein conjugate and the goat anti-mouse secondary antibody (or Biotin-BSA) on a reaction membrane as detection lines T1, T2, T3 and a quality control line C to prepare a reaction membrane coated with the detection line T1 of the methamphetamine hapten-carrier protein conjugate, the detection line T2 of the morphine hapten-carrier protein conjugate, the detection line T3 of the ketamine 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, a morphine antibody and a ketamine 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 0.02MpH7.4 PBS (containing 0.1-1% BSA and 0.1-5% trehalose), thus obtaining 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, the morphine antibody and the ketamine 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 each 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:
coating a methamphetamine hapten-carrier protein conjugate, a morphine hapten-carrier protein conjugate, a ketamine hapten-carrier protein conjugate and a goat anti-mouse secondary antibody (or Biotin-BSA) on an NC membrane respectively: respectively adjusting the methamphetamine hapten-carrier protein conjugate, the morphine hapten-carrier protein conjugate and the ketamine hapten-carrier protein conjugate to 0.5-2mg/mL by using 0.02M PBS (pH7.4), coating the conjugates on an NC membrane to form a detection line (T1, T2 and T3), 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, morphine hapten-carrier protein conjugate and ketamine hapten-carrier protein conjugate as a detection line and goat anti-mouse secondary antibody (or Biotin-BSA) as a quality control line, 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 and a detection window are respectively reserved on the part 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, morphine and ketamine 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 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, T2/C and T3/C, the analyzer can calculate the concentration of methamphetamine, morphine and ketamine in the sample through a built-in standard curve and judge the negative and positive, as shown in figure 3.
Test example 1 measurement of contents of methamphetamine, morphine and ketamine in hair samples
1. Establishment of methamphetamine standard curve
Blank hair samples are taken, cut into pieces and subjected to ultrasonic treatment, methamphetamine is added into the ultrasonic solution to the final concentration of 0.1ppb, 0.25ppb, 0.5ppb, 1ppb, 2.5ppb, 5ppb, 10ppb, 25ppb, 50ppb and 100ppb respectively, 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 subjected to ultrasonic treatment, morphine is added into the ultrasonic solution to the final concentration of 0.1ppb, 0.25ppb, 0.5ppb, 1ppb, 2.5ppb, 5ppb, 10ppb, 25ppb, 50ppb and 100ppb respectively, 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.
3. Establishment of ketamine detection standard curve
Blank hair samples are taken, cut into pieces and ultrasonically treated or vortexed or ground, ketamine is respectively added into the treated solution until the final concentration is 1ppb, 2.5ppb, 5ppb, 10ppb, 25ppb, 50ppb, 100ppb, 250ppb and 500ppb, 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.
4. Detection of methamphetamine, morphine and ketamine 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 an immunochromatography analyzer is used for reading the fluorescence intensity of the detection line and the quality control line and providing T1/C, T2/C and T3/C, the analyzer can simultaneously calculate the concentration of methamphetamine, morphine and ketamine in the sample through a built-in standard curve and judge the negative and positive.
Test example 2 detection of contents of methamphetamine, morphine and ketamine 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) Establishment of ketamine 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 ketamine into a hemolyzed blood sample solution after dilution to the final concentration of 1ppb, 2.5ppb, 5ppb, 10ppb, 25ppb, 50ppb, 100ppb, 250ppb and 500ppb respectively, taking a test strip for detection, and repeating the detection for three times for each sample. And averaging the three repeated results of the determination, and calibrating on an immunofluorescence analyzer.
(3) Detection of methamphetamine, morphine and ketamine 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 methamphetamine, morphine and ketamine in the sample by using a built-in standard curve by using the analyzer, and judging whether the sample is negative or positive.
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 contents of methamphetamine, morphine and ketamine in urine samples
In this example, all preparation methods and sample detection methods of the test strip were the same as those of examples 1 and 2.
Test example 4 measurement of contents of methamphetamine, morphine and ketamine in saliva sample
In this embodiment, all preparation methods and sample detection methods of the test strip are the same as those of embodiments 1 and 2, and a saliva collecting device is used for collecting saliva, as shown in fig. 6, and includes a collecting groove cover 12, a collecting groove 13, a collecting tube 14, and a collecting tube cover 15, wherein a lowest collecting line 16 is arranged on the collecting tube 14.
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 measurement was repeated 20 times with blank samples, the mean M and standard deviation SD of the 20 results were calculated, and the detection limit of the method was reported as blank mean plus two times standard deviation (M +2SD), with the results being 1ppb for methamphetamine and morphine and 2ppb for ketamine.
Linear range: taking concentration values of methamphetamine and morphine such as 0.1ppb, 0.25ppb, 0.5ppb, 1ppb, 2.5ppb, 5ppb, 10ppb, 25ppb, 50ppb and 100ppb and concentration values of ketamine such as 1ppb, 2.5ppb, 5ppb, 10ppb, 25ppb, 50ppb, 100ppb, 250ppb and 500ppb for detection, measuring the concentration values three times repeatedly, and carrying out linear analysis on the measured concentration average value and the theoretical value to obtain a linear equation y of-1.2232 x +0.1987, R is the linear equation of methamphetamine20.9808. (the experimental results and analysis are shown in table 1 and fig. 4); the linear equation for morphine was found to be-1.912 x +0.7607, R20.9815. (the experimental results and analysis are shown in Table 2 and FIG. 5); the linear equation for ketamine was found to be-1.3508 x +1.9065, R20.9804. (results and analysis are shown in Table 3 and FIG. 6)
TABLE 1 Methylamphetamine Standard test results
TABLE 2 morphine standards test results
TABLE 3 Ketamine Standard 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, a ketamine standard substance with the concentration of 10ppb and a mixed solution of the methamphetamine, morphine and ketamine with the concentration of 10ppb respectively, the test paper strip prepared in the embodiment 1 is used for detection, the detection is repeated for three times, and the average value of the detection results is taken for calculation. The recovery rate is detected concentration/actual added concentration multiplied by 100 percent, and the calculated methamphetamine recovery rate is 98.67 percent; the recovery rate of morphine was 106.43%; the recovery rate of ketamine was 109.78%; the recovery rate of methamphetamine in the mixed solution is 110.3%, the recovery rate of morphine is 107.53% and the recovery rate of ketamine is 101.44%.
Precision: taking three batches of the time-resolved fluorescence quantitative triple test strips prepared in the example 1, detecting mixed liquor of methamphetamine, morphine and ketamine with the detection concentration of 10ppb, and carrying out parallel detection on the standard products of each batch of test strips for 10 times, wherein the results show that the CV values of the methamphetamine in the three batches are 4.263%, 4.786% and 3.470% respectively, and the CV value among the three batches is 3.683%; CV values for morphine 3.979%, 3.343%, 3.455%, respectively; the CV value between batches was 3.863%; the CV values of ketamine were 3.992%, 3.667%, 4.045%, respectively, and the CV value between three batches was 4.287%.
From the data, compared with the existing method for detecting methamphetamine, morphine and ketamine, the test strip provided 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 triple test strip for detecting methamphetamine, morphine and ketamine 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 a methamphetamine antibody, a morphine antibody and a ketamine 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, a detection line T3 of ketamine 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 is characterized in that rare earth ions with the diameter of 100nm-500nm are used as markers, and the surface of the time-resolved fluorescent microsphere is modified with functional groups and used for covalent coupling of proteins, antibodies and nucleic acids.
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: 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 a methamphetamine antibody, a morphine antibody and a ketamine antibody by using fluorescent microspheres, and spraying the mixed solution of the three on a conjugate release pad to prepare the conjugate release pad sprayed with a methamphetamine monoclonal antibody-fluorescent microsphere marker, a morphine monoclonal antibody-fluorescent microsphere marker and a ketamine monoclonal antibody-fluorescent microsphere marker;
2) respectively spraying methamphetamine hapten-carrier protein conjugate, morphine hapten-carrier protein conjugate, ketamine hapten-carrier protein conjugate and goat anti-mouse secondary antibody (or Biotin-BSA) on a reaction membrane to serve as a detection line T1, a detection line T2, a detection line T3 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, the morphine antibody and the ketamine 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 or a ketamine 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 for preparing the test strip of claim 10 or 11, wherein the conjugate release pad in 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, morphine and ketamine in a sample using the triple strip of any one of claims 1-9, 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 a T1/C, T2/C value and a T3/C value, calculating the concentrations of methamphetamine, morphine and ketamine in the sample by using a built-in standard curve by using the analyzer, and judging whether the sample is negative or positive.
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