A calibration strip and its preparation method and application

Abstract

The present invention provides a calibration strip and a preparation method and application thereof, wherein the calibration strip comprises polydimethylsiloxane and oil-soluble quantum dots. In the present invention, the calibration strip prepared by combining polydimethylsiloxane (PDMS) and oil-soluble quantum dots has the characteristics of good transparency, high and stable fluorescence intensity, plastic structure, and stable storage, and can be used for the calibration of a portable fluorescent immunoassay analyzer.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis, and relates to a calibration strip and a preparation method and application thereof, and in particular to a calibration strip for a portable fluorescent immunoassay analyzer and a preparation method thereof. Background Art

[0002] Fluorescence immunochromatography analysis combines the specific immune reaction between antigen and antibody with chromatographic technology. Generally, fluorescent substances are used as tracers and marked on antigens (or antibodies). When the analyte is present, it reacts with the corresponding antibody (or antigen) and the concentration of the analyte is analyzed by the output fluorescence intensity.

[0003] Portable fluorescence immunoassay analyzer is a common type of fluorescence immunochromatographic analyzer. It is manually loaded with samples on the matching reagent card and then placed in the analyzer for testing. It has the advantages of small size, instantaneousness and convenience. Its basic principle is that the fluorescent substances in the test line and quality control line area of ​​the reagent card will produce a stable light intensity fluorescence signal under the excitation of the excitation light source, which is converted into an electrical signal by the sensor, and the concentration of the analyte is determined by analysis and fitting.

[0004] To date, there are nearly 100 IVD manufacturers related to fluorescent immunoassay analyzers at home and abroad. However, the fluorescent materials (excitation light, emission light), test strip size parameters, and instrument excitation light sources (wavelength, power) used in the portable fluorescent immunoassay analyzers and fluorescent immunochromatographic test strips of each manufacturer are not the same. The measurement process is also affected by factors such as the equipment's excitation light source, the non-uniform photobleaching of the fluorescence itself, and the detection limit of the detection equipment.

[0005] To this end, some manufacturers in the industry have established some calibration and evaluation methods for the performance parameters of such instruments and equipment through independent research and development, including the following: (1) Various synthetic fluorescent materials (such as fluorescent microsphere solutions, LED fluorescent powders, etc.) are solidified on test strips by methods such as streaking, printing or gelling to participate in the preparation of calibration cards; (2) Two specific wavelength LED light sources are fixed on the internal circuit board of the calibration card as the detection line and quality control line light sources, and their brightness is traced back to the standard light source to determine the calibration card; (3) The prepared fluorescent solution is mixed with the photocurable fluorescent material and poured into the calibration card designed with groove channels or a container with a specific structure. These methods have their own advantages, but there are also some technical problems, including: 1) After multiple excitations, fluorescent substances are prone to fluorescence bleaching, resulting in fluorescence signal attenuation; 2) Portable fluorescent immunoassay analyzers of different brands need to match different card shells, which have poor applicability to equipment from different manufacturers.

[0006] In order to ensure the reliability of the measurement results, in this field, it is expected to develop a set of calibration strips with stable luminescence, plastic structure, covering three types of fluorescence intensities: high, medium and low, suitable for portable fluorescent immunoassay analyzers, which can be used to test the repeatability, linearity, channel consistency, stability and other related indicators of such instruments. Summary of the invention

[0007] In view of the deficiencies in the prior art, an object of the present invention is to provide a calibration strip and a preparation method and application thereof, and in particular to provide a calibration strip for a portable fluorescent immunoassay analyzer and a preparation method thereof.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a calibration strip, wherein the calibration strip comprises polydimethylsiloxane and oil-soluble quantum dots.

[0010] In the present invention, the calibration strip prepared by combining polydimethylsiloxane (PDMS) and oil-soluble quantum dots has the characteristics of good transparency, high and stable fluorescence intensity, plastic structure, and stable storage, and can be used for the calibration of portable fluorescent immunoassay analyzers. PDMS has the characteristics of good optical properties, transparency at optical frequencies (240nm-1100nm), and good toughness (7.1Mpa). PDMS and soluble quantum dots are evenly mixed and solidified in capillaries, which can effectively avoid the problem of quantum dots being affected by environmental factors such as water and oxygen, thereby causing unstable fluorescence signals. Compared with ordinary fluorescent molecules, oil-soluble quantum dots have the advantages of wide excitation wavelength, narrow emission wavelength, strong fluorescence signal and high stability, and are less disturbed by background fluorescence, so they have high sensitivity.

[0011] Preferably, the polydimethylsiloxane is Dow Corning SYLGARD 184 silicone rubber. Dow Corning SYLGARD 184 silicone rubber is a two-component kit product composed of liquid components, including a basic component (liquid A) and a curing agent (liquid B). When used, liquid A and liquid B are mixed in a weight ratio of 10:1 to obtain the polydimethylsiloxane described in the present application.

[0012] Preferably, the oil-soluble quantum dots include fluorescent nanomaterials encapsulated by hydrophobic ligands.

[0013] Preferably, the oil-soluble quantum dots include oil-soluble CdTe / CdSe / ZnS quantum dots and / or oil-soluble CdSe quantum dots. Oil-soluble CdTe / CdSe / ZnS quantum dots are core / shell / shell fluorescent nanomaterials with CdTe as the core, CdSe and ZnS as the shell, and the surface coated with hydrophobic ligands.

[0014] In a second aspect, the present invention provides a method for preparing the calibration strip according to the first aspect, the preparation method comprising the following steps:

[0015] Three different ratios of polydimethylsiloxane and oil-soluble quantum dots were mixed to obtain three mixed liquids, and bubbles were removed. The three mixed liquids were then pumped into mold tubes and solidified to obtain three calibration strips containing oil-soluble quantum dots at different concentrations.

[0016] Preferably, the preparation method comprises the following steps:

[0017] Liquid A and liquid B of Dow Corning SYLGARD 184 silicone rubber were mixed in a weight ratio of 10:1 to obtain a silicone rubber premix, and then different amounts of oil-soluble quantum dots were added to the silicone rubber premix to obtain three mixed solutions. After removing bubbles, the three mixed solutions were respectively pumped into mold tubes and cured to obtain three calibration strips containing oil-soluble quantum dots at different concentrations.

[0018] It should be noted that the present invention does not impose any specific restrictions on the ratio of polydimethylsiloxane and oil-soluble quantum dots (i.e., the ratio of silicone rubber premix to oil-soluble quantum dots), which can be determined according to actual application requirements. For example, if the amount of polydimethylsiloxane (i.e., silicone rubber premix) added is 1g, the amount of oil-soluble quantum dots added can be 1μg, 3μg, and 5μg, thereby obtaining three calibration strips containing oil-soluble quantum dots of different concentrations.

[0019] Preferably, the step of pumping the three mixed liquids into the mold tube respectively comprises: using a circulating water vacuum pump to pump the three mixed liquids into the mold tube respectively.

[0020] Preferably, the mould tube comprises a capillary tube.

[0021] Preferably, the curing temperature is 70-90°C, for example 70°C, 73°C, 75°C, 78°C, 80°C, 83°C, 85°C, 88°C or 90°C, and the curing time is 0.5-1.5h, for example 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h or 1.5h, etc.

[0022] In a third aspect, the present invention provides use of the calibration strip described in the first aspect in a portable fluorescent immunoassay analyzer.

[0023] Preferably, the application method comprises: sticking the calibration strip on the test paper and clamping it in the middle of the card shell, and then performing a calibration test.

[0024] Preferably, the application method comprises: attaching three calibration strips containing oil-soluble quantum dots of different concentrations to the test paper in sequence, sandwiching them between the card shells, and then performing a calibration test.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] In the present invention, PDMS has the characteristics of good optical properties, transparency at optical frequencies (240nm-1100nm), and good toughness (7.1Mpa). PDMS and soluble quantum dots are evenly mixed and solidified in capillaries, which can effectively avoid the problem of unstable fluorescence signals caused by the influence of environmental factors such as water and oxygen on quantum dots. Compared with ordinary fluorescent molecules, oil-soluble quantum dots have the advantages of wide excitation wavelength, narrow emission wavelength, strong fluorescence signal and high stability, and are less disturbed by background fluorescence, thus having high sensitivity. The calibration strip prepared by combining polydimethylsiloxane and oil-soluble quantum dots has the characteristics of good transparency, high and stable fluorescence intensity, plastic structure, and stable storage, and can be used for the calibration of portable fluorescent immunoassays. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic diagram of the structure of a calibration strip installed in a cartridge provided in Example 1, wherein: (left): a front view of the cartridge; (middle): a side view of the cartridge; (right): a calibration strip attached to a test paper.

[0028] FIG. 2( a ) and FIG. 2( b ) are respectively a physical picture of the calibration strip provided in Example 1 and a picture of the test result under ultraviolet excitation.

[0029] Figure 3 This is a graph showing the optical density test results of the calibration strip provided in Example 1.

[0030] Figure 4 This is a graph showing the fluorescence bleaching test results of the calibration strip 1 provided in Example 1.

[0031] Figure 5 This is a graph showing the optical density intensity stability test results of the calibration strip 1 provided in Example 1.

[0032] Figure 6 This is a graph showing the optical density test results of the calibration strip provided in Example 2.

[0033] Figure 7 This is a test result diagram of the calibration strip provided for Comparative Example 1 under ultraviolet excitation.

[0034] Figure 8 This is a test result diagram of the calibration strip provided for Comparative Example 2 under ultraviolet excitation.

[0035] Fig. 9This is a graph of the optical density test results of the calibration strip provided for Comparative Example 2. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0037] Example 1

[0038] In this embodiment, a calibration strip for a portable fluorescent immunoassay analyzer is provided, and the preparation method comprises the following steps:

[0039] (1) Preparing a polydimethylsiloxane mixed liquid: Dow Corning SYLGARD 184 silicone rubber basic component (liquid A) and a curing agent (liquid B) were stirred and mixed at a mass ratio of 10:1 to obtain a polydimethylsiloxane mixed liquid.

[0040] (2) Weigh 1 g of the polydimethylsiloxane mixed solution prepared in step (1), add three different masses (1 μg, 3 μg, and 5 μg, respectively) of oil-soluble CdTe / CdSe / ZnS quantum dots, mix and stir them evenly to obtain three mixed solutions.

[0041] (3) The three mixed solutions prepared in step (2) are then placed in a vacuum dryer and the bubbles are removed by vacuuming to ensure that there are no bubbles in the system.

[0042] (4) The three mixed liquids evacuated in step (3) are respectively and evenly pumped into a capillary tube with a diameter of 1.5 mm and a length of about 15 cm using a circulating water vacuum pump, while avoiding the generation of bubbles.

[0043] (5) The capillary filled with the mixed solution is placed in a culture dish, and the culture dish is placed in a constant temperature vacuum drying oven and cured at 80°C for 1 hour. The cured sample is then peeled off the capillary and cut to obtain three calibration strips containing oil-soluble quantum dots with different concentrations. The diameter of the calibration strip is about 1 mm and the length is about 8 mm. They are recorded as calibration strip 1, calibration strip 2, and calibration strip 3 in descending order according to the concentration of oil-soluble quantum dots.

[0044] The three calibration strips containing different concentrations of oil-soluble quantum dots provided in this embodiment are attached to the test paper from top to bottom in the order of oil-soluble quantum dot concentration from high to low, and are sandwiched in the middle of the card shell. The structural schematic diagram of the obtained calibration strip installed in the card shell is shown in FIG. Figure 1 shown.

[0045] The actual image of the calibration strip provided in this embodiment is shown in FIG2( a ), and it is placed in a UV gel imaging analysis system (BIO-RAD Gel Doc XR, USA) for imaging and photographing, and the test result is shown in FIG2( b ).

[0046] The optical density test of the calibration strip provided in this embodiment is carried out. The specific test conditions are: the excitation wavelength of the fluorescence spectrophotometer is set to (305±10)nm, and the emission wavelength is set to (600±10)nm. The test results are as follows: Figure 3 shown.

[0047] The calibration strip 3 provided in this embodiment was tested for fluorescence bleaching. The specific test conditions were: the excitation wavelength of the fluorescence spectrophotometer was set to (305±10) nm, the emission wavelength was set to (600±10) nm, and the number of cycle scans was set to 10. The test results are shown in FIG. Figure 4 shown.

[0048] from Figure 4 It can be seen that after the calibration strip is excited 10 times continuously by the fluorescence spectrophotometer light source, the fluorescence intensity of the calibration strip remains almost unchanged.

[0049] The optical density intensity stability test of the calibration strip 3 provided in this embodiment is carried out. The test method is as follows: the prepared fluorescence standard strip is stored at room temperature and protected from light, and the fluorescence signal is measured by a fluorescence spectrophotometer at certain intervals. The test results are as follows: Figure 5 shown.

[0050] from Figure 5 It can be seen that the calibration strip can be stored for at least 60 days, and the fluorescence signal intensity remains basically unchanged.

[0051] In practical applications, since the fluorescence signal intensity is easily affected by the wavelength and power of the excitation light source, the fluorescence signal intensity of the same fluorescent gel calibration strip measured by portable fluorescence immunoassay analyzers of different manufacturers and models varies greatly. Therefore, only the calibration strips are used to investigate the repeatability and stability of portable fluorescence immunoassay analyzers of different manufacturers.

[0052] The calibration strip is used to verify the repeatability of portable fluorescent immunoassay analyzers from various manufacturers:

[0053] The fluorescent gel calibration strips 1, 2 and 3 (fluorescence intensity from high to low) provided in Example 1 are cut into appropriate lengths to fit on the reagent cartridges of portable fluorescent immunoassay analyzers from different manufacturers. The fluorescent gel calibration strip 1 is used as the C line of each calibration test card. At the same time, the fluorescent gel calibration strips 1, 2 and 3 are placed at the T line position of each calibration test card to form three calibration test cards with different gradients. Each card is measured 10 times in a row, and the relative standard deviation (RSD) of the fluorescence signal intensity of the T and C lines is calculated as the repeatability (%) index of the portable fluorescent immunoassay analyzer. The repeatability (%) of the fluorescence signal intensity of the T and C lines was measured using 6 different portable fluorescent immunoassay analyzers, and the results are shown in Table 1:

[0054] Table 1

[0055]

[0056] As can be seen from Table 1, the calibration test card can be used for portable fluorescent immunoassay analyzers from different manufacturers to verify the repeatability of the instrument's T-line and C-line fluorescent signal. The standard deviation of the reading results of the fluorescence signal of the calibration test card by the manufacturer's instrument in Table 1 is less than 3%.

[0057] Example 2

[0058] The only difference between this embodiment and embodiment 1 is that the oil-soluble CdTe / CdSe / ZnS quantum dots in embodiment 1 are replaced with an equal amount of oil-soluble quantum dots CdSe.

[0059] The optical density test was performed on the calibration strip provided in this embodiment. The specific test conditions were the same as those in Example 1. The test results are shown in FIG. Figure 6 As shown, it can be seen that the calibration strip prepared with CdSe quantum dots can also stimulate good fluorescence intensity.

[0060] Comparative Example 1

[0061] The only difference between this comparative example and Example 1 is that the oil-soluble quantum dots in Example 1 are replaced with an equal amount of water-soluble quantum dots (CdSe / ZnS). It is found that since PDMS is a hydrophobic elastomer, it is difficult for the water-soluble quantum dots to wet PDMS, that is, CdSe / ZnS cannot be uniformly mixed with PDMS, and the quantum dots are disordered and dot-shapedly distributed inside the calibration strip, so they cannot emit a uniform fluorescent signal. The test results of the calibration strip provided in this comparative example under ultraviolet excitation are shown in the figure below. Figure 7 shown.

[0062] Comparative Example 2

[0063] The only difference between this comparative example and Example 1 is that the oil-soluble CdTe / CdSe / ZnS quantum dots in Example 1 are replaced with an equal amount of oil-soluble quantum dots (InP / ZnS). It is found that InP / ZnS quantum dots can be mixed with PDMS. However, under the same preparation conditions, the fluorescence intensity differentiation of the three calibration strips containing different concentrations of oil-soluble quantum dots is weaker than that of the calibration strips in Example 1. The test results of the calibration strips provided in this comparative example under ultraviolet excitation are shown in the figure below. Figure 8 As shown, the optical density test results of the calibration strip provided in this comparative example are shown in FIG. Fig. 9 shown.

[0064] The applicant declares that the present invention illustrates the calibration strip and its preparation method and application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A test paper containing a calibration strip, characterized in that: The calibration strip comprises polydimethylsiloxane and oil-soluble quantum dots; The oil-soluble quantum dots include oil-soluble CdTe / CdSe / ZnS quantum dots or oil-soluble CdSe quantum dots; The calibration strip is prepared by the following method, which comprises the following steps: respectively mixing three different ratios of polydimethylsiloxane and oil-soluble quantum dots to obtain three mixed solutions, removing bubbles, and then respectively pumping the three mixed solutions into a mold tube, curing at 70-90° C. for 0.5-1.5 h, to obtain three calibration strips containing oil-soluble quantum dots of different concentrations; Three calibration strips containing oil-soluble quantum dots of different concentrations are attached to the test paper and sandwiched between the card shells to obtain the test paper containing the calibration strips.

2. The test paper containing a calibration strip according to claim 1, characterized in that: The polydimethylsiloxane is Dow Corning SYLGARD 184 silicone rubber.

3. The test paper containing a calibration strip according to claim 1, characterized in that: The oil-soluble quantum dots include fluorescent nanomaterials wrapped by hydrophobic ligands.

4. The method for preparing a test paper containing a calibration strip according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: Three different ratios of polydimethylsiloxane and oil-soluble quantum dots were mixed to obtain three mixed solutions, and bubbles were removed. The three mixed solutions were then pumped into mold tubes and cured at 70-90° C. for 0.5-1.5 h to obtain three calibration strips containing oil-soluble quantum dots at different concentrations. Three calibration strips containing oil-soluble quantum dots of different concentrations are attached to the test paper and sandwiched between the card shells to obtain the test paper containing the calibration strips.

5. The preparation method according to claim 4, characterized in that: The step of pumping the three mixed liquids into the mold tube respectively comprises: using a circulating water vacuum pump to pump the three mixed liquids into the mold tube respectively.

6. The preparation method according to claim 4, characterized in that: The mold tube includes a capillary tube.

7. Use of the test paper containing the calibration strip according to any one of claims 1 to 3 in a portable fluorescent immunoassay analyzer.

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