Fluorescent calibration card and preparation method thereof

Abstract

A disclosed fluorescence calibration card comprises a card shell internally provided with a calibration strip in a clamping manner, the calibration strip comprises a supporting plate, a nitrocellulose membrane and absorbent paper, the nitrocellulose membrane and the absorbent paper are located on the supporting plate and are in end-to-end lap joint, a fluorescence band is fixed on the nitrocellulose membrane, and the fluorescence band comprises a test band and a quality control band; according to the fluorescent band, fluorescent liquid is printed on a nitrocellulose membrane through a silk-screen printing method, the fluorescent liquid comprises LED fluorescent powder and a diluent, the diluent comprises water-based color matching paint and double distilled water, and the invention discloses a preparation method of the calibration card. The water-based toning paint and the double distilled water are blended into the diluent for adjusting the concentration of the LED fluorescent powder, and the fluorescent tape is printed by adopting a silk-screen printing technology, so that the fluorescence sensitivity and stability are greatly improved, and the storage life is prolonged.

Description

Technical Field

[0001] The invention relates to the technical field of immunological detection devices, and in particular to a fluorescence calibration card and a preparation method thereof. Background Art

[0002] Fluorescence immunoassay analyzer is a device used to detect and analyze specific molecules in biological samples. Its principle is to use fluorescent substances to mark the substances to be tested, and then collect the fluorescent signals emitted by the substances to be tested to perform quantitative analysis of the substances to be tested, especially for the analysis of low-concentration or trace substances in biological samples. Due to the high sensitivity and wide detection range of fluorescence immunoassay analyzers, they are easily affected by the instrument itself (such as sensor stability, electronic component life, etc.) and the external environment (temperature, humidity or noise, etc.) during use, and the instrument needs to be calibrated frequently using fluorescence calibration cards.

[0003] Existing fluorescence calibration cards have the problems of single fluorescence wavelength and fluorescence signal attenuation. For example, Chinese patent CN202110765213.3 discloses a method for preparing a fluorescent microsphere calibration card, which adds fluorescein and a coupling agent to microspheres to prepare a fluorescent microsphere solution, and then uses a film scribing instrument to scribble the fluorescent microsphere solution diluted to different multiples to form C lines and T lines. However, in this scheme, after multiple excitations of the fluorescent microspheres, the fluorescence signal is unstable, and fluorescence bleaching is prone to occur, which leads to fluorescence signal attenuation and inaccurate continuous measurement results; and the fluorescence wavelength excited by the fluorescent microspheres is single, and fluorescent immunoassay analyzers with different receiving wavelengths need to find microspheres with different wavelengths, which is cumbersome to operate. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a fluorescence calibration card and a preparation method thereof, so as to solve the problems of single fluorescence wavelength and fluorescence signal attenuation existing in the existing fluorescence calibration card.

[0005] The present invention adopts the following technical solutions:

[0006] A fluorescent calibration card includes a card shell with a calibration strip clamped inside, the calibration strip includes a support plate and a nitrocellulose membrane and absorbent paper located on the support plate and overlapped end to end, a fluorescent tape is fixed on the nitrocellulose membrane, the fluorescent tape includes a test tape and a quality control tape, the fluorescent tape is screen-printed to print fluorescent liquid on the nitrocellulose membrane, the fluorescent liquid includes LED fluorescent powder and a diluent, and the diluent includes water-based color varnish and double distilled water.

[0007] Furthermore, the support plate is made of any one or more materials selected from the group consisting of polyvinyl chloride, acrylonitrile-butadiene-styrene copolymer, polyvinylidene fluoride, polypropylene and silica gel.

[0008] Furthermore, the concentration of the LED fluorescent powder in the fluorescent liquid is 0.3 mg / ml to 70 mg / ml.

[0009] Furthermore, the LED phosphor is any one of red LED phosphor, yellow LED phosphor, orange LED phosphor, green LED phosphor and blue LED phosphor.

[0010] Furthermore, the red LED phosphor is a nitride doped with Sm and Eu, comprising SmOSi 2 O 2 N 2 :Yb, Eu, NaGd(WO 4 ) 2 :Eu, Sm and SrIn 2 O 4 :Eu 3+ , Sm 3+ , where the maximum emission wavelength range of red LED phosphor is 590nm~650nm;

[0011] The yellow LED phosphor is a nitride doped with Sr and Eu, including Sr 7 AlLi(PO 4 ) 7 Eu 2 :Ce 3+ and Sr 3 SiO 5 :Eu 2+ , where the maximum emission wavelength range of yellow LED phosphor is 530nm ~ 590nm.

[0012] Furthermore, the proportion of water-based tinting paint in the diluent is 50% to 80%.

[0013] A method for preparing a fluorescence calibration card, the method is used to prepare the fluorescence calibration card as described above, comprising the following steps:

[0014] S1. Making film negatives: setting a straight line of drug film on two film drawings as the test strip and the quality control strip respectively;

[0015] S2. Making the screen: evenly apply diazonium salt photosensitive glue on the stretched screen, stick the prepared film on the photosensitive glue, and expose it in a vacuum exposure machine;

[0016] S3, preparing water-based tinting paint and double distilled water in different proportions, stirring at room temperature for 2 hours to prepare diluents with different viscosities;

[0017] S4, using a diluent to prepare a plurality of fluorescent solutions with different concentration gradients;

[0018] S5. Use a screen printer to print 10.0 mg / ml fluorescent liquid on the quality control strip of cellulose acetate membrane, and print other fluorescent liquids of different concentration gradients on the test strip of cellulose acetate membrane, and dry them at 45° C. for 2 hours to obtain fluorescent calibration strips with different ratios of the peak area of ​​the test strip to the peak area of ​​the quality control strip;

[0019] S6. Cut the fluorescence calibration strips into card strips of the same width, and then put the card strips into a plastic card shell to make a fluorescence calibration card.

[0020] Furthermore, the linear medicine film is 0.8 mm wide and 20-25 cm long, and two linear medicine films are at the same height of two film drawings, with a left-right spacing of 6 mm.

[0021] Furthermore, the ratio of the water-based tinting paint to double distilled water includes 4:1, 3:1, 2:1, and 1:1.

[0022] Furthermore, the concentration gradient of the fluorescent liquid includes 0.3 mg / ml, 0.6 mg / ml, 1.0 mg / ml, 3.0 mg / ml, 5.0 mg / ml, 10.0 mg / ml, 15.0 mg / ml, 30.0 mg / ml, 50.0 mg / ml, and 70.0 mg / ml.

[0023] After adopting the above technical solution, the present invention has the following advantages compared with the background technology:

[0024] 1. The present invention mixes water-based tinting paint and double distilled water into a diluent for adjusting the concentration of LED fluorescent liquid. The LED fluorescent powder is in a stable state in the water-based tinting paint with a certain viscosity, which is conducive to the stable adhesion of the LED fluorescent powder to the nitrocellulose membrane, so that the paths of the excitation light and the emission light remain consistent for a long time, thereby greatly improving the fluorescence sensitivity and stability and extending the shelf life. Different LED fluorescent powders can correspond to different excitation light wavelengths, so that they are suitable for different fluorescent immunoassay analyzers to solve the problems of single fluorescence wavelength and fluorescence signal attenuation.

[0025] 2. The present invention adopts screen printing technology to replace the traditional film drawing instrument to screen print the fluorescent liquid onto the nitrocellulose membrane. On the one hand, since two straight line films with fixed positions and widths are precisely set on the screen, the fluorescent band after screen printing can be accurately positioned and uniform in thickness. On the other hand, the screen printing technology can achieve precise quantification, thereby improving the accuracy of the fluorescence calibration strip. For example, when the ratio of the test peak area to the quality control peak area is required to be 1.0, the same concentration of fluorescent liquid can be used for printing. In addition, screen printing can be used to prevent viscous water-based toning paint from sticking to the tube wall when the film is drawn with a gold spray film drawing instrument. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1It is a schematic diagram of the structure of the fluorescence calibration card of the present invention.

[0027] Description of reference numerals:

[0028] 1. Test tape; 2. Quality control tape; 3. Support plate; 4. Jam. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] Embodiment 1

[0031] like Figure 1 As shown, a fluorescent calibration card includes a calibration strip, which is composed of a support plate 3 and a nitrocellulose membrane and absorbent paper overlapped end to end above the support plate 3, a fluorescent strip is fixed on the nitrocellulose membrane, and the fluorescent strip includes a test strip 1 and a quality control strip 2. The fluorescent strip is printed on the nitrocellulose membrane by screen printing with fluorescent liquid, and the fluorescent liquid includes LED fluorescent powder and a diluent, and the diluent includes water-based color varnish and double distilled water. The above-mentioned calibration strip is clamped inside the card shell 4.

[0032] The support plate 3 is made of polyvinyl chloride, the concentration of LED fluorescent powder in the fluorescent liquid is 3 mg / ml, the LED fluorescent powder is red LED fluorescent powder and yellow LED fluorescent powder, the red LED fluorescent powder is a nitride doped with Sm and Eu, and contains 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 of the red LED phosphor is in the range of 590nm to 650nm; the yellow LED phosphor is a nitride doped with Sr and Eu, including 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 phosphor is 530nm to 590nm; and the proportion of water-based tinting paint in the diluent is 60%.

[0033] Adding a certain amount of double distilled water to the water-based tinting paint can adjust the viscosity and make a diluent suitable for dissolving LED fluorescent powder. By stirring, the LED fluorescent powder is evenly dispersed in the solution. The LED fluorescent powder is stable in the water-based tinting paint with a certain viscosity, which can ensure that the paths of the excitation light and the emission light remain consistent for a long time, greatly improving the fluorescence sensitivity and stability and extending the shelf life. In addition, different LED fluorescent powders can correspond to different excitation light wavelengths, so that they are suitable for different machines. At the same time, the screen printing technology is introduced to print the fluorescent liquid onto the nitrocellulose membrane, which can ensure that the position of the fluorescent band is fixed, the thickness is uniform and can be quantified.

[0034] Embodiment 2

[0035] A method for preparing a fluorescence calibration card, the method comprising the following steps:

[0036] S1. Making film negatives: Make two film linear films, which are 0.8 mm wide and 20 cm long. The two linear films are at the same height on two film drawings and the left and right spacing is 6 mm, which are used as the test strip and the quality control strip respectively;

[0037] S2. Make screen: tighten the screen (the screen can be made of silk, nylon, polyester fiber or stainless steel wire), glue the four sides of the screen to the screen frame, and evenly apply diazonium salt photosensitive glue on the printing surface of the stretched screen, stick the prepared film on the photosensitive glue, and expose it in a vacuum exposure machine to form a screen;

[0038] S3, preparing water-based tinting paint and double distilled water in proportions of 4:1, 3:1, 2:1 and 1:1, respectively, stirring in a blender at room temperature for 2 hours to prepare diluents of different viscosities;

[0039] S4, using a diluent to prepare a plurality of fluorescent solutions with different concentration gradients, the concentration gradients including 0.3 mg / ml, 0.6 mg / ml, 1.0 mg / ml, 3.0 mg / ml, 5.0 mg / ml, 10.0 mg / ml, 15.0 mg / ml, 30.0 mg / ml, 50.0 mg / ml, and 70.0 mg / ml;

[0040] S5. Use a screen printer to print 10.0 mg / ml fluorescent liquid on the quality control strip of cellulose acetate membrane, and print the other 10 concentrations of fluorescent liquid on the test strip of cellulose acetate membrane respectively, and dry them in a drying oven at 45° C. for 2 hours to obtain fluorescent calibration strips with different ratios of the peak area of ​​the test strip to the peak area of ​​the quality control strip;

[0041] S6. Put the fluorescent calibration strip into a strip cutting machine and cut it into card strips with the same width of 3.88 mm. Then put the card strip into a plastic card shell and press the shell to make a fluorescent calibration card.

[0042] This embodiment uses screen printing technology to replace the traditional film marking instrument to screen print the fluorescent liquid onto the nitrocellulose membrane. On the one hand, because two straight lines with fixed positions and widths are precisely set on the screen, the fluorescent band after screen printing can be accurately positioned and uniform in thickness. On the other hand, the use of screen printing technology can achieve precise quantification, thereby improving the accuracy of the fluorescence calibration strip. For example, when the ratio of the test peak area to the quality control peak area is required to be 1.0, the same concentration of fluorescent liquid can be used for printing. In addition, the method of marking with a film marking instrument used in the prior art is prone to cause pipeline blockage and value deviation, while the viscous water-based toning paint in this embodiment is screen printed to avoid adhesion to the wall of the film marking instrument tube during film marking.

[0043] The fluorescence calibration card prepared by the above method can be used for the calibration of the fluorescence immunoassay analyzer. The fluorescence calibration card is sequentially placed in the fluorescence immunoassay analyzer for detection, and the test peak area of ​​each calibration card (i.e., the test band area S T ) and quality control peak area (quality control band area S C ), recorded as the ratio of the peak area of ​​the test band to the peak area of ​​the quality control band T 实际 , the theoretical S T With S C The ratio is recorded as T 理论 , the deviation formula from the theoretical value is: |T 实际 -T 理论 ∣ / T 理论 ×100%; after 100 consecutive measurements, the peak area S of the first test band is calculated. 1 and the peak area S of the 100th test band 100 , the attenuation formula of the calibration card is: |S 100 -S 1 ∣ / S 1 ×100%; Test 100 times in a row and count the number of times T 1-100 The ratio of the standard deviation of the result to the mean is CV. The formula for CV is: SD 1-100 / M 1-100 × 100%, where M 1-100 is the average of these 100 statistics, SD 1-100 is the standard deviation of these 100 statistics.

[0044] Table 1 shows the deviation, attenuation and CV data of the fluorescence calibration card;

[0045] Calibration card number 1 2 3 4 5 6 7 8 9 10 <![CDATA[T 理论 ]]> 0.030 0.060 0.100 0.300 0.500 1.000 1.500 3.000 5.000 7.000 <![CDATA[T 实际 ]]> 0.029 0.058 0.098 0.299 0.502 1.003 1.505 3.006 4.997 6.991 Relative deviation (%) 3.333 3.333 2.000 0.333 0.400 0.300 0.333 0.200 0.060 0.129 100 times attenuation (%) 0.301 0.220 0.215 0.201 0.110 0.156 0.080 0.094 0.055 0.056 100 times CV (%) 0.499 0.287 0.150 0.189 0.146 0.101 0.009 0.008 0.007 0.005

[0046] It can be concluded from Table 1 that the relative deviations between the actual detection values ​​and the theoretical values ​​are all below 5%, indicating that the detection results of the fluorescent immunoassay analyzer are reliable and the instrument can be used normally.

[0047] The 10 different fluorescence calibration cards prepared were tested repeatedly 100 times. The average relative deviation between the peak area value of the 100th test band and the peak area value of the 1st test band was less than 0.5%, and the maximum relative deviation was also less than 0.5%, indicating that the fluorescence signal tested by the fluorescence calibration card is stable and not easy to attenuate. Moreover, after 100 tests, the CV of each fluorescence calibration strip calculated was less than 0.5%, indicating that the fluorescence calibration card can be used stably for multiple times.

[0048] The prepared fluorescence calibration card was sealed in an aluminum foil bag at 37°C, and four fluorescence calibration cards were taken out in the third, sixth, ninth and twelfth months, respectively, to test two normal fluorescence instruments and two abnormal fluorescence instruments, respectively, to test the stability of the fluorescence calibration card. In the test, the relative deviations of all test values ​​and the standard values ​​were less than 5%, and the results showed that the effective shelf life of the fluorescence standard card at room temperature was at least 12 months.

[0049] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A fluorescence calibration card, characterized in that: A card shell is provided with a calibration strip inside, wherein the calibration strip comprises a support plate, a nitrocellulose membrane and absorbent paper which are overlapped end to end and are located on the support plate, a fluorescent strip is fixed on the nitrocellulose membrane, the fluorescent strip comprises a test strip and a quality control strip, the fluorescent strip is screen-printed to print fluorescent liquid onto the nitrocellulose membrane, the fluorescent liquid comprises LED fluorescent powder and a diluent, and the diluent comprises water-based color varnish and double distilled water.

2. A fluorescence calibration card as claimed in claim 1, characterized in that: The support plate is made of any one or more materials selected from the group consisting of polyvinyl chloride, acrylonitrile-butadiene-styrene copolymer, polyvinylidene fluoride, polypropylene and silica gel.

3. A fluorescence calibration card as claimed in claim 2, characterized in that: The concentration of the LED fluorescent powder in the fluorescent liquid is 0.3 mg / ml to 70 mg / ml.

4. A fluorescence calibration card as claimed in claim 3, characterized in that: The LED phosphor is any one of red LED phosphor, yellow LED phosphor, orange LED phosphor, green LED phosphor and blue LED phosphor.

5. A fluorescence calibration card as claimed in claim 4, characterized in that: The red LED phosphor is a nitride doped with Sm and Eu, including SmOSi2O2N2:Yb, Eu, NaGd(WO4)2:Eu, Sm and SrIn2O4:Eu. 3+ , Sm 3+ , where the maximum emission wavelength range of red LED phosphor is 590nm~650nm; The yellow LED phosphor is a nitride doped with Sr and Eu, comprising Sr7AlLi(PO4)7Eu2:Ce 3+ and Sr3SiO5:Eu 2 + , where the maximum emission wavelength range of yellow LED phosphor is 530nm ~ 590nm.

6. A fluorescence calibration card as claimed in claim 5, characterized in that: The proportion of water-based tinting paint in the diluent is 50% to 80%.

7. A method for preparing a fluorescence calibration card, characterized in that: The method is used to prepare the fluorescence calibration card according to any one of claims 1 to 6, comprising the following steps: S1. Making film negatives: setting a straight line of drug film on two film drawings respectively for making a test strip and a quality control strip; S2. Making the screen: evenly apply diazonium salt photosensitive glue on the stretched screen, stick the prepared film on the photosensitive glue, and expose it in a vacuum exposure machine; S3, preparing water-based tinting paint and double distilled water in different proportions, stirring at room temperature for 2 hours to prepare diluents with different viscosities; S4, using a diluent to prepare a plurality of fluorescent solutions with different concentration gradients; S5. Use a screen printer to print 10.0 mg / ml fluorescent liquid on the quality control strip of cellulose acetate membrane, and print other fluorescent liquids of different concentration gradients on the test strip of cellulose acetate membrane, and dry them at 45° C. for 2 hours to obtain fluorescent calibration strips with different ratios of the peak area of ​​the test strip to the peak area of ​​the quality control strip; S6. Cut the fluorescence calibration strips into card strips of the same width, and then put the card strips into a plastic card shell to make a fluorescence calibration card.

8. The method for preparing a fluorescence calibration card according to claim 7, characterized in that: The linear medicine film is 0.8 mm wide and 20-25 cm long, and two linear medicine films are at the same height of two film drawings, with a left-right spacing of 6 mm.

9. The method for preparing a fluorescence calibration card according to claim 8, characterized in that: The ratios of the water-based tinting paint and double distilled water include 4:1, 3:1, 2:1, and 1:

1.

10. A fluorescence calibration card and a method for preparing the same as claimed in claim 9, characterized in that: The concentration gradient of the fluorescent liquid includes 0.3 mg / ml, 0.6 mg / ml, 1.0 mg / ml, 3.0 mg / ml, 5.0 mg / ml, 10.0 mg / ml, 15.0 mg / ml, 30.0 mg / ml, 50.0 mg / ml, and 70.0 mg / ml.

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