An ultrasensitive IFN-γ detection probe and its application

Abstract

The present invention relates to an IFN-γ ultrasensitive detection probe and application, wherein the probe comprises a SERS tag and an aptamer, wherein the SERS tag is a gold core-silver shell alloy nanoparticle embedded with a SERS probe molecule 4-MBA, and the aptamer is a nucleic acid aptamer, and the probe has a high SERS enhancement effect and stability. The lateral flow test strip prepared by the present invention using the probe has high sensitivity, strong specificity, and is convenient and quick to use. It can not only be observed with the naked eye but also can be matched with a portable detection instrument, and has great potential. It can not only become a reliable on-site IFN-γ detection system, but also can become a universal detection platform for a pair of aptamers and complementary DNA, and can realize qualitative detection and highly sensitive quantitative detection of IFN-γ visualization within 20 minutes, and can be widely used for clinical rapid diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to an IFN-γ ultrasensitive detection probe and application thereof. Background Art

[0002] Interferon-γ (IFN-γ) is an inflammatory cytokine produced primarily by natural killer (NK) cells and thymus-derived (T) cells to determine disease-specific innate immune responses and serves as an active diagnostic biomarker for a range of diseases, such as autoinflammatory diseases, viral and bacterial infections, parasitic infections, and tumor control. The identification and evaluation of IFN-γ in body fluids can provide insights into the body's immune response mechanisms to inflammation or disease progression, which in turn facilitates personalized diagnosis and treatment. However, for most diseases, such as tuberculosis and lung cancer, physiological concentrations of IFN-γ at picogram levels require highly sensitive and selective detection methods.

[0003] In order to solve the above problems, there are mainly two solutions in the prior art:

[0004] Solution 1: Enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT) are the traditional gold standard methods for cytokine quantification, with high detection sensitivity and a detection limit of pg / mL. However, this solution has the following disadvantages: high detection time consumption, high antibody cost, and the need for highly skilled operators, which hinder its on-site clinical application.

[0005] Solution 2: Use gold nanoparticles or fluorescent molecules and antibodies to prepare traditional immunoassay test strips. However, the disadvantage of this solution is that the determination result is related to the depth of color, and it can only semi-quantitate the test object, and cannot achieve precise quantification, which affects the accuracy of the analysis result.

[0006] Existing methods: For example, ichroma developed by Boditech Med Inc. TM The IGRA-TB kit uses an immunofluorescence flow measurement system to detect IFN-γ, but the detection limit is not as good as the gold standard ELISA method, and this method relies on antibodies, which are expensive, difficult to preserve, and easily affected by the environment.

[0007] Based on the above-mentioned prior art, how to obtain a low-cost detection method for interferon-γ detection that is both visualized and has high sensitivity and high specificity has become a technical problem that needs to be solved urgently. Summary of the invention

[0008] In order to solve the above-mentioned technical problems, the present invention provides an IFN-γ ultra-sensitive detection probe, that is, an IFN-γ ultra-sensitive detection probe based on surface enhanced Raman spectroscopy (SERS) technology, wherein the probe comprises a SERS tag and an aptamer, wherein the SERS tag is a gold core-silver shell alloy nanoparticle embedded with a SERS probe molecule 4-mercaptobenzoic acid (4-MBA), and the aptamer is a nucleic acid aptamer. The probe has a high SERS enhancement effect, high sensitivity and stability, wherein the minimum IFN-γ concentration that the probe can detect is 5.4 pg / mL, and the detection range is 10 pg / mL-2000 pg / mL.

[0009] The present invention also provides a method for preparing the above-mentioned IFN-γ ultrasensitive detection probe, and the specific steps are as follows:

[0010] (1) Preparation of SERS tags: i.e., preparation of gold core silver shell alloy nanoparticles (Au / MBA / Ag alloy NPs) embedded with SERS probe molecule 4-mercaptobenzoic acid (4-MBA), including synthesis of gold nanoparticles (AuNPs), synthesis of gold nanoparticles-mercaptobenzoic acid (AuNPs-MBA), and synthesis of gold core silver shell alloy nanoparticles (Au / MBA / Ag alloy NPs);

[0011] (2) Aptamer-labeled SERS tag is used to prepare the probe. The aptamer is a nucleic acid aptamer, i.e., a thiol-modified IFN-γ aptamer, which is covalently coupled to the SERS probe surface via an Ag-S bond between the Ag surface of the SERS probe and the terminal thiol group in the thiol-modified IFN-γ aptamer.

[0012] Furthermore, the specific steps of synthesizing gold nanoparticles (AuNPs) in step (1) are as follows: adding deionized water to a chloroauric acid solution (HAuCl4·4H2O), heating to boiling, adding a sodium citrate solution, continuing heating and stirring, cooling to room temperature after stirring to obtain an AuNPs solution, filtering with a filter membrane to remove large particles of AuNPs, obtaining a synthesized AuNPs solution, and storing it.

[0013] Furthermore, the concentration of the chloroauric acid solution (HAuCl4·4H2O) is 8-12 mM, the concentration of the sodium citrate solution is 0.5-1.5 wt %, the pore size of the filter membrane is 0.22 μm, and the storage temperature is 4-8°C.

[0014] Furthermore, the specific steps for synthesizing AuNPs-MBA in step (1) are: adding 4-mercaptobenzoic acid (4-MBA) to the synthesized AuNPs solution, incubating and centrifuging, discarding the supernatant, and washing the precipitate with deionized water to remove unbound 4-mercaptobenzoic acid (4-MBA), thereby forming an AuNPs-MBA solution.

[0015] Furthermore, the concentration of 4-mercaptobenzoic acid (4-MBA) is 0.5-2 mM, the incubation time is 2 h-3 h, the centrifugal speed is 6000-10000 rpm, and the centrifugation time is 10-30 min.

[0016] Furthermore, the synthesis of Au / MBA / Ag alloy NPs in step (1) specifically comprises the following steps: mixing the synthesized AuNPs-MBA solution with a polyvinyl pyrrolidone solution, sequentially adding a silver nitrate solution, an ammonia solution and a chloroauric acid solution (HAuCl4·4H2O) solution to obtain a mixture one, then adding an L-ascorbic acid solution to the mixture one, mixing and reacting at room temperature, centrifuging and discarding the supernatant, washing the precipitate with deionized water, and finally dispersing the washed precipitate in deionized water to obtain Au / MBA / Ag alloy NPs, i.e., a SERS tag.

[0017] Furthermore, the concentration of the polyvinyl pyrrolidone solution is 1.0-1.5wt%, the concentration of the silver nitrate solution is 0.5-2mM, the concentration of the HAuCl4·4H2O solution is 0.5-1.5mM, the concentration of the L-ascorbic acid solution is 15-25mM, the shaking time is 1h-2h, the centrifugal speed is 6000-8000rpm, and the centrifugal time is 10-30min.

[0018] Furthermore, the sequence of the thiol-modified IFN-γ aptamer in step (2) is: 5'-SH-C6-GGG GTT GGTTGT GTT GGG TGT TGT GT-TTT TTT TTT TTT TTT TTT TTT TT-3',

[0019] The specific steps are as follows: first, using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to activate the disulfide bond-modified IFN-γ aptamer, i.e., the thiol-modified IFN-γ aptamer, and after activation, transferring it to the SERS tag prepared in step (1), incubating it overnight at room temperature to obtain a second mixture, adding sodium dodecyl sulfate (SDS) to the second mixture, adjusting the concentration of SDS, and performing the first incubation, and then adding tris(hydroxymethylaminomethane) hydrochloride (Tris-HCl) of a sodium chloride solution, performing the second incubation at room temperature, and then centrifuging to remove the unbound aptamer and resuspending it, thereby obtaining a SERS ultrasensitive probe suitable for IFN-γ detection, i.e., an IFN-γ ultrasensitive detection probe.

[0020] Furthermore, the concentration of SDS is 0.01-0.05wt%, the first incubation time is 30-60min, the concentration of TCEP is 5-15mg / mL, the concentration of sodium chloride solution is 0.1-0.25M, the concentration of Tris-HCl is 10-20mM, the second incubation time is 16-24h, the centrifugal speed is 6000-8000rpm, and the centrifugation time is 5-30 minutes.

[0021] The present invention also provides an application of the above-mentioned IFN-γ ultrasensitive detection probe, for example, the IFN-γ ultrasensitive detection probe is applied to prepare a lateral flow test strip.

[0022] Furthermore, the preparation process of the lateral flow test strip is as follows: the streptavidin solution is incubated with DNA1 and DNA2 at room temperature to obtain a streptavidin-DNA 1 conjugate and a streptavidin-DNA 2 conjugate, the streptavidin-DNA 1 conjugate or the streptavidin-DNA 2 conjugate is diluted with a PBS solution (phosphate buffer solution), the streptavidin-DNA 1 conjugate is sprayed on the NC membrane (nitrocellulose membrane) in the form of a detection line (T line), and the streptavidin-DNA 2 conjugate is sprayed on the NC membrane (nitrocellulose membrane) in the form of a control line (C line), and dried under a constant temperature environment, and then the dried NC membrane, sample pad and absorption pad are pasted on a PVC (polyvinyl chloride) back plate, overlapped with each other, and then cut to obtain a finished test strip, i.e., a lateral flow test strip, and finally the lateral flow test strip is loaded into a card shell and placed in a dry environment and sealed for standby use.

[0023] The sample pad is a region for absorbing the test sample and contains the IFN-γ ultrasensitive detection probe, and the absorption pad is a region for absorbing the aptamer in the IFN-γ ultrasensitive detection probe during the detection process.

[0024] Furthermore, the DNA1 and DNA2 are complementary DNAs complementary to the sequence of the aptamer, and the aptamer is a nucleic acid aptamer, that is, a thiol-modified IFN-γ aptamer, wherein the sequences of the DNA1 and DNA2 may be the same or different.

[0025] Furthermore, the sequence of the DNA1 is: 5'-ACA ACC AAC CCC C6-biotin-3', and the sequence of the DNA2 is: 5'-AAA AAA AAA AAA AAA AAA AA-biotin-3'.

[0026] Furthermore, the concentration of the streptavidin solution is 0.5-1 mg / mL, the concentration of the DNA1 or DNA2 is 20-100 μM, the volume ratio of the streptavidin solution to DNA1 or DNA2 is 0.5:1-2:1, the incubation time is 1-2.5 h, and the constant temperature environment temperature is 20-40° C.

[0027] Furthermore, the overlapping width is 1.5-2.5 mm, and the width of the finished test strip is 3-5 mm.

[0028] Furthermore, the lateral flow test strip has two detection methods, namely visual detection and quantitative detection. The specific detection methods are as follows:

[0029] (1) Visual detection: The color depth of the T line in the lateral flow test strip containing 0 pg / mL of the analyte, i.e., a blank sample containing 0 pg / mL IFN-γ, was used as a control.

[0030] If both the T line and C line of the lateral flow test strip of the test sample are colored, and the color of the T line is darker than that of the T line in the blank sample, the test sample does not contain the analyte, that is, the test sample is negative and does not contain IFN-γ;

[0031] If both the T line and the C line of the lateral flow test strip of the test sample are colored, and the color of the T line is lighter than that of the T line in the blank sample, the test sample contains the analyte, that is, the test sample is positive and contains IFN-γ;

[0032] If the C line of the lateral flow test strip of the test sample is colored, but the T line is not colored, then the test sample contains the analyte, that is, the test sample is positive and contains IFN-γ;

[0033] If the C line of the lateral flow test strip of the test sample does not show color, it means that the lateral flow test strip is invalid and a new lateral flow test strip needs to be replaced and retested;

[0034] Among them, the lighter the color of the T line, the higher the IFN-γ concentration in the test sample; when the color of the T line is not observed, it means that the IFN-γ concentration of the test sample is higher than 2000pg / mL.

[0035] (2) Quantitative detection: The Raman intensities of the T-line and C-line of the sample lateral flow test strip were scanned and detected using a portable Raman spectrometer to examine the sensitivity, specificity, repeatability and stability of the prepared lateral flow test strip, including sensitivity detection, specificity detection, repeatability detection and stability detection, where sensitivity refers to the lowest concentration of the analyte that can be detected by the lateral flow test strip.

[0036] Furthermore, the detection time of the visual detection or quantitative detection is less than 20 minutes.

[0037] The beneficial effects of the present invention are:

[0038] 1. The IFN-γ ultrasensitive detection probe of the present invention comprises a SERS tag and an aptamer, wherein the SERS tag is a gold core-silver shell alloy nanoparticle embedded with a SERS probe molecule 4-MBA, and the aptamer is a nucleic acid aptamer, i.e., a thiol-modified IFN-γ aptamer. The probe has a high SERS enhancement effect, high sensitivity and stability. The sensitivity of the present invention, i.e., the lowest detectable IFN-γ concentration is 5.4 pg / mL, which is lower than the lowest IFN-γ concentration (5.69 pg / mL) in the gold standard ELISA method in the prior art, and the detection range is 10 pg / mL-2000 pg / mL, which is lower and wider than the detection range of the ELISA method, wherein the ELISA detection range is 17.6 pg / mL-1000 pg / mL.

[0039] 2. The present invention adopts aptamers as recognition elements and utilizes the competitive effect of target protein, aptamer and complementary DNA sequence to achieve highly sensitive and highly specific detection, so that the IFN-γ ultrasensitive detection probe of the present invention has high stability, strong specificity, low cost, easy storage, and has broad application and promotion prospects.

[0040] 3. The lateral flow test strip prepared by the present invention using the IFN-γ ultrasensitive detection probe has high sensitivity, strong specificity, is easy and quick to use, can be observed by the naked eye and can be matched, can become a reliable on-site IFN-γ detection system, and can also become a universal detection platform for a pair of aptamers and complementary DNA, and can achieve visual qualitative detection and highly sensitive quantitative detection of IFN-γ within 20 minutes. Compared with the ELISA method (4.5h), the detection speed is fast and can be widely used for rapid clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1The schematic diagram of the structure of the lateral flow test strip prepared by using the IFN-γ ultrasensitive detection probe of the present invention;

[0042] Figure 2 The experimental principle of the lateral flow test strip prepared by the present invention using the IFN-γ ultrasensitive detection probe;

[0043] Figure 3 for Figure 2 Enlarged view of area C in the middle;

[0044] Figure 4 This is a blank control of the test strip of the present invention, where the T line does not develop color and the C line does not develop color;

[0045] Figure 5 It is a comparison diagram of visual detection and a curve diagram of quantitative detection in Example 3 of the present invention;

[0046] Figure 6 This is the specific detection based on B / B0 in Example 3 of the present invention;

[0047] Figure 7 This is the repeatability test of Example 3 of the present invention;

[0048] Figure 8 This is a stability test of the test strip of Example 3 of the present invention;

[0049] Fig. 9 The test results of the actual sample (serum) in the test example of the present invention are compared with the test strip prepared in Example 2 of the present invention and the ELISA kit in Comparative Example 1.

[0050] The names of the labels in the figure are:

[0051] 1. Sample pad; 2. Absorption pad; 3. NC membrane; 4. T line; 5. C line; 6. PVC backboard; 7. Portable Raman spectrometer; 8. Interferon-γ; 9. Thiol-modified IFN-γ aptamer; 10. DNA 1 sequence; 11. DNA 2 sequence; A. Negative sample; B. Positive sample; C. Raman intensity curve; D. Visualized detection result; E. Quantitative detection result; a. Blank control test strip; b. T line non-color test strip; c. C line non-color test strip; d. ELISA kit detection performance; e. Detection performance of the test strip prepared by the present invention. DETAILED DESCRIPTION

[0052] Example 1

[0053] This embodiment provides a method for preparing an ultrasensitive IFN-γ detection probe, and the specific steps are as follows:

[0054] (1) Preparation of SERS tags: i.e., preparation of gold core silver shell alloy nanoparticles (Au / MBA / Ag alloy NPs) embedded with SERS probe molecule 4-mercaptobenzoic acid (4-MBA), including synthesis of gold nanoparticles (AuNPs), synthesis of gold nanoparticles-mercaptobenzoic acid (AuNPs-MBA), and synthesis of gold core silver shell alloy nanoparticles (Au / MBA / Ag alloy NPs);

[0055] (2) Aptamer-labeled SERS tag is used to prepare the probe. The aptamer is a nucleic acid aptamer, i.e., a thiol-modified IFN-γ aptamer, which is covalently coupled to the SERS probe surface via an Ag-S bond between the Ag surface of the SERS probe and the terminal thiol group in the thiol-modified IFN-γ aptamer.

[0056] The specific steps for synthesizing gold nanoparticles (AuNPs) in step (1) are as follows: 2.5 ml of 10 mM chloroauric acid solution (HAuCl4·4H2O) is added to 97.5 ml of deionized water, heated to boiling, and then 1.5 ml of 1 wt% sodium citrate solution is quickly added, and heating and stirring are continued for 30 minutes. After stirring, the mixture is cooled to room temperature to obtain an AuNPs solution, and large particles of AuNPs are removed by filtering with a 0.22 μm filter membrane to obtain a synthesized AuNPs solution, which is then stored in a refrigerator at 4°C for further use.

[0057] The specific steps for synthesizing AuNPs-MBA in step (1) are as follows: taking 1 ml of the synthesized AuNPs solution, adding 20 μL of 1 mM 4-mercaptobenzoic acid (4-MBA), incubating for 2 hours under slight shaking, and then centrifuging at 6000 rpm for 10 minutes, discarding the supernatant, and washing the precipitate with deionized water to remove unbound 4-mercaptobenzoic acid (4-MBA), thereby forming an AuNPs-MBA solution.

[0058] The specific steps of synthesizing Au / MBA / Ag alloy NPs in step (1) are as follows: gently mixing 500 μL of the synthesized AuNPs-MBA solution with 500 μL of 1wt% polyvinyl pyrrolidone solution, adding 125 μL of 1mM silver nitrate, 40 μL of ammonia water and 375 μL of 1mM chloroauric acid solution (HAuCl4·4H2O) solution in sequence to obtain a mixture one, and then immediately adding 500 μL of 20mM L-ascorbic acid solution to the mixture one, gently shaking the mixture for 1h at room temperature for mixing reaction, centrifuging and discarding the supernatant, and washing the precipitate twice with deionized water, and finally dispersing the washed precipitate in deionized water to obtain Au / MBA / Agalloy NPs, i.e., SERS tags.

[0059] The nucleic acid sequence of the thiol-modified IFN-γ aptamer in step (2) is: 5'-SH-C6-GGG GTT GGT TGTGTT GGG TGT TGT GT-TTT TTT TTT TTT TTT TTT TTT TT-3',

[0060] The specific steps are as follows: first, 10 mg / mL tris(2-carboxyethyl)phosphine hydrochloride (TCEP) is used to activate the disulfide bond-modified IFN-γ aptamer, i.e., the thiol-modified IFN-γ aptamer, and after activation, it is transferred to the 1 mL SERS tag prepared in step (1), and incubated overnight at room temperature to obtain mixture 2, sodium dodecyl sulfate (SDS) is added to mixture 2, and the concentration of SDS is adjusted to 0.01 wt %, and after the first incubation, the incubation time is 30 minutes, and 100 μL of 1 M sodium chloride solution of tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) is added, and the second incubation is carried out at room temperature for 24 hours, and then the aptamer is centrifuged at 6000 rpm for 10 minutes to remove the unbound aptamer and resuspend it to obtain a SERS ultrasensitive probe suitable for IFN-γ detection, i.e., an IFN-γ ultrasensitive detection probe.

[0061] Example 2

[0062] This embodiment is an application of the IFN-γ ultrasensitive detection probe in the above-mentioned embodiment 1, that is, the IFN-γ ultrasensitive detection probe is applied to prepare a lateral flow test strip.

[0063] The preparation process of the lateral flow test strip is as follows: 1 mg / mL streptavidin solution is incubated with DNA1 and DNA2 at room temperature, respectively, the concentration of DNA1 and DNA2 is 100 μM, the volume ratio of the streptavidin solution to DNA1 or DNA2 is 1:1, and streptavidin-DNA 1 conjugate and streptavidin-DNA 2 conjugate are obtained, and the streptavidin-DNA 1 conjugate or streptavidin-DNA 2 conjugate is diluted with PBS solution, as shown in FIG. Figure 1-2 As shown, the NC membrane 3 is sprayed in the form of T line 4 and C line 5, respectively, and dried in a constant temperature environment of 37°C. Then, the dried NC membrane 3, sample pad 1 and absorption pad 2 are pasted on a PVC backboard 6, overlapping each other by 2mm, and then cut to obtain a 5mm wide finished test strip, i.e., a lateral flow test strip. Finally, the lateral flow test strip is placed in a card shell and sealed in a dry environment for standby use.

[0064] The DNA1 and DNA2 are complementary DNAs complementary to the sequence of the IFN-γ aptamer.

[0065] The sequence of the DNA1 is: 5'-ACA ACC AAC CCC C6-biotin-3', and the sequence of the DNA2 is: 5'-AAAAAA AAA AAA AAA AAA AA-biotin-3'.

[0066] The experimental principle of the lateral flow test strip prepared using the IFN-γ ultrasensitive detection probe is as follows Figure 1-2 As shown, after the test sample is dropped onto the sample pad 1, competition between the interferon-γ8 in the test sample and the DNA1 sequence 10 on the T line 4 for the thiol-modified IFN-γ aptamer 9 sequence in the IFN-γ ultrasensitive detection probe and the absorption pad 2 for the thiol-modified IFN-γ aptamer 9 in the IFN-γ ultrasensitive detection probe during the detection process.

[0067] In the test sample not containing interferon-γ8 (negative sample A), the thiol-modified IFN-γ aptamer 9 sequence of the IFN-γ ultrasensitive detection probe can be captured by the DNA 1 sequence 10 contained on the T line 4, and the thiol-modified IFN-γ aptamer 9 in the excess IFN-γ ultrasensitive detection probe can be captured by the DNA 2 sequence 11 contained on the C line 5. At this time, two lines, T line 4 and C line 5, are displayed on the lateral flow test strip.

[0068] However, when interferon-γ8 is present in the sample (positive sample B), the thiol-modified IFN-γ aptamer 9 sequence in the IFN-γ ultrasensitive detection probe will first bind to interferon-γ8 and weakly bind to the DNA 1 sequence 10 contained on T line 4, causing the color of T line 4 to become lighter or darker relative to the color of T line 4 in the negative sample. The visual color and Raman intensity of T line 4 depend on the concentration of interferon-γ8 in the sample.

[0069] Regardless of whether interferon-γ8 is present in the sample, the DNA 2 sequence 11 contained in line C 5 can always capture the thiol-modified IFN-γ aptamer 9 sequence in the IFN-γ ultrasensitive detection probe and appear red, thereby proving the effectiveness and feasibility of this scheme. If no red line is observed on line C 5, it means that the lateral flow test strip cannot work properly and a new lateral flow test strip needs to be replaced for re-testing.

[0070] At the same time, a portable Raman spectrometer 7 at 785 nm was used to scan and detect the Raman intensity of the T line 4 of the sample lateral flow test strip. The Raman intensity curve C is as shown in FIG. Figure 2 As shown in area C, more specifically Figure 2 shown.

[0071] Example 3

[0072] This embodiment is the detection of the lateral flow test strip of the above embodiment 2. The lateral flow test strip has two detection modes, namely visual detection and quantitative detection. The specific detection method is as follows:

[0073] (1) Visual detection: Figure 4 , 5 As shown, the color depth of the T line in the lateral flow test strip of the blank sample containing 0 pg / mL of the analyte IFN-γ is used as a control, wherein a is a blank control test strip, b is a T line 4 non-color test strip, and c is a control diagram of the C line 5 non-color test strip.

[0074] If both T line 4 and C line 5 of the lateral flow test strip of the test sample are colored, and the color of T line 4 is darker than or the same as the color of T line in the blank sample, the test sample does not contain the analyte IFN-γ, that is, the test sample is negative;

[0075] If both T line 4 and C line 5 of the lateral flow test strip of the test sample are colored, and the color of T line 4 is lighter than that of T line in the blank sample, the test sample contains the analyte IFN-γ, that is, the test sample is positive;

[0076] If the C line 5 of the lateral flow test strip of the test sample is colored, and the T line 4 is not colored or the color is lighter than the C line 5, the test sample contains the analyte, that is, the test sample is positive;

[0077] If the C line 5 of the lateral flow test strip of the test sample does not show color, it means that the lateral flow test strip is invalid and a new lateral flow test strip needs to be replaced and retested;

[0078] The lighter the color of T line 4, the higher the concentration of IFN-γ in the test sample. Figure 5 As shown in the visualization test result D; when the color of T line 4 is not observed, it means that the IFN-γ concentration of the test sample is higher than 2000pg / mL.

[0079] (2) Quantitative detection: The Raman intensities of T line 4 and C line 5 of the sample lateral flow test strip were scanned and detected by a portable Raman spectrometer to examine the sensitivity, specificity, repeatability and stability of the prepared lateral flow test strip. The sensitivity is the lowest concentration of the analyte that can be detected by the lateral flow test strip.

[0080] like Figure 5As shown in the quantitative detection result E, the sensitivity detection method is: dilute the IFN-γ standard to 2000pg / mL, 1000pg / mL, 500pg / mL, 250pg / mL, 100pg / mL, 50pg / mL, 25pg / mL, 10pg / mL, 0pg / mL, pre-mix it with the IFN-γ ultra-sensitive detection probe for 1 minute, obtain the mixture, take 70μL of each mixture and drop it on the sample pad 1, and use the portable Raman spectrometer to record the IFN-γ ultra-sensitive detection probe signal on the T line 4 and the C line 5. After 20 minutes, the color change on the T line 4 and the C line 5 can be seen by the naked eye; in addition, the IFN-γ ultra-sensitive detection probe signal on the T line 4 and the C line 5 is recorded by the portable Raman spectrometer, which is marked as I T and I C Calculate the I of the negative control (0 ng / mL) and the positive standard T / I C The values ​​were recorded as B0 and B, respectively, to establish a standard curve. By drawing a calibration curve between B / B0 and the log value of IFN-γ concentration, a good linear relationship (R 2 =0.978), and according to the formula LOD = V blank +3SD blank , the minimum detection limit was 5.4 pg / mL.

[0081] like Figure 6 As shown, the specific detection method is: dilute the IFN-γ standard to 100pg / mL, dilute the IL-6, IL-1, and BSA standards to 1μg / mL, premix them with the IFN-γ ultrasensitive detection probe for 1 minute, and take 70μL of each and drop it on the sample pad 1. After 20 minutes, the color changes on the T line 4 and the C line 5 are visible to the naked eye, and the IFN-γ ultrasensitive detection probe signal on the T line and the C line is recorded using a portable Raman spectrometer. The B / B0 of IL-6, IL-1β and BSA is significantly different from the B / B0 of IFN-γ, indicating that the specificity of this test strip is good.

[0082] like Figure 7 As shown in the figure, the repeatability test method is: dilute the IFN-γ standard to 1000pg / mL, 100pg / mL, 10pg / mL, and 0pg / mL, premix it with the IFN-γ ultrasensitive detection probe for 1min, and take 70μL of each and drop it on the sample pad 1. Ten repeated tests were performed for each concentration. The IFN-γ ultrasensitive detection probe signals on T line 4 and C line 5 were recorded using a portable Raman spectrometer to evaluate its repeatability. The results showed that the test strip had good repeatability.

[0083] like Figure 8As shown, the method of stability testing is: from the test strips prepared above, the stability at 0 days, 14 days and 28 days is tested with B / B0, and the results show that the stability within 28 days is good.

[0084] Comparative Example 1

[0085] This comparative example is a commercial IFN-γ ELISA kit (DIF50C), purchased from R&D Company in the United States.

[0086] Test Case

[0087] The lateral flow test strip of the present invention and the commercial ELISA kit were tested and compared:

[0088] like Fig. 9 As shown, the detection performance e of the test strip prepared by the present invention is compared with the detection performance d of the ELISA kit by using actual samples (serum), so as to comprehensively evaluate the detection performance of the test strip prepared by the present invention. The serum sample is diluted 10 times before the test, and 70 μL is added dropwise to the test strip during the test, and the Raman signal is detected by the Raman spectrometer after standing for 20 minutes. At the same time, the sample is tested using a commercial ELISA kit and compared with the results of the test strip. The results show that the test results of the test strip are in good agreement with the test results of the ELISA kit (the sample recovery rate is: 90% to 109%), and the detection recovery rate of the actual sample is 90% to 105%.

[0089] It should be understood that the present invention is not limited to what has been described above and shown in the accompanying drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the present invention is limited only by the appended claims.

Claims

1. An ultrasensitive IFN-γ detection probe, characterized in that: The probe includes a SERS tag and an aptamer, wherein the SERS tag is a gold core-silver shell alloy nanoparticle embedded with a SERS probe molecule 4-MBA, and the aptamer is a nucleic acid aptamer; The preparation method of the IFN-γ ultrasensitive detection probe comprises the following specific steps: (1) Preparation of SERS tags: that is, the preparation of gold core-silver shell alloy nanoparticles embedded with the SERS probe molecule 4-mercaptobenzoic acid, including the synthesis of AuNPs, the synthesis of AuNPs-MBA, and the synthesis of Au / MBA / Ag alloy NPs; (2) preparing a probe by labeling an aptamer with a SERS tag, wherein the aptamer is a nucleic acid aptamer, i.e., a thiol-modified IFN-γ aptamer, and is immobilized on the surface of the SERS probe by covalent coupling via a strong Ag-S bond between the Ag surface of the SERS probe and the terminal thiol group of the thiol-modified IFN-γ aptamer; The sequence of the thiol-modified IFN-γ aptamer in step (2) is: 5'-SH-C6-GGG GTT GGT TGT GTTGGG TGT TGT GT-TTT TTT TTT TTT TTT TTT TTT TT-3'.

2. The IFN-γ ultrasensitive detection probe according to claim 1, characterized in that: The specific steps of synthesizing AuNPs in step (1) are as follows: adding deionized water to chloroauric acid solution, heating to boiling, adding sodium citrate solution, continuing heating and stirring, cooling to room temperature after stirring to obtain AuNPs solution, filtering with a filter membrane to remove large particles of AuNPs, obtaining a synthesized AuNPs solution, and storing it.

3. The IFN-γ ultrasensitive detection probe according to claim 2, characterized in that: The specific steps of synthesizing AuNPs-MBA in step (1) are as follows: adding 4-mercaptobenzoic acid to the synthesized AuNPs solution, incubating and centrifuging, discarding the supernatant, and washing the precipitate with deionized water to remove unbound 4-mercaptobenzoic acid, thereby forming an AuNPs-MBA solution.

4. The IFN-γ ultrasensitive detection probe according to claim 3, characterized in that: The specific steps of synthesizing Au / MBA / Agalloy NPs in step (1) are as follows: mixing the synthesized AuNPs-MBA solution with a polyvinyl pyrrolidone solution, adding silver nitrate solution, ammonia water and chloroauric acid solution in sequence to obtain a mixture one, and then immediately adding L-ascorbic acid solution to the mixture one, mixing and reacting at room temperature, centrifuging and discarding the supernatant, washing the precipitate with deionized water, and finally dispersing the washed precipitate in deionized water to obtain Au / MBA / Ag alloy NPs, i.e., SERS tags.

5. The IFN-γ ultrasensitive detection probe according to claim 1, characterized in that: The specific steps are as follows: first, use tris(2-carboxyethyl)phosphine hydrochloride to activate the disulfide bond-modified IFN-γ aptamer, i.e., the thiol-modified IFN-γ aptamer, and after activation, transfer it to the SERS tag prepared in step (1), and incubate it overnight at room temperature to obtain a mixture 2, and then add SDS to the mixture 2, adjust the concentration of SDS, and after the first incubation, add tris(hydroxymethyl)aminomethane hydrochloride of sodium chloride solution, and after the second incubation at room temperature, centrifuge to remove the unbound aptamer and resuspend it to obtain a SERS ultrasensitive probe suitable for IFN-γ detection, i.e., an IFN-γ ultrasensitive detection probe.

6. A use of the IFN-γ ultrasensitive detection probe according to any one of claims 1 to 5, characterized in that: The IFN-γ ultrasensitive detection probe is used to prepare a lateral flow test strip.

7. The use of the IFN-γ ultrasensitive detection probe according to claim 6, characterized in that: The preparation process of the lateral flow test strip is as follows: a streptavidin solution is incubated with DNA1 and DNA2 at room temperature to obtain a streptavidin-DNA1 conjugate and a streptavidin-DNA2 conjugate; the streptavidin-DNA1 conjugate or the streptavidin-DNA2 conjugate is diluted with a PBS solution; the streptavidin-DNA1 conjugate is sprayed on the NC membrane in the form of a T line; the streptavidin-DNA2 conjugate is sprayed on the NC membrane in the form of a C line; and the conjugate is dried under a constant temperature environment; the dried NC membrane, the sample pad and the absorption pad are then pasted on a PVC back plate, overlapped with each other, and then cut to obtain a finished test strip, i.e., a lateral flow test strip; and finally, the lateral flow test strip is placed in a card shell and sealed in a dry environment for standby use. The sample pad is a region for absorbing the test sample and contains the IFN-γ ultrasensitive detection probe, and the absorption pad is a region for absorbing the aptamer in the IFN-γ ultrasensitive detection probe during the detection process; The sequence of the DNA1 is: 5'-ACA ACC AAC CCC C6-biotin-3', and the sequence of the DNA2 is: 5'-AAA AAA AAA AAA AAA AAA AA-biotin-3'.

8. The use of the IFN-γ ultrasensitive detection probe according to claim 7, characterized in that: The DNA1 and DNA2 are complementary DNAs complementary to the IFN-γ aptamer nucleic acid sequence, wherein the sequences of the DNA1 and DNA2 may be the same or different.

9. The use of the IFN-γ ultrasensitive detection probe according to claim 8, characterized in that: The lateral flow test strip has two detection modes, namely visual detection and quantitative detection.

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