CN111366720B - Fluorescent immunoassay analyzer calibration card and preparation method thereof - Google Patents
Fluorescent immunoassay analyzer calibration card and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a fluorescent immunoassay instrument calibration card and a preparation method thereof, wherein the bottom layer of the calibration card is a supporting plate, a fluorescent strip and a quality control strip are fixed on the supporting plate, the fluorescent strip and the quality control strip both contain fluorescent compositions, and the fluorescent compositions comprise porous resin, filler, silane coupling agent and LED fluorescent powder. The fluorescent substance on the calibration card can meet the calibration of a plurality of wave band fluorescent analyzers, after 40 times of excitation, the fluorescent signal can still keep a stable fluorescent value, the coefficient of variation CV is less than or equal to 0.5%, the fluorescent substance can be stored for more than 12 months for a long time, the stability is good, the sensitivity is high, the storage time is greatly prolonged, the detection efficiency and the reliability of the result are improved, the application range is enlarged, and therefore the problems of instability of the fluorescent substance and single excitation wavelength are solved. The fluorescent analyzer has potential popularization and application values in factory inspection of the fluorescent analyzer and quality control in the using process.
Description
Technical Field
The invention relates to the technical field of instrument calibration, in particular to a fluorescent immunoassay instrument calibration card and a preparation method thereof.
Background
Because of high fluorescence sensitivity and wider detection range, the fluorescent dye can be widely applied to the field of medical inspection, can be used for marking substances to be detected, the method can be used for quantitatively analyzing substances to be detected, in particular to analyze low-concentration or trace substances in biological samples. However, due to the high sensitivity and wide detection range of the fluorescence analyzer, the fluorescence analyzer is often affected by factors such as stability of the sensor, life of the electronic component, and external environment (temperature, humidity or noise), and the result of the fluorescence analyzer is greatly fluctuated to some extent, so as to interfere with the test result. It is therefore of great importance to carry out quality testing of the fluorescence analysis instrument prior to its use.
Current fluorescence analyzers face the inability to standardize calibration measurement methods. The fluorescent substances for biological labeling mostly adopt fluorescein such as FITC, phycoerythrin, quantum dots and the like, firstly, after the fluorescein is excited for a plurality of times, the fluorescent signal is unstable, and fluorescence bleaching easily occursWhite, resulting in a decay of the fluorescent signal; secondly, the fluorescence wavelength is single, and instruments with different receiving wavelengths need to find different wavelengths, so that the operation is complex. As disclosed in patent CN 104614357A, a method for preparing a fluorescent standard card is disclosed, wherein the fluorescent material of the standard card is white phenol N- (5-chloro-2-methoxyphenyl) -3-hydroxy-2-naphthamide and Ba 3 MgSi 2 O 8 ：Eu 2+ ，Mn 2+ And NaOH is used for dissolving two fluorescent substances, white phenol forms a sodium salt of the phenol in a sodium hydroxide solution, but the sodium salt of the phenol is easy to hydrolyze in the solution and is easy to oxidize in the air, so that the influence factor in the preparation process is large. Patent CN107037215a discloses a quality control detection card for a fluorescence immunochromatography instrument, wherein a fluorescence microsphere solution is marked on a nitrocellulose membrane, but the nitrocellulose membrane is not easy to store for a long time due to poor stability, and the preparation process is complicated.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a fluorescent immunoassay analyzer calibration card (quality control card) and a preparation method thereof, which solve the problems of poor stability of fluorescent substances, single fluorescence excitation wavelength, complex operation and the like of the existing fluorescent standard card.
In order to solve the technical problems, the invention adopts the following technical scheme: a bottom layer of the calibration card is a supporting plate, a fluorescent strip and a quality control strip are fixed on the supporting plate, the fluorescent strip and the quality control strip both contain fluorescent compositions, and the fluorescent compositions comprise porous resin, filler, (nano) silane coupling agent and LED fluorescent powder.
Thus, the nanoscale silane coupling agent can form a compact three-dimensional reticular crystal structure with the porous resin, and the oxygen atoms in the LED fluorescent powder and the polyphenol rigid structure in the porous resin form a stronger interaction force, so that the LED fluorescent powder presents orderly arranged crystals, the long-term consistency of the paths of excitation light and emitted light can be ensured, the fluorescence sensitivity and stability are greatly improved, and the storage life is prolonged.
Further, the supporting plate is made of one or more materials of PVC, ABS, PVDF, PP and silica gel.
Further, the porous resin is one or more of polyurethane rubber, polyvinyl chloride resin, polyacrylic resin and epoxy resin.
Further, the nanoscale silane coupling agent is one or more of 3-chloropropyl trimethoxysilane, butadienyl phenyl dimethoxy silane and p-aminophenyl dimethoxy chlorosilane.
Further, the LED fluorescent powder is red LED fluorescent powder or/and yellow LED fluorescent powder.
Preferably, the red LED phosphor is Sm and Eu doped nitrides, including but not limited to SmOSi 2 O 2 N 2 :Yb,/Eu、NaGd(WO 4 ) 2 Eu, sm and SrIn 2 O 4 ∶Eu 3+ ,Sm 3+ Wherein the maximum emission wavelength range of the red LED fluorescent powder is 590 nm-650 nm.
Preferably, the yellow LED phosphor is a nitride doped with Sr and Eu, including but not limited to Sr 7 AlLi(PO 4 ) 7 Eu 2 ,Ce( 3+ ) And Sr 3 SiO 5 :Eu 2+ Wherein the maximum emission wavelength range of the yellow LED fluorescent powder is 530 nm-590 nm.
Thus, the red LED fluorescent powder and the yellow LED fluorescent powder can contain the calibration of fluorescent signals of a plurality of wave bands of the fluorescent analyzer, so that the standard card is suitable for different fluorescent immunoassays.
The mass ratio of the porous resin to the total amount of the nano-scale silane coupling agent to the LED fluorescent powder to the filler is 7-9:0.5-2:0.05-0.6.
Further, the filler is one or more of fumed silica, and alumina.
Thus, the filler can uniformly disperse the LED fluorescent powder in the pores of the porous resin, and fully react with the porous resin under the catalysis of the nano-scale silane coupling agent.
The LED fluorescent powder accounts for 0.01% -4% of the total amount of the fluorescent composition.
The preparation method of the fluorescent immunoassay analyzer calibration card specifically comprises the following steps:
1) Uniformly stirring and mixing the porous resin, the filler and the LED fluorescent powder in a stirrer, slowly dripping a nano-scale silane coupling agent dissolved in acetone at 60 ℃, continuously stirring and reacting for 5-30 min, and carrying out ice water bath for 2-5 min to form uniform and stable porous resin-LED fluorescent powder polycrystal colloid;
2) Scribing the porous resin-LED fluorescent powder polycrystal colloid obtained in the step 1) on a supporting plate by using a scribing instrument to form a uniform fluorescent belt and a quality control belt, and naturally drying for later use;
3) And cutting the dried support plate into fluorescent card strips with proper width by using a slitter, and embedding the fluorescent card strips into or adhering the fluorescent card strips onto the plastic card shell.
The detection method of the fluorescent immunoassay analyzer calibration card comprises the following steps:
s1: drawing a standard curve by taking the concentration of fluorescent substances as an abscissa and the fluorescence area as an ordinate; the concentration of the fluorescent substance is the ratio of the mass of the LED fluorescent powder to the total mass of the fluorescent composition;
s2: placing the calibration card in a detection window of a fluorescence analysis instrument to be detected, calculating the ratio of the fluorescence area of the obtained fluorescence band to the fluorescence area of the quality control band to obtain a test value, and comparing the test value with a standard value obtained under the corresponding fluorescent substance concentration in a standard curve;
s3: setting a relative deviation N, and if the relative deviation result of the test value and the standard value is smaller than N, the measurement result of the instrument is reliable and can be normally used; if the relative deviation result of the test value and the standard value is larger than N, the reliability of the measurement result of the instrument is low, and the instrument cannot be used normally; wherein N is more than 0 and less than or equal to 5 percent.
Compared with the prior art, the invention has the following beneficial effects:
1. the fluorescent composition on the calibration card can meet the calibration of the fluorescent analyzers with multiple wave bands, after 40 times of excitation continuously, the fluorescent signal can still keep a stable fluorescent value, the variation coefficient CV is less than or equal to 0.5%, the fluorescent analyzer calibration card can be stored for 12 months for a long time, has good stability and high sensitivity, greatly prolongs the storage time, improves the detection efficiency and the reliability of the result, expands the application range, and solves the problems of unstable fluorescent substances and single excitation wavelength. The fluorescent analyzer has potential popularization and application values in factory inspection of the fluorescent analyzer and quality control in the using process.
2. The preparation method of the fluorescent analyzer calibration card has the advantages of simple operation, high detection speed, wide and easily obtained raw material sources, low cost and easy realization of large-scale industrial production, provides new theoretical guidance and technical support for preparing the calibration card with high stability and multiple wave bands, and has good economic benefit.
Drawings
FIG. 1 is a schematic diagram of the structure of a calibration card for a fluorescence analyzer.
Detailed Description
The present invention will be described in further detail with reference to examples. The experimental procedure described in the examples is not particularly specified, i.e. it is carried out in a conventional manner.
1. Fluorescent immunity analyzer calibration card
As shown in fig. 1, the bottom layer of the calibration card is a supporting plate 3, and a fluorescent strip (T line) 1 and a quality control strip (C line) 2 are fixed on the supporting plate 3 and embedded into a plastic card case 4.
2. Preparation method of fluorescent immunoassay instrument calibration card
1. Preparation of samples
And (3) uniformly stirring and mixing the porous resin, the filler and the LED fluorescent powder in a stirrer, slowly dripping acetone at 60 ℃ to dissolve the nano-scale silane coupling agent, continuously stirring and reacting for 5min, and carrying out ice water bath for 2min to form porous resin-LED fluorescent powder polycrystal colloid with different LED fluorescent powder contents for standby. Wherein the fluorescent compositions in each sample are shown in table 1.
TABLE 1 content of principal Components of each sample
2. Scribing fixture
Adjusting scribing parameters of a X, Y, Z axis of a scribing instrument, wherein the Z axis is 70mm, the X axis is 50mm, the Y axis moving distance is 250mm, the Y axis moving speed is 50mm/s, the pressure is 0.2MPa, injecting porous resin-LED fluorescent powder polycrystal colloid prepared by a sample 4 into a sample tube, opening an air pump and a starting button, placing a PVC plate on a platform of the scribing instrument, and starting the scribing button to obtain a quality control belt, namely a C line; then adjusting the scribing parameters of X, Y, Z axes of a scribing instrument, setting the Z axis position to 70mm, setting the X axis position to 55mm, setting the Y axis moving distance to 250mm, setting the Y axis moving speed to 50mm/s, and setting the pressure to 0.2MPa, injecting the porous resin-LED fluorescent powder polycrystal colloid prepared by the sample 1 into a sample tube, starting a scribing button, and scribing on the same PVC plate to obtain a fluorescent strip, namely a T line; wherein, the linewidth of fluorescence area and matter accuse area is 1mm, and fluorescence area and matter accuse area are 5mm apart. After natural drying, obtaining a fluorescent immunoassay analyzer calibration card I for standby;
adjusting scribing parameters of a X, Y, Z axis of a scribing instrument, wherein the Z axis is 70mm, the X axis is 50mm, the Y axis moving distance is 250mm, the Y axis moving speed is 50mm/s, the pressure is 0.2MPa, injecting porous resin-LED fluorescent powder polycrystal colloid prepared by a sample 4 into a sample tube, opening an air pump and a starting button, placing a PVC plate on a platform of the scribing instrument, and starting the scribing button to obtain a quality control belt, namely a C line; then adjusting the scribing parameters of X, Y, Z axes of a scribing instrument, setting the Z axis position to 70mm, setting the X axis position to 55mm, setting the Y axis moving distance to 250mm, setting the Y axis moving speed to 50mm/s, setting the pressure to 0.2MPa, injecting the porous resin-LED fluorescent powder polycrystal colloid prepared by the sample 2 into a sample tube, starting a scribing button, and scribing on the same PVC plate to obtain a fluorescent band, namely a T line, wherein the line width of the fluorescent band and the quality control band is 1mm, and the distance between the fluorescent band and the quality control band is 5mm; after natural drying, obtaining a fluorescent immunoassay analyzer calibration card II for standby;
adjusting scribing parameters of a X, Y, Z axis of a scribing instrument, wherein the Z axis is 70mm, the X axis is 50mm, the Y axis moving distance is 250mm, the Y axis moving speed is 50mm/s, the pressure is 0.2MPa, injecting porous resin-LED fluorescent powder polycrystal colloid prepared by a sample 4 into a sample tube, opening an air pump and a starting button, placing a PVC plate on a platform of the scribing instrument, and starting the scribing button to obtain a quality control belt, namely a C line; then adjusting the scribing parameters of X, Y, Z axes of a scribing instrument, setting the Z axis position to 70mm, setting the X axis position to 55mm, setting the Y axis moving distance to 250mm, setting the Y axis moving speed to 50mm/s, setting the pressure to 0.2MPa, injecting the porous resin-LED fluorescent powder polycrystal colloid prepared by the sample 3 into a sample tube, starting a scribing button, and scribing on the same PVC plate to obtain a fluorescent band, namely a T line, wherein the line width of the fluorescent band and the quality control band is 1mm, and the distance between the fluorescent band and the quality control band is 5mm; after natural drying, obtaining a fluorescent immunoassay analyzer calibration card III for standby;
adjusting scribing parameters of a X, Y, Z axis of a scribing instrument, wherein the Z axis is 70mm, the X axis is 50mm, the Y axis moving distance is 250mm, the Y axis moving speed is 50mm/s, the pressure is 0.2MPa, injecting porous resin-LED fluorescent powder polycrystal colloid of a sample 4 into a sample tube, opening an air pump and a starting button, placing a PVC plate on a platform of the scribing instrument, and starting the scribing button to obtain a quality control belt, namely a C line; then adjusting the scribing parameters of X, Y, Z axes of a scribing instrument, setting the Z axis position to 70mm, setting the X axis position to 55mm, setting the Y axis moving distance to 250mm, setting the Y axis moving speed to 50mm/s, setting the pressure to 0.2MPa, injecting the porous resin-LED fluorescent powder polycrystal colloid prepared by the sample 4 into a sample tube, starting a scribing button, and scribing on the same PVC plate to obtain a fluorescent band, namely a T line, wherein the line width of the fluorescent band and the line width of the quality control band are 1mm, and the distance between the fluorescent band and the quality control band is 5mm; after natural drying, obtaining a fluorescent immunoassay analyzer calibration card IV for standby;
adjusting scribing parameters of a X, Y, Z axis of a scribing instrument, wherein the Z axis is 70mm, the X axis is 50mm, the Y axis moving distance is 250mm, the Y axis moving speed is 50mm/s, the pressure is 0.2MPa, injecting porous resin-LED fluorescent powder polycrystal colloid prepared by a sample 4 into a sample tube, opening an air pump and a starting button, placing a PVC plate on a platform of the scribing instrument, and starting the scribing button to obtain a quality control belt, namely a C line; then adjusting the scribing parameters of X, Y, Z axes of a scribing instrument, setting the Z axis position to 70mm, setting the X axis position to 55mm, setting the Y axis moving distance to 250mm, setting the Y axis moving speed to 50mm/s, setting the pressure to 0.2MPa, injecting the porous resin-LED fluorescent powder polycrystal colloid prepared by the sample 5 into the sample tube, starting a scribing button, and scribing on the same PVC plate to obtain a fluorescent band, namely a T line, wherein the line width of the fluorescent band and the quality control band is 1mm, and the distance between the fluorescent band and the quality control band is 5mm; after natural drying, obtaining a fluorescent immunoassay analyzer calibration card V for standby;
3. slitting and preserving
And sequentially placing the calibration cards of the fluorescence immunoassay analyzer into a slitter, slitting according to a start button to obtain clamping strips with the width of 4mm, placing the clamping strips into a plastic clamping shell, pressing the clamping strips into the shell for detection, and placing the clamping strips after detection of the pressing shell into an aluminum foil bag for sealing and preservation.
4. Detection of
The calibration cards are sequentially put into a fluorescence analyzer for detection, and each calibration card Q is sequentially calculated T And Q is equal to C The same fluorescent calibration card was tested 40 more times in succession to obtain stability data for the calibration card, the results are shown in table 2.
TABLE 2 relative deviation of fluorescence calibration cards and fluorescence stability test results
As can be seen from Table 2, the relative deviation between the detection value and the standard value is below 5%, which indicates that the measurement result of the fluorescence instrument is reliable, and the instrument can be used normally. And (3) carrying out 40 times of repeated tests on different prepared calibration cards respectively, wherein the average value of the obtained relative deviation between the test value and the first test value is less than 0.5%, and the maximum relative deviation is less than or equal to 0.5%, so that the fluorescent signal tested by the calibration card is stable and has no fluorescent bleaching phenomenon.
And (3) placing the prepared standard cards in an aluminum foil bag for sealing and storing at 37 ℃, respectively taking out 4 standard cards at 4 months, 6 months, 8 months and 12 months, respectively detecting 2 normal fluorescent instruments and 2 abnormal fluorescent instruments, and detecting the stability of the standard cards. In the test, the relative deviation between all the test values and the standard values is less than 5%, and the result shows that the effective retention period of the standard card under the normal temperature condition is at least 12 months, and the fluorescent composition has high stability.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (7)
1. The fluorescent immunoassay instrument calibration card is characterized in that the bottom layer of the calibration card is a supporting plate, a fluorescent strip and a quality control strip are fixed on the supporting plate, the fluorescent strip and the quality control strip both contain fluorescent compositions, and the fluorescent compositions comprise porous resin, filler, nanoscale silane coupling agent and LED fluorescent powder;
the porous resin is one or more of polyurethane rubber, polyvinyl chloride resin, polyacrylic resin and epoxy resin;
the nanoscale silane coupling agent is one or more of 3-chloropropyl trimethoxysilane, butadienyl phenyl dimethoxy silane and p-aminophenyl dimethoxy chlorosilane;
the LED fluorescent powder is red LED fluorescent powder or/and yellow LED fluorescent powder; wherein the red LED fluorescent powder is Sm and Eu doped nitride and comprises SmOSi 2 O 2 N 2 ：Yb,Eu、NaGd(WO 4 ) 2 Eu, sm and SrIn 2 O 4 :Eu 3+ ,Sm 3+ The maximum emission wavelength range of the red LED fluorescent powder is 650 nm-650 nm; the yellow LED fluorescent powder is nitride doped with Sr and Eu and contains Sr 7 AlLi(PO 4 ) 7 Eu 2 :Ce 3+ And Sr 3 SiO 5 :Eu 2+ The maximum emission wavelength range of the yellow LED fluorescent powder is 530 nm-780 nm.
2. The calibration card of claim 1, wherein the support plate is made of one or more of PVC, ABS, PVDF, PP and silica gel.
3. The fluoroimmunoassay instrument calibration card of claim 1, wherein the filler is one or more of fumed silica, silica and alumina.
4. The calibration card of the fluorescence immunoassay analyzer according to claim 1, wherein the mass ratio of the porous resin, the nanoscale silane coupling agent to the total amount of the LED fluorescent powder and the filler is 7-9:0.5-2:0.05-0.6.
5. The calibration card of claim 1, wherein the LED phosphor comprises 0.01% -4% of the total amount of the fluorescent composition.
6. The method for preparing the calibration card for the fluorescence immunoassay analyzer according to any one of claims 1 to 5, which is characterized by comprising the following steps:
1) Uniformly stirring and mixing the porous resin, the filler and the LED fluorescent powder in a stirrer, slowly dripping a nano-scale silane coupling agent dissolved in acetone at 60 ℃, continuously stirring and reacting for 5-30 min, and carrying out ice water bath for 2-5 min to form uniform and stable porous resin-LED fluorescent powder polycrystalline colloid;
2) Scribing the porous resin-LED fluorescent powder polycrystal colloid obtained in the step 1) on a supporting plate by using a scribing instrument to form a uniform fluorescent belt and a quality control belt, and naturally drying for later use;
3) And cutting the dried support plate into fluorescent card strips with proper width by using a slitter, and embedding the fluorescent card strips into or adhering the fluorescent card strips onto the plastic card shell.
7. The method for detecting a calibration card of a fluorescence immunoassay analyzer according to any one of claims 1 to 5, wherein: the method comprises the following steps:
s1: drawing a standard curve by taking the concentration of fluorescent substances as an abscissa and the fluorescence area as an ordinate;
s2: placing the calibration card in a detection window of a fluorescence analysis instrument to be detected, calculating the ratio of the fluorescence area of the obtained fluorescence band to the fluorescence area of the quality control band to obtain a test value, and comparing the test value with a standard value obtained under the corresponding fluorescent substance concentration in a standard curve;
s3: setting a relative deviation N, and if the relative deviation result of the test value and the standard value is smaller than N, the measurement result of the instrument is reliable and can be normally used; if the relative deviation result of the test value and the standard value is larger than N, the reliability of the measurement result of the instrument is low, and the instrument cannot be used normally; wherein N is more than 0 and less than or equal to 5 percent.
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| JP2015152306A (en) * | 2014-02-10 | 2015-08-24 | 古河電気工業株式会社 | Fluorescence immuno-chromatography and detector thereof |
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| US9726605B2 (en) * | 2013-03-14 | 2017-08-08 | Furukawa Electric Co., Ltd. | Fluorescence immuno-chromatography, kit and test strip for the same |
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| CN105358981A (en) * | 2013-07-25 | 2016-02-24 | 古河电气工业株式会社 | Test strip for immunochromatography, developing fluid used therefor, and immunochromatography using same |
| JP2015152306A (en) * | 2014-02-10 | 2015-08-24 | 古河電気工業株式会社 | Fluorescence immuno-chromatography and detector thereof |
| CN107037215A (en) * | 2016-12-31 | 2017-08-11 | 必欧瀚生物技术（合肥）有限公司 | A kind of fluorescence immune chromatography instrument Quality Control detection card, detection suit, its preparation method and detection method |
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