CN113391062A - Morphine immunofluorescence chromatography rapid detection test paper strip, preparation method and detection method thereof - Google Patents
Morphine immunofluorescence chromatography rapid detection test paper strip, preparation method and detection method thereof Download PDFInfo
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9486—Analgesics, e.g. opiates, aspirine
Abstract
The invention relates to a morphine immunofluorescence chromatography rapid detection test paper strip, a preparation method and a detection method thereof. The morphine fluorescent immune quick-detection test paper strip adopts the principle of immune competition, morphine and morphine antigen compete together to bind morphine antibodies coupled with fluorescent microspheres on a pad, when the morphine content in a lysate is sequentially increased, more antibodies are required to be bound with the morphine antibody, so that the content of the antibodies capable of being bound with the antigen on a T line is reduced, the brightness of the T line is reduced, a C line is a quality detection line, and the brightness is not changed along with the increase and decrease of the morphine content. The morphine immunofluorescence rapid detection test paper strip has the advantages of small volume, light weight, convenience in carrying, high analysis speed, simple operation steps and the like, and breaks through the limitation of the traditional detection method. Powerful technical support is provided for on-site rapid detection with huge sample size. Meanwhile, great convenience is brought to on-site drug investigation and detection for public security personnel.
Description
Technical Field
The invention relates to the technical field of quantitative identification, in particular to a morphine immunofluorescence chromatography rapid detection test paper strip, a preparation method and a detection method thereof.
Background
The qualitative detection of the drugs in the hair is a detection technology which is raised in the discipline of forensic toxicant identification, and is mainly used for qualitatively detecting drugs such as opium, amphetamine, ketamine and the like. Because the hair has the advantages of easy preservation, stable property, easy repeated sampling, long monitoring time and the like, the rapid detection of the drugs in the hair is widely applied to the fields of drug judicial identification, drug crime fighting, drug case detection, drug spreading inhibition and the like.
At present, the main methods for detecting the content of the toxic substances in the hair are available; the method can be used for measuring the morphine content in human hair, but the used instruments and equipment have overlarge volume, high equipment maintenance cost, long detection time and complicated detection operation steps, and the methods are difficult to realize for the field rapid detection with large sample quantity.
Disclosure of Invention
The invention researches and establishes an analysis method for rapidly detecting drugs in hair, so as to solve the limitations of long detection time, complicated operation steps and the like of the traditional method.
The technical scheme adopted by the invention is as follows:
a morphine immunofluorescence chromatography rapid examination test paper strip comprises a sample pad, a combination pad, an NC membrane, absorbent paper and a rubber plate, wherein the preparation method of the combination pad comprises the following steps:
(1) placing the bonding pad in a bonding pad soaking solution for soaking for 2 hours, taking out the soaked bonding pad, and placing the bonding pad in an oven to be dried for 2 hours for later use under 45 ℃;
(2) activating the fluorescent microspheres: taking 100 microliters of the fluorescent microspheres, adding 1ml of 0.05mol/LMES (PH is 5.0) solution into the 100 microliters of the fluorescent microspheres, uniformly mixing, centrifuging for 13000r/min, 20min, discarding the supernatant, adding 1ml of 0.05mol/LMES, uniformly mixing, ultrasonically treating for 5min, centrifuging for 13000r/min, 20min, discarding the supernatant, adding 200 microliters of 0.05mol/LMES solution, uniformly mixing, ultrasonically treating for 5min, preparing NHS10mg/ml by using 0.05mol/LMES solution, taking 15 microliters, adding into the fluorescent microspheres, preparing EDC15mg/ml by using pure water, taking 2 microliters, adding into the fluorescent microspheres, wrapping the microspheres by using tinfoil, and reacting for 15min by using a light-shielding shaking table;
(3) preparing a fluorescent microsphere and morphine antibody conjugate, namely adding 20 microliters of 100 microliters per milligram of morphine antibody into activated fluorescent microspheres for uniformly mixing, carrying out shaking table reaction in a dark place for 2 hours, adding 10 microliters of 0.1mol/L ethanolamine solution for uniformly mixing, carrying out shaking table reaction in a dark place for 30 minutes, centrifuging for 13000r/min and 10 minutes, discarding supernatant, uniformly mixing with 500 microliters of microsphere diluent (0.01mol/LPBS + 1% BSA), carrying out ultrasonic treatment for 5 minutes, carrying out shaking table reaction in a dark place for 1 hour, centrifuging for 13000r/min and 10 minutes, discarding supernatant, uniformly mixing with 200 microliters of microsphere diluent, carrying out ultrasonic treatment for 5 minutes, and storing at 2-8 ℃;
(4) taking out the microsphere morphine antibody conjugate, carrying out ultrasonic treatment for 5 minutes, taking 1 microliter of the ultrasonically treated microsphere antibody conjugate, adding 100 microliters of gold spraying diluent to prepare a gold spraying mixed solution, carrying out ultrasonic treatment on the diluted gold spraying mixed solution for 5 minutes again to fully and uniformly mix the microsphere antibody conjugate and prevent the microsphere antibody conjugate from aggregating, taking out the ultrasonically treated gold spraying mixed solution, carrying out gold spraying on the treated bonding pad at the speed of 1 microliter per centimeter by adopting an XYZ three-dimensional gold spraying scriber, and drying the bonded pad subjected to gold spraying for 1 hour at 45 ℃ in an oven for later use.
Preferably: the bonding pad soaking solution is Tris-HCl buffer solution with pH of 7.4 and 0.05mol/L, and the bonding pad soaking solution comprises 5% of trehalose, 0.5% of BSA and 0.1% of Tween;
preferably: the formula of the metal spraying diluent comprises the following components: 30mmol/l PBS, 0.05% PEG20000, 1% BSA, 0.01% SDS, 10% sucrose, 3% Tween.
Preferably: the sample pad is CB06, SB06, RB45, KB50, SB08, the combination pad is VL98, VL78, SAP-Z70, the NC membrane is JJ140, CN140, the absorbent paper is CH37, SH27, CH27, SH37, the offset plate is PVC, SM31-40, SMNF31-40, SMA 31-40.
Preferably: the sample pad is SB06, the combination pad is SAP-Z70, the NC film is JJ140, the absorbent paper is CH37, and the offset plate is PVC.
The preparation method of the morphine immunofluorescence chromatography rapid detection test paper strip comprises the following steps:
(1) treatment of the sample pad: placing the sample pad in the sample pad soaking solution, soaking for 10 minutes, taking out, placing in an oven, and drying at 37 ℃ for 2 hours for later use;
(2) treatment of the bonding pad: soaking the bonding pad in the bonding pad soaking solution for 2 hours, taking out the soaked bonding pad, and drying in a drying oven at 37 ℃ for 2 hours for later use;
(3) the preparation of the metal spraying diluent and the concrete operation steps of metal spraying are as follows: taking out the microsphere morphine antibody conjugate, carrying out ultrasonic treatment for 5 minutes, taking 1 microliter of the ultrasonic microsphere antibody conjugate, and adding 100 microliters of gold spraying diluent to prepare a gold spraying mixed solution; carrying out ultrasonic treatment on the diluted gold spraying mixed solution for 5 minutes again to fully and uniformly mix the microsphere antibody conjugate and prevent the microsphere antibody conjugate from aggregating, taking out the ultrasonic gold spraying mixed solution, carrying out gold spraying on the treated bonding pad at the speed of 1 microliter per centimeter by adopting an X Y Z three-dimensional gold spraying scriber, and drying the bonding pad subjected to gold spraying for 1 hour at 45 ℃ in an oven for later use;
(3) respectively diluting a T line and a C line on an NC membrane with MOP + BSA solution and an IgG solution by using a diluent of 1% sucrose +10mmol/l PBS, respectively diluting the two solutions to the concentrations of 0.5mg/ml and 1mg/ml, then scribing on the NC membrane at the speed of 1 microliter per centimeter by using an XYZ three-dimensional gold spraying scriber, and putting the scribed NC membrane into an oven to be dried for 15 minutes at 45 ℃ for later use;
(4) will handle sample pad, combination pad, NC membrane, absorbent paper and laminate on the PVC offset plate in proper order, the laminating order is: firstly pasting an NC film, pressing the NC film by a combination pad, pressing the combination pad by a sample pad, pressing the NC film by absorbent paper, overlapping the NC film and the sample pad with each other with the length of about 1mm, and cutting the test paper into test strips with the width of 4mm by a cutter after the pasting is finished.
Preferably: the formula of the sample pad soak solution is as follows: 30mmol/l PBS, 0.5% SDS, 1% BSA, 0.5% PVA; the formula of the diluent of the T, C line is as follows: 1% sucrose, 10 mmol/LPBS.
The invention also provides a method for detecting the morphine content in hair, the test strip prepared by the invention is used for detecting the morphine content in hair, and the specific steps are as follows
(1) Preparing a series of morphine standard solution with concentration gradient, adding 100 microliters of morphine standard solution to the test strip, running the test strip for 5 minutes, measuring T, C line peak by using a dry immunofluorescence detector and integrating peak areas to obtain a T/C value;
(2) and establishing a standard curve by taking the ratio of the T/C as an ordinate and the concentration of the morphine standard as an abscissa. Writing the drawn standard curve into an ID chip, introducing the ID chip into a fluorescence tester, and measuring the morphine content in the hair by using the introduced standard curve when measuring by using the tester;
(3) taking a proper amount of hair of a drug addict, cutting the hair into about 1mm in length, adding 1ml of hair lysate into the cut hair, shaking for 2-3 times, standing for 5 minutes, taking 100 microliters after 5 minutes, adding the 100 microliters onto a sample pad of a test strip, and after the hair runs for 5 minutes, adopting a micro-fluorometer to measure the morphine content and judge whether the hair is negative or positive.
The invention has the beneficial effects that:
the morphine immunofluorescence rapid detection test paper strip has the advantages of small volume, light weight, convenience in carrying, high analysis speed, simple operation steps and the like, and breaks through the limitation of the traditional detection method. Powerful technical support is provided for on-site rapid detection with huge sample size. Meanwhile, great convenience is brought to on-site drug investigation and detection for public security personnel.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Figure 1 is a standard curve plotted for morphine standards;
FIG. 2 is a particle size screening diagram of fluorescent microspheres;
FIG. 3 is a screening chart of the gold spraying dilution;
FIG. 4 is a screening diagram of the mating of the conjugate pad and the sample pad;
FIG. 5 is a screening chart of the gold spraying drying temperature;
FIG. 6 is a comparison graph of the drying effect of the bonding pad with and without soaking;
FIG. 7 is a graph of bond pad drying time screening;
FIG. 8 is a graph comparing the soaking and non-soaking effects of the sample pad;
FIG. 9 is a graph showing the results of T, C at line concentrations of 0.5mg/ml and 1mg/ml, respectively
FIG. 10 is a graph showing the results of T, C at line concentrations of 0.5mg/ml and 0.1mg/ml, respectively;
FIG. 11 is a graph showing the results of line T, C concentrations of 1mg/ml and 0.5mg/ml, respectively;
FIG. 12 is a graph showing the results of T, C at line concentrations of 1mg/ml and 0.2mg/ml, respectively;
FIG. 13 is a graph showing the results of T, C at line concentrations of 1mg/ml and 0.15mg/ml, respectively.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Examples 1 to 3
A morphine immunofluorescence chromatography rapid examination test paper strip comprises a sample pad, a combination pad, an NC membrane, absorbent paper and a rubber plate, and the preparation method can be carried out according to the following steps:
(1) activating the fluorescent microspheres: and (3) carrying out ultrasonic treatment on the fluorescent microspheres for 5 minutes, (wherein the fluorescent microspheres are respectively selected to be 300nm, 200nm and 100 nm). 100 microliter of the well-sonicated fluorescent microspheres are added with 1ml of 0.05mol/LMES (PH 5.0) (prepared by pure water) solution and mixed evenly. Centrifuging at 13000r/min for 20min, discarding the supernatant, adding 1ml of 0.05mol/LMES, mixing uniformly and ultrasonically treating for 5min. Centrifuging at 13000r/min for 20min, discarding the supernatant, adding 200 microliters of 0.05mol/LMES solution, mixing uniformly, and performing ultrasonic treatment for 5min. NHS10mg/ml was prepared with 0.05mol/LMES solution, and 15. mu.l was added to the sonicated fluorescent microspheres. Then 2. mu.l of EDC15mg/ml prepared with pure water was added to the above fluorescent microspheres. Wrapping the mixture with tinfoil paper and reacting for 15 minutes by a light-proof shaking table.
(2) The preparation of the fluorescent microsphere and morphine antibody conjugate comprises the steps of adding 20 microliters of morphine antibody per milligram of 100 microliters into activated fluorescent microspheres, uniformly mixing, carrying out shaking table reaction in a dark place for 2 hours, adding 10 microliters of 0.1mol/L ethanolamine solution, uniformly mixing, carrying out shaking table reaction in the dark place for 30 minutes, centrifuging for 13000r/min and 10 minutes, discarding supernatant, uniformly mixing with 500 microliters of microsphere diluent (0.01mol/LPBS + 1% BSA), carrying out ultrasonic treatment for 5 minutes, carrying out shaking table reaction in the dark place for 1 hour, centrifuging for 13000r/min and 10 minutes, and discarding supernatant. And uniformly mixing with 200 microliter of microsphere diluent, and carrying out ultrasonic treatment for 5min, and storing at 2-8 ℃.
(3) Treatment of the sample pad: placing a sample pad in a sample pad soaking solution for soaking for 10 minutes, taking out the sample pad, placing the sample pad in an oven for drying for 2 hours at 37 ℃, wherein the formula of the sample pad soaking solution is as follows: 30mmol/l PBS, 0.5% SDS, 1% BSA, 0.5% PVA.
(4) Treatment of the bonding pad: soaking the bonding pad in a bonding pad soaking solution for 2 hours, taking out the soaked bonding pad, and drying the bonding pad in an oven for 2 hours at 37 ℃ for later use, wherein the bonding pad soaking solution is a 0.05mol/L Tris-HCl buffer solution with the pH of 7.4, and comprises 5% of trehalose, 0.5% of BSA and 0.1% of Tween;
(5) the preparation of the metal spraying diluent and the concrete operation steps of metal spraying are as follows: taking out the microsphere morphine antibody conjugate, carrying out ultrasonic treatment for 5 minutes, taking 1 microliter of the ultrasonically treated microsphere antibody conjugate, adding 100 microliters of gold spraying diluent to prepare a gold spraying mixed solution, carrying out ultrasonic treatment on the diluted gold spraying mixed solution for 5 minutes again to fully and uniformly mix the microsphere antibody conjugate and prevent the microsphere antibody conjugate from aggregating, taking out the ultrasonically treated gold spraying mixed solution, carrying out gold spraying on the treated bonding pad at the speed of 1 microliter per centimeter by adopting an XYZ three-dimensional gold spraying scriber, and drying the bonded pad subjected to gold spraying for 1 hour at 45 ℃ in an oven for later use. (the metal spraying speed and the metal spraying width can be adjusted according to the requirement); the formula of the metal spraying diluent comprises the following components: 30mmol/l PBS, 0.05% PEG20000, 1% BSA, 0.01% SDS, 10% sucrose, 3% Tween;
(6) the T line and the C line on the NC membrane are respectively MOP + BSA solution and IgG solution, and the two are respectively; diluting the T, C diluted solution, wherein the concentration of the diluted solution is 0.5mg/ml and 1mg/ml respectively, then scribing on an NC film by using an XYZ three-dimensional metal spraying scriber at the speed of 1 microliter per centimeter, and putting the scribed NC film into an oven to be dried for 15 minutes at the temperature of 45 ℃ for later use, wherein the formula of the T, C diluted solution is as follows: 1% sucrose, 10 mmol/LPBS;
(7) will handle sample pad, combination pad, NC membrane, absorbent paper and laminate on the PVC offset plate in proper order, the laminating order is: firstly pasting an NC film, pressing the NC film by a combination pad, pressing the combination pad by a sample pad, and pressing the NC film by absorbent paper. The overlap length between each other is generally about 1 mm. After the completion of the attachment, the test paper was cut into test paper strips each having a width of 4mm by a cutter. (the T line on the NC film is habitually brought close to the conjugate pad during fitting and the C line is brought close to the absorbent paper, since then the values of X1/X2 correspond to the values of T/C during measurement, facilitating the drawing of a standard curve).
The method for detecting the morphine content in the hair by using the test strip prepared by the invention comprises the following specific steps
(1) Preparing a series of morphine standard solution with concentration gradient, adding 100 microliters of morphine standard solution to the test strip, running the test strip for 5 minutes, measuring T, C line peak by using a dry immunofluorescence detector and integrating peak areas to obtain a T/C value;
(2) and establishing a standard curve by taking the ratio of the T/C as an ordinate and the concentration of the morphine standard as an abscissa. Writing the drawn standard curve into an ID chip, introducing the ID chip into a fluorescence tester, and measuring the morphine content in the hair by using the introduced standard curve when measuring by using the tester;
(3) taking a proper amount of hair of a drug addict, cutting the hair into about 1mm in length, adding 1ml of hair lysate into the cut hair, shaking for 2-3 times, standing for 5 minutes, taking 100 microliters after 5 minutes, adding the 100 microliters onto a sample pad of a test strip, and after the hair runs for 5 minutes, adopting a micro-fluorometer to measure the morphine content and judge whether the hair is negative or positive.
The standard curve drawn for morphine standards is shown in figure 1, and the measured T/C values are shown in table 1.
TABLE 1 morphine standards assay data
| Morphine concentration (ng/ml) | T/C (ratio of T line peak area to C line peak area) |
| 0.1 | 1.324 |
| 0.2 | 1.161 |
| 0.5 | 0.8734 |
| 1 | 0.5396 |
| 2 | 0.4029 |
| 5 | 0.1639 |
| 10 | 0.0279 |
The particle size of different fluorescent microspheres is screened, the phenomenon that the agglomeration sensitivity of the microspheres is reduced can occur when the particle size of the microspheres is too large, and the centrifugal effect during marking can be influenced when the particle size of the microspheres is too small, so that the release effect of the gold spraying liquid of the bonding pad is poor. The microspheres of the same manufacturer with different particle sizes are selected for screening in the experiment, the result is shown in figure 2, and the screening result shows that the microspheres with the particle size of 300nm in Thermo Fisher company have good effect.
Examples 4 to 8
Basically the same as example 1, except that: the fluorescent microsphere is 300nm, and the formula of the gold spraying diluent is sequentially (1): 30mmol/l PBS, 0.05% PEG20000, 1% BSA, 0.01% SDS, 10% sucrose, 2% Tween.
(2): 30mmol/l PBS, 0.05% PEG20000, 1% BSA, 0.01% SDS, 10% sucrose, 0.5% Tween.
(3): 30mmol/l PBS, 0.05% PEG20000, 1% BSA, 0.01% SDS, 10% sucrose, 1% Tween.
(4): 30mmol/l PBS, 0.05% PEG20000, 1% BSA, 0.01% SDS, 10% sucrose, no Tween.
(5): 30mmol/l PBS, 0.05% PEG20000, 1% BSA, 0.01% SDS, 10% sucrose, 3% Tween.
The results are shown in FIG. 3, which shows that the effect of Tween content of 3% is best when the dilution is No. 1-5 metal spraying dilution from the top to the bottom.
Examples 9 to 15
Basically the same as example 1, except that: the fluorescent microsphere is 300nm, and the matching of the sample pad and the combination pad is as follows in sequence: (1) SB06 sample pad and SAP-Z70 conjugate pad. (2): SB06 sample pad and RB45 conjugate pad. (3): SB06 sample pad and RB65 conjugate pad. (4): SB06 sample pad and SB06 conjugate pad. (5): SB06 sample pad and VL78 conjugate pad. (6) SB06 sample pad and KB50 conjugate pad.
The results are shown in FIG. 4, which shows numbers 1-6 from top to bottom, and the comparison of the effects shows that the sample pad and the conjugate pad have the best effects of SB06 and SAP-Z70, respectively. Other matches all have the conditions of incomplete release and poor running board effect.
Examples 16 to 18
Basically the same as example 1, except that: the fluorescent microspheres are 300nm, and the drying temperature after gold spraying is (1)37 ℃ and drying for 1 hour; (2) drying for 1 hour at the temperature of 45 ℃; (3) drying at 50 ℃ for 1 hour. The specific result is shown in FIG. 5, which is numbered from top to bottom in sequence from number 1 to number 3. Fig. 5 shows that incomplete release of the conjugate pad occurs after drying at 37 degrees for 1 hour and drying at 50 degrees for 1 hour, the binding effect of the morphine antibody on the conjugate pad to T, C lines is poor, and the determination effect is poor. Although the effect of the 50-degree drying for 1 hour is improved relative to the 37-degree release and the running board, compared with the 45-degree drying for 1 hour, the incomplete release is not existed. Therefore, the optimal experimental condition is that the sample is dried for 1 hour at the temperature of 45 ℃ by screening.
Examples 19 to 20
Basically the same as example 1, except that: fluorescent microspheres with the size of 300nm, and (1) soaking the bonding pads; (2) no soaking is performed. Specific results fig. 6 shows that the run-out effect of the bonding pad soaking and non-soaking is sequentially performed from top to bottom, and the gold-sprayed liquid of the bonding pad is not substantially released after the bonding pad is not soaked and dried, so that the sensitivity of the measurement of the antibody which can be linearly bonded with T, C is greatly reduced.
Examples 21 to 23
Basically the same as example 1, except that: the fluorescent microspheres are 300nm, the drying temperature of the bonding pad after soaking is 45 ℃, the drying time is 2h, 1h and 15min, and the result is shown in fig. 7, wherein the left image in the figure is dried for 2 hours at 45 ℃ and 1 hour at 45 ℃ from top to bottom. The right graph shows that the mixture is dried for 15 minutes at 45 ℃. The drying effect at 45 degrees for 2 hours is not much different from the drying effect at 45 degrees for 1 hour, but the drying effect at 45 degrees for 1 hour is more efficient from the viewpoint of working aging. After the bonding pad is dried for 15 minutes at the temperature of 45 ℃, the gold spraying liquid of the bonding pad is not released, the bonding effect of T, C lines on an antibody on the bonding pad is poor, and a large error occurs in a measurement result.
Examples 24 to 25
Basically the same as example 1, except that: the fluorescent microspheres are 300nm, the soaking effect and the non-soaking effect of the sample pad are compared, the result is shown in fig. 8, the non-soaked sample pad and the soaked sample pad are sequentially arranged from top to bottom in the figure, the effect of the sample pad subjected to soaking pretreatment is better through the comparison effect, and the problems that the release of the gold spraying liquid on the combination pad is incomplete, the morphine content measurement is inaccurate and the like can be caused if the sample pad is not subjected to soaking pretreatment.
Examples 26 to 30
Basically the same as example 1, except that: the concentration of the fluorescent microsphere is 300nm, and the T, C linear concentration is 0.5mg/ml and 1mg/ml respectively; 0.5mg/ml, 0.1 mg/ml; 1mg/ml, 0.5 mg/ml; 1mg/ml, 0.2mg/ml, 1mg/ml, 0.15 mg/ml. The results are shown in FIGS. 9-13.
The T/C is obviously changed when morphine is dripped (namely the test strip has certain sensitivity to the concentration of the morphine), the peak of the T line is higher than the peak of the C line when a blank board is run, and the height has certain difference, so that the T/C is a value larger than zero when the blank board is run, when morphine is measured, the morphine antigen in a sample to be measured is combined with the antibody on the combination pad, the antibody amount capable of being combined with the T line antigen is greatly reduced, the peak of the T line is obviously reduced, the value of the T/C is obviously reduced (generally smaller than zero), and the test strip has higher sensitivity to the concentration of the morphine in the sample to be measured. The concentrations of T, C were set to satisfy the measurement sensitivity and to achieve a certain economic benefit by comparing the measured values and by an experimental protocol in which the concentrations were 1mg/ml and 0.15mg/ml, respectively, which is preferable from the viewpoint of economic benefit.
Test strip effect experiment
EXAMPLE 1 (test paper precision)
The test strip prepared in example 1 was used, morphine with a concentration of 1ng/ml was added to the test strip in 100. mu.l, and after running the plate for 5 minutes, the peak at line T, C was measured by a dry immunofluorescence detector and the peak area was integrated to obtain the T/C value, which was repeated 5 times, and the specific results are shown in Table 2.
TABLE 2 test paper strip precision test results
| Morphine concentration (1ng/ml) | Value of T/ |
| 1 | 0.5294 |
| 1 | 0.5466 |
| 1 | 0.5113 |
| 1 | 0.5523 |
| 1 | 0.5391 |
As shown in Table 2, the test strip of the present invention has high precision.
EXAMPLE 2 test paper specificity assay
The test strip prepared in example 1 was used, 1ng/ml of K powder, cannabis sativa and cocaine were used, purified water was used as a blank, 100 microliters of each was added to the test strip, after running the plate for 5 minutes, the peak at line T, C was measured by a dry immunofluorescence detector and the peak area was integrated, and then the value of T/C was obtained, and the specific results were repeated 5 times and are shown in table 3.
TABLE 3 test paper strip specificity test results
| Drug name | Value of T/C |
| Blank space | 2.135 |
| K powder | 1.932 |
| Cannabis sativa (Cannabis sativa L.) Linne | 2.194 |
| Cocaine | 2.094 |
As shown in Table 3, the test strip of the present invention has excellent specificity, and can rapidly distinguish different drugs.
EXAMPLE 3 test paper strip stability test
The test strip prepared in example 1 was used, morphine with a concentration of 1ng/ml was added to the test strip in 100 microliters, and after different running times, the peak at line T, C was measured by a dry immunofluorescence detector and the peak area was integrated to obtain the value of T/C, which was repeated 5 times, with the specific results shown in table 4.
Table 4 test paper strip stability test results
| Running time/min | Value of T/C (ten averaging per time measurement) |
| 5 | 0.3325 |
| 7 | 0.3075 |
| 9 | 0.2954 |
| 12 | 0.2933 |
| 14 | 0.2930 |
| 16 | 0.2914 |
| 18 | 0.2912 |
| 20 | 0.2902 |
As can be seen from Table 4, the test strip of the present invention has good stability.
Experimental example 4
Taking a proper amount of hair of a drug addict, cutting the hair into about 1mm in length, adding 1ml of hair lysate into the cut hair, shaking for 2-3 times, standing for 5 minutes, taking 100 microliters of the hair lysate after 5 minutes, adding the hair lysate to a sample pad of a test strip, and after the hair lysate runs for 5 minutes, adopting a micro-fluorometer to measure morphine content and judge whether the hair is negative or positive, wherein specific results are shown in table 5.
TABLE 5 test paper strips for measuring morphine content in hair samples
As can be seen from the above table, the morphine immunofluorescence rapid detection test paper strip has the advantages of small volume, light weight, portability, high analysis speed, simple operation steps and the like, and breaks the limitation of the traditional detection method.
Although embodiments of the present invention have been described above, it would be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the claims.
Claims (8)
1. A morphine immunofluorescence chromatography rapid examination test strip is characterized in that: the preparation method of the bonding pad comprises the following steps:
(1) placing the bonding pad in a bonding pad soaking solution for soaking for 2 hours, taking out the soaked bonding pad, and placing the bonding pad in an oven to be dried for 2 hours for later use under 45 ℃;
(2) activating the fluorescent microspheres: taking 100 microliters of the fluorescent microspheres, adding 1ml of 0.05mol/LMES (PH is 5.0) solution into the 100 microliters of the fluorescent microspheres, uniformly mixing, centrifuging for 13000r/min, 20min, discarding the supernatant, adding 1ml of 0.05mol/LMES, uniformly mixing, ultrasonically treating for 5min, centrifuging for 13000r/min, 20min, discarding the supernatant, adding 200 microliters of 0.05mol/LMES solution, uniformly mixing, ultrasonically treating for 5min, preparing NHS10mg/ml by using 0.05mol/LMES solution, taking 15 microliters, adding into the fluorescent microspheres, preparing EDC15mg/ml by using pure water, taking 2 microliters, adding into the fluorescent microspheres, wrapping the microspheres by using tinfoil, and reacting for 15min by using a light-shielding shaking table;
(3) preparing a fluorescent microsphere and morphine antibody conjugate: adding 20 microliters of morphine antibody per milligram into activated fluorescent microspheres for uniformly mixing, carrying out shaking table reaction in a dark place for 2 hours, adding 10 microliters of 0.1mol/L ethanolamine solution for uniformly mixing, carrying out shaking table reaction for 30 minutes in the dark place, centrifuging for 13000r/min and 10 minutes, discarding the supernatant, uniformly mixing with 500 microliters of microsphere diluent (0.01mol/LPBS + 1% BSA), carrying out ultrasonic treatment for 5 minutes, carrying out shaking table reaction in the dark place for 1 hour, centrifuging for 13000r/min and 10 minutes, discarding the supernatant, uniformly mixing with 200 microliters of microsphere diluent, and carrying out ultrasonic treatment for 5 minutes and storing at 2-8 ℃;
(4) taking out the microsphere morphine antibody conjugate, carrying out ultrasonic treatment for 5 minutes, taking 1 microliter of the ultrasonically treated microsphere antibody conjugate, adding 100 microliters of gold spraying diluent to prepare a gold spraying mixed solution, carrying out ultrasonic treatment on the diluted gold spraying mixed solution for 5 minutes again to fully and uniformly mix the microsphere antibody conjugate and prevent the microsphere antibody conjugate from aggregating, taking out the ultrasonically treated gold spraying mixed solution, carrying out gold spraying on the treated bonding pad at the speed of 1 microliter per centimeter by adopting an XYZ three-dimensional gold spraying scriber, and drying the bonded pad subjected to gold spraying for 1 hour at 45 ℃ in an oven for later use.
2. The morphine immunofluorescence chromatography rapid-examination test strip as claimed in claim 1, wherein: the soaking solution of the bonding pad is Tris-HCl buffer solution with pH 7.4 and 0.05mol/L, and the Tris-HCl buffer solution comprises 5% of trehalose, 0.5% of BSA and 0.1% of Tween.
3. The morphine immunofluorescence chromatography rapid-examination test strip as claimed in claim 1, wherein: the formula of the metal spraying diluent comprises the following components: 30mmol/l PBS, 0.05% PEG20000, 1% BSA, 0.01% SDS, 10% sucrose, 3% Tween.
4. The morphine immunofluorescence chromatography rapid-examination test strip as claimed in claim 1, wherein: the sample pad is CB06, SB06, RB45, KB50, SB08, the combination pad is VL98, VL78, SAP-Z70, the NC membrane is JJ140, CN140, the absorbent paper is CH37, SH27, CH27, SH37, the offset plate is PVC, SM31-40, SMNF31-40, SMA 31-40.
5. The morphine immunofluorescence chromatography rapid-examination test strip as claimed in claim 4, wherein: the sample pad is SB06, the combination pad is SAP-Z70, the NC film is JJ140, the absorbent paper is CH37, and the offset plate is PVC.
6. A method for preparing a morphine immunofluorescence chromatography rapid test strip as claimed in any one of claims 1 to 5, wherein: the method comprises the following steps:
(1) treatment of the sample pad: placing the sample pad in the sample pad soaking solution, soaking for 10 minutes, taking out, placing in an oven, and drying at 37 ℃ for 2 hours for later use;
(2) treatment of the bonding pad: soaking the bonding pad in the bonding pad soaking solution for 2 hours, taking out the soaked bonding pad, and drying in a drying oven at 37 ℃ for 2 hours for later use;
(3) the preparation of the metal spraying diluent and the concrete operation steps of metal spraying are as follows: taking out the microsphere morphine antibody conjugate, carrying out ultrasonic treatment for 5 minutes, taking 1 microliter of the ultrasonic microsphere antibody conjugate, and adding 100 microliters of gold spraying diluent to prepare a gold spraying mixed solution; carrying out ultrasonic treatment on the diluted gold spraying mixed solution for 5 minutes again to fully and uniformly mix the microsphere antibody conjugate and prevent the microsphere antibody conjugate from aggregating, taking out the ultrasonic gold spraying mixed solution, carrying out gold spraying on the treated bonding pad at the speed of 1 microliter per centimeter by adopting an XYZ three-dimensional gold spraying scribing instrument, and drying the bonding pad subjected to gold spraying for 1 hour at 45 ℃ in an oven for later use;
(3) respectively diluting a T line and a C line on an NC membrane with MOP + BSA solution and an IgG solution by using a diluent of 1% sucrose +10mmol/l PBS, respectively diluting the two solutions to the concentrations of 0.5mg/ml and 1mg/ml, then scribing on the NC membrane at the speed of 1 microliter per centimeter by using an XYZ three-dimensional gold spraying scriber, and putting the scribed NC membrane into an oven to be dried for 15 minutes at 45 ℃ for later use;
(4) will handle sample pad, combination pad, NC membrane, absorbent paper and laminate on the PVC offset plate in proper order, the laminating order is: firstly pasting an NC film, pressing the NC film by a combination pad, pressing the combination pad by a sample pad, pressing the NC film by absorbent paper, overlapping the NC film and the sample pad with each other with the length of about 1mm, and cutting the test paper into test strips with the width of 4mm by a cutter after the pasting is finished.
7. The method for preparing the morphine immunofluorescence chromatography rapid-examination test paper strip as claimed in claim 6, wherein: the formula of the sample pad soak solution is as follows: 30mmol/l PBS, 0.5% SDS, 1% BSA, 0.5% PVA; the formula of the diluent of the T, C line is as follows: 1% sucrose, 10 mmol/LPBS.
8. A method for detecting the morphine content in hair by using the test strip prepared by the method of claim 6 comprises the following steps
(1) Preparing a series of morphine standard solution with concentration gradient, adding 100 microliters of morphine standard solution to the test strip, running the test strip for 5 minutes, measuring T, C line peak by using a dry immunofluorescence detector and integrating peak areas to obtain a T/C value;
(2) and establishing a standard curve by taking the ratio of the T/C as an ordinate and the concentration of the morphine standard as an abscissa. Writing the drawn standard curve into an ID chip, introducing the ID chip into a fluorescence tester, and measuring the morphine content in the hair by using the introduced standard curve when measuring by using the tester;
(3) taking a proper amount of hair of a drug addict, cutting the hair into about 1mm in length, adding 1ml of hair lysate into the cut hair, shaking for 2-3 times, standing for 5 minutes, taking 100 microliters after 5 minutes, adding the 100 microliters onto a sample pad of a test strip, and after the hair runs for 5 minutes, adopting a micro-fluorometer to measure the morphine content and judge whether the hair is negative or positive.
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