A metal nanoparticle reagent kit, its preparation method, and its application in the detection of eczema biomarkers.

Abstract

This invention discloses a metal nanoparticle reagent kit, its preparation method, and its application in the detection of eczema biomarkers. The metal nanoparticle reagent kit utilizes a chemical self-assembly method to deposit metal nanoparticles onto the surface of the kit's well plates. ADNA is modified onto the surface of the metal nanoparticles through Ag-S bonds. Subsequently, through a complementary pairing process between the ADNA and cDNA chains, a cDNA chain with a Cy3 terminus at one end is brought close to the metal nanoparticles to synthesize a nucleic acid aptamer sensor. The metal nanoparticle reagent kit can specifically recognize and rapidly capture eczema biomarkers using nucleic acid aptamers. Combined with surface-enhanced Raman spectroscopy, it achieves rapid, sensitive, and accurate qualitative and quantitative detection of eczema biomarkers. Simultaneously, the internally encapsulated internal standard molecule signal can dynamically correct the Raman signal in the metal nanoparticle reagent kit.

Description

Technical Field

[0001] This invention belongs to the field of biochemical analysis technology, specifically relating to a metal nanoparticle reagent kit, its preparation method, and its application in the detection of eczema biomarkers. Background Technology

[0002] Eczema is essentially a skin inflammation reaction characterized by intense itching, caused by a variety of internal and external factors. Inflammation is a complex and highly regulated process through which the body responds to external stimuli and infections in a self-protective manner. However, excessive inflammation is a precursor to various long-term inflammatory diseases and even cancers, such as diabetes, arthritis, asthma, and colon cancer. When inflammation occurs, white blood cells, including macrophages, lymphocytes, and neutrophils, are activated and produce pro-inflammatory factors such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), triggering apoptosis and necrosis of normal tissues. Interleukin-6 (IL-6) is a polypeptide widely involved in physiological activities such as chronic inflammation, tissue regeneration, and hematopoiesis, especially in acute inflammation. Elevated levels of IL-6 are often used to determine inflammatory responses triggered by bacterial infections; therefore, it is an important diagnostic tool for most inflammatory diseases.

[0003] A series of methods for detecting interleukin-6 (IL-6) have been developed, such as chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay, colorimetric assay, and electrochemical immunoassay. Among these, ELISA is considered the most routine and standard method for detecting IL-6, with a specificity and sensitivity of approximately 5-10 pg / ml. However, this method is cumbersome and time-consuming. Therefore, there is an urgent need to develop a more advanced, reliable, and rapid technology for detecting IL-6 in complex biological systems.

[0004] In recent years, surface-enhanced Raman scattering (SERS) technology has shown great potential in biomolecular analysis due to its unique fingerprint vibrational spectra and ultra-narrow spectral linewidth. However, proteins have low Raman scattering cross sections and lack chromophores in their molecular structure, failing to provide strong SERS signals, thus preventing direct protein detection using SERS. Indirect sensing techniques combining SERS with highly specific biomolecules can significantly improve the application of SERS in protein quantification and characterization.

[0005] Nucleic acid aptamers are a class of single-stranded DNA or RNA molecules obtained through in vitro screening. These molecules can perform the same affinity recognition function as antibodies, but possess superior characteristics. They can be synthesized in large quantities in vitro, shortening the preparation cycle. DNA molecules are less affected by environmental factors and have higher stability than antibodies. Furthermore, DNA molecules are easily modified with functional groups; the introduced amino, carboxyl, thiol, biotin, and fluorescein groups facilitate the construction of sensing and detection methods. Therefore, with the development of nanosensor technology, nanosensors constructed by combining nanomaterials with aptamers are playing an increasingly important role in the field of biosensoring. Among these, the most widely used nanoparticles are noble metal nanoparticles such as gold and silver nanoparticles, which possess advantages such as ease of synthesis, small size effect, surface effect, optical effect, and good biocompatibility.

[0006] The patent with publication number CN110261621A, entitled "An Interleukin-6 Detection Kit," achieves accurate quantification of interleukin-6 by introducing a streptavidin-biotin signal amplification system. However, this invention requires the use of sensitizers, preservatives, and surfactants to avoid interference from certain substances (such as lipids) in the sample to be tested, reducing non-specific binding and affecting the accuracy of the results. The patent with publication number CN107727632A discloses "A Method for Detecting Thrombin Using SERS-Enhanced Controllable Chain Assemblies," which utilizes the recognition and binding of aptamer molecules with thrombin to achieve quantitative detection of thrombin. However, this method requires Au@Ag nanoparticles and Ag nanoparticle molecules to modify the two aptamer DNA molecules of thrombin, and it is necessary to control the amount of aptamer DNA molecules modified on the nanoparticles. Summary of the Invention

[0007] This invention introduces a metal nanoparticle kit, its preparation method, and its application in the detection of eczema biomarkers. The invention utilizes Ag-S bonds to prepare an Au@MBN@Ag-ADNA-cDNA structure on the surface of the kit wells, forming a nucleic acid aptamer sensor. In the presence of eczema biomarkers, the ADNA chain specifically binds to the biomarker, forming a 3D conformation to achieve capture. A stable protein-aptamer complex is formed through van der Waals forces, hydrogen bonds, and electrostatic interactions. Simultaneously, because the affinity between the ADNA chain and the target protein is much greater than that of the aptamer, the cDNA chain carrying the Cy3 signal will be competitively replaced by the target protein, leading to cDNA chain shedding and a decrease in the Cy3 Raman signal. As the concentration of the target protein increases, more cDNA chain detaches, and the Cy3 Raman signal is negatively correlated with the target protein concentration. Furthermore, using 4MBN as an internal standard molecule allows for dynamic calibration of the SERS signal, forming a self-calibrating aptamer sensor.

[0008] The technical solution of the present invention is as follows:

[0009] One of the objectives of this invention is to provide a method for preparing a metal nanoparticle reagent kit. The method involves depositing metal nanoparticles onto the surface of a reagent kit well plate using a chemical self-assembly method. ADNA is then modified onto the surface of the metal nanoparticles through the action of Ag-S bonds. Subsequently, through the complementary pairing process of the ADNA chain and the cDNA chain, a cDNA chain with Cy3 at one end is brought close to the metal nanoparticles to form a nucleic acid aptamer sensor.

[0010] Furthermore, the preparation method is as follows:

[0011] Preparation of S1 and Au NPs: Sodium citrate solution was added to boiling chloroauric acid aqueous solution under vigorous stirring. After the solution boiled again, it was kept in this state for 10-20 minutes under stirring. Then the solution was allowed to cool to room temperature naturally. The gold nanoparticles were stored at 4℃ for future use.

[0012] Preparation of S2 Au@MBN@Ag NPs solution: Add Au NPs to MBN solution, stir and centrifuge to remove excess MBN solution, resuspend in distilled water, then add silver nitrate solution and ascorbic acid solution, and stir for 30 min;

[0013] S3. Preparation of well plates modified with metal nanoparticles: Wash the well plates of the kit with ethanol, immerse them in 3-mercaptopropyltriethoxysilane ethanol solution at 2-6℃ for 24h, then wash the well plates three times with ethanol and dry them under nitrogen, and finally immerse them in Au NPs or Au@MBN@Ag NPs.

[0014] S4. Preparation of Au@MBN@Ag-ADNA-CDNA structure: Add ADNA and CDNA solutions to the prepared well plate respectively, and incubate in a shaker;

[0015] Furthermore, in S1, the concentration of sodium citrate solution is 1 wt% and the volume is 1-3 mL, and the concentration of chloroauric acid aqueous solution is 0.01 wt% and the volume is 80-120 mL.

[0016] Furthermore, in S2, the concentration of the MBN solution is 1×10⁻⁶. -5 M, volume 20 μL; silver nitrate solution concentration 1 mM, volume 100-150 μL; ascorbic acid solution concentration 0.01 mM, volume 80 μL; stirring time 30 min.

[0017] Furthermore, the immersion time in S3 is 6-24 hours.

[0018] Furthermore, in S4, the shaking speed of the shaker is 120 rpm, the temperature of the shaker is 37°C, and the incubation time is 12 hours.

[0019] Furthermore, in S4

[0020] The ADNA sequence is 5'-SH-(CH2)6-CTTCCAACGCTCGTATTGTAGTCTTTAGT-3', and the CDNA sequence is 5'-AAATACGAGCGTTGGAAGTA-Cy3-3', where Cy3 is anthocyanin dye 3. The concentrations of the ADNA and CDNA solutions are 2-12 mM.

[0021] The second objective of this invention is to provide a metal nanoparticle reagent kit.

[0022] The third objective of this invention is to provide a metal nanoparticle reagent kit and its preparation method, as well as its application in the detection of eczema biomarkers. The metal nanoparticle reagent kit can utilize nucleic acid aptamers to specifically identify and rapidly capture eczema biomarkers, and combined with surface-enhanced Raman spectroscopy, it can achieve qualitative and quantitative detection of eczema biomarkers.

[0023] Furthermore, the eczema marker is interleukin-6.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1. The internal standard molecule encapsulated within the Au@MBN@Ag NPs structure in the metal nanoparticle reagent kit provided by this invention can dynamically calibrate the Raman signal in the kit, thereby reducing experimental errors caused by measurements. Simultaneously, the internal standard molecule is located at 2227 cm⁻¹. -1 The Raman signal is located in the Raman silent region, which can avoid interference from other biomolecules and further improve the stability of calibration.

[0026] 2. The metal nanoparticle reagent kit provided by this invention can achieve interleukin-6 (IL-6) at 10 -4 -10 -9 Quantitative detection is possible within the mg / mL range, with a limit of detection (LOD) of 0.056 pg / mL. Due to the high specificity of nucleic acid aptamers in recognizing analytes, the sensor exhibits good selectivity, even with weaker responses to other similar proteins, enabling high-precision and high-specificity SERS detection of eczema biomarkers.

[0027] 3. The metal nanoparticle reagent kit provided by this invention can be widely used in the detection of eczema markers in clinical body fluid samples. The body fluid can be any one of plasma, serum, whole blood, urine, sweat, saliva or tears. The measurement operation is simple, and the accuracy and sensitivity are high. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the working principle of the metal nanoparticle reagent kit for detecting IL-6 of the present invention;

[0029] Figure 2 This is a partially enlarged schematic diagram illustrating the working principle of the metal nanoparticle kit for detecting IL-6 of the present invention;

[0030] Figure 3 A is a TEM image of the Au NPs obtained in Embodiment 1 of the present invention;

[0031] Figure 3 B is a TEM image of the Au@MBN@Ag NPs prepared in Example 2 of this invention;

[0032] Figure 4 To establish a standard working curve for IL-6 concentration using the metal nanoparticle kit in Example 3 of this invention;

[0033] Figure 5 The results of the anti-interference experiment of the metal nanoparticle reagent kit in Example 4 of this invention are shown. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and preferred embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0037] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0038] The DNA sequences involved in this invention are as follows:

[0039]

[0040] Example 1

[0041] S1. Under vigorous stirring, first, add 1 mL of 1 wt% Nact to 80 mL of 0.01 wt% boiling chloroauric acid aqueous solution. After the solution boils again, maintain this state for 10 min under stirring. Then, allow the final wine-red solution to cool naturally to room temperature. Finally, store the prepared gold nanoparticles at 4°C for future use.

[0042] S2. First, add 2 mL of purified Au NPs to 20 μL of a solution with a concentration of 1×10⁻⁶.-5 M MBN solution was stirred for 1 h, then centrifuged to remove excess MBN and resuspended in 2 mL of distilled water. Then, 200 μL of 1 mM silver nitrate solution and 80 μL of 0.01 mM ascorbic acid solution were added and stirred for 30 min to finally obtain an orange-red Au@MBN@Ag NPs solution.

[0043] S3. First, the well plate was washed with ethanol and then immersed in a 10% (v / v) 3-mercaptopropyltriethoxysilane ethanol solution at 2°C for 24 hours. Next, the well plate was washed three times with ethanol and dried under nitrogen. Finally, it was immersed in an Au NPs or Au@MBN@AgNPs solution for 6 hours. This allows the metal nanoparticles to be chemically adsorbed onto the surface of the well plate, forming a well plate with an enhancing effect.

[0044] S4. First, add 10 μL of 2 mM IL-6 aptamer (ADNA) and complementary strand (CDNA) aqueous solution to the prepared well plate, respectively; then, incubate in a shaker at 120 rpm and 37°C for 12 h to modify Au@MBN@Ag NPs particles with ADNA and CDNA, thus preparing Au@MBN@Ag-ADNA-CDNA.

[0045] Example 2

[0046] S1. Under vigorous stirring, first, take 3 mL of 1 wt% Nact and add it to 120 mL of 0.01 wt% boiling chloroauric acid aqueous solution. After the solution boils again, maintain this state for 20 min under stirring. Then, let the final wine-red solution cool naturally to room temperature. Finally, store the prepared gold nanoparticles at 4 °C for future use.

[0047] S2. First, add 2 mL of purified Au NPs to 20 μL of a solution with a concentration of 1×10⁻⁶. -5 M MBN solution was stirred for 1 h, then centrifuged to remove excess MBN and resuspended in 2 mL of distilled water. Then, 300 μL of 1 mM silver nitrate solution and 80 μL of 0.01 mM ascorbic acid solution were added and stirred for 30 min to finally obtain an orange-red Au@MBN@Ag NPs solution.

[0048] S3. First, wash the well plate with ethanol and immerse it in a 10% (v / v) 3-mercaptopropyltriethoxysilane ethanol solution at 6°C for 24 hours. Then, wash the well plate three times with ethanol and dry it under nitrogen. Finally, immerse it in Au NPs or Au@MBN@AgNPs solution for 24 hours. This allows the metal nanoparticles to be chemically adsorbed onto the surface of the well plate, forming a well plate with an enhancing effect.

[0049] S4. First, add 10 μL of 12 mM IL-6 aptamer (ADNA) and complementary strand (CDNA) aqueous solution to the prepared well plate, respectively; then, incubate in a shaker at 120 rpm and 37°C for 12 h to modify Au@MBN@Ag NPs particles with ADNA and CDNA, thus preparing Au@MBN@Ag-ADNA-CDNA.

[0050] Example 3: Establishing the standard operating curve for IL-6

[0051] S1. Under vigorous stirring, first, take 2 mL of 1 wt% Nact and add it to 100 mL of 0.01 wt% boiling chloroauric acid aqueous solution. After the solution boils again, maintain this state for 15 min under stirring. Then, let the final wine-red solution cool naturally to room temperature. Finally, store the prepared gold nanoparticles at 4 °C for future use.

[0052] S2. First, add 2 mL of purified Au NPs to 20 μL of a solution with a concentration of 1×10⁻⁶. -5 M MBN solution was stirred for 1 h, then centrifuged to remove excess MBN and resuspended in 2 mL of distilled water. Then, 240 μL of 1 mM silver nitrate solution and 80 μL of 0.01 mM ascorbic acid solution were added and stirred for 30 min to finally obtain an orange-red Au@MBN@Ag NPs solution.

[0053] S3. First, wash the well plate with ethanol and immerse it in a 10% (v / v) 3-mercaptopropyltriethoxysilane ethanol solution at 4°C for 24 hours. Then, wash the well plate three times with ethanol and dry it under nitrogen. Finally, immerse it in Au NPs or Au@MBN@AgNPs solution for 12 hours. This allows the metal nanoparticles to be chemically adsorbed onto the surface of the well plate, forming a well plate with an enhancing effect.

[0054] S4. First, add 10 μL of 10 mM IL-6 aptamer (ADNA) and complementary strand (CDNA) aqueous solution to the prepared well plate, respectively; then, incubate in a shaker at 120 rpm and 37°C for 12 h to modify Au@MBN@Ag NPs particles with ADNA and CDNA, thus preparing Au@MBN@Ag-ADNA-CDNA.

[0055] S5. Mix IL-6 standard solutions of different concentrations with Au@MBN@Ag-ADNA-cDNA, incubate at 37℃ for 90 min, and construct a standard working curve for IL-6 concentration based on the results, such as... Figure 4 As shown.

[0056] Example 4 Anti-interference test

[0057] S1. Under vigorous stirring, first, take 2 mL of 1 wt% Nact and add it to 100 mL of 0.01 wt% boiling chloroauric acid aqueous solution. After the solution boils again, maintain this state for 15 min under stirring. Then, let the final wine-red solution cool naturally to room temperature. Finally, store the prepared gold nanoparticles at 4 °C for future use.

[0058] S2. First, add 2 mL of purified Au NPs to 20 μL of a solution with a concentration of 1×10⁻⁶. -5 M MBN solution was stirred for 1 h, then centrifuged to remove excess MBN and resuspended in 2 mL of distilled water. Then, 240 μL of 1 mM silver nitrate solution and 80 μL of 0.01 mM ascorbic acid solution were added and stirred for 30 min to finally obtain an orange-red Au@MBN@Ag NPs solution.

[0059] S3. First, wash the well plate with ethanol and immerse it in a 10% (v / v) 3-mercaptopropyltriethoxysilane ethanol solution at 4°C for 24 hours. Then, wash the well plate three times with ethanol and dry it under nitrogen. Finally, immerse it in Au NPs or Au@MBN@AgNPs solution for 12 hours. This allows the metal nanoparticles to be chemically adsorbed onto the surface of the well plate, forming a well plate with an enhancing effect.

[0060] S4. First, add 10 μL of 10 mM IL-6 aptamer (ADNA) and complementary strand (CDNA) aqueous solution to the prepared well plate, respectively; then, incubate in a shaker at 120 rpm and 37°C for 12 h to modify Au@MBN@Ag NPs particles with ADNA and CDNA, thus preparing Au@MBN@Ag-ADNA-CDNA.

[0061] S5. First, mix IL-6 standard solutions of different concentrations with Au@MBN@Ag-ADNA-CDNA and incubate at 37℃ for 90 min. Use the results to create a standard working curve for IL-6 concentration. Then, mix IL-6, PCT, CEA, 5-HT and IgG of the same concentration (0.1 μg / mL) with Au@MBN@Ag-ADNA-CDNA and incubate at 37℃ for 90 min. Collect the corresponding curves using a Raman spectroscopy instrument.

[0062] like Figure 5 As shown, at 1470 cm⁻¹ in the SERS spectrum -1 At the site of observation, the intensity of Cy3 did not change significantly with the addition of other interfering substances but without the addition of IL-6; only with the addition of IL-6 did the intensity decrease significantly. These results indicate that the kit can better exclude the influence of other protein molecules in the environment, achieving highly specific recognition of IL-6.

[0063] Example 5 Reliability Test

[0064] S1. Under vigorous stirring, first, take 2 mL of 1 wt% Nact and add it to 100 mL of 0.01 wt% boiling chloroauric acid aqueous solution. After the solution boils again, maintain this state for 15 min under stirring. Then, let the final wine-red solution cool naturally to room temperature. Finally, store the prepared gold nanoparticles at 4 °C for future use.

[0065] S2. First, add 2 mL of purified Au NPs to 20 μL of a solution with a concentration of 1×10⁻⁶. -5 M MBN solution was stirred for 1 h, then centrifuged to remove excess MBN and resuspended in 2 mL of distilled water. Then, 240 μL of 1 mM silver nitrate solution and 80 μL of 0.01 mM ascorbic acid solution were added and stirred for 30 min to finally obtain an orange-red Au@MBN@Ag NPs solution.

[0066] S3. First, the well plate was washed with ethanol and then immersed in a 10% (v / v) 3-mercaptopropyltriethoxysilane ethanol solution at 4°C for 24 hours. Next, the well plate was washed three times with ethanol and dried under nitrogen. Finally, it was immersed in Au NPs or Au@MBN@AgNPs solution for 12 hours. This allows the metal nanoparticles to be chemically adsorbed onto the surface of the well plate, forming a well plate with an enhancing effect.

[0067] S4. First, add 10 μL of 10 mM IL-6 aptamer (ADNA) and complementary strand (CDNA) aqueous solution to the prepared well plate, respectively; then, incubate in a shaker at 120 rpm and 37°C for 12 h to modify Au@MBN@Ag NPs particles with ADNA and CDNA, thus preparing Au@MBN@Ag-ADNA-CDNA.

[0068] S5. 100 μL of serum from healthy individuals was mixed with different concentrations of IL-6 (5 pg / mL, 10 pg / mL, 50 pg / mL) to obtain the spiked recovery rates, and the results are shown in Table 1.

[0069] Table 1 Recovery rate of IL-6 in clinical serum samples

[0070]

[0071] The recovery rates of clinical serum samples were 93.01%, 109.28%, and 98.36%, respectively, with RSD values ​​far less than 20%. Therefore, the IL-6 concentration obtained from the test results is close to the IL-6 concentration after spiking, indicating that the kit has good accuracy and reliability and can be applied to the detection of IL-6 in real human serum.

[0072] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing a metal nanoparticle reagent kit, characterized in that: Metal nanoparticles were deposited on the surface of the reagent kit well plate using a chemical self-assembly method. ADNA was modified on the surface of the metal nanoparticles through Ag-S bond interaction. Then, through the complementary pairing process of ADNA and cDNA strands, a cDNA strand with Cy3 at one end was brought close to the metal nanoparticles to form a nucleic acid aptamer sensor. Specifically, the following steps are included: Preparation of S1 and Au NPs: Sodium citrate solution was added to boiling chloroauric acid aqueous solution under vigorous stirring. After the solution boiled again, it was kept in this state for 10-20 min under stirring. Then the solution was allowed to cool to room temperature naturally and the prepared gold nanoparticles were stored at 4℃. Preparation of S2, Au@MBN@Ag NPs solution: Add the Au NPs obtained in S1 to MBN solution, stir and centrifuge to remove excess MBN solution, resuspend in distilled water, and then add silver nitrate solution and ascorbic acid solution and stir. S3. Preparation of well plates modified with metal nanoparticles: Wash the well plates of the kit with ethanol, immerse them in 3-mercaptopropyltriethoxysilane ethanol solution at 2-6℃ for 24h, then wash the well plates three times with ethanol and dry them under nitrogen, and finally immerse them in Au@MBN@Ag NPs; S4. Preparation of Au@MBN@Ag-ADNA-CDNA structure: Add ADNA and CDNA solutions to the prepared well plate respectively, and incubate in a shaker; The ADNA sequence is 5'-SH-(CH2)6-CTTCCAACGCTCGTATTGTAGTCTTTAGT-3', and the CDNA sequence is 5'-AAATACGAGCGTTGGAAGTA-Cy3-3', where Cy3 is anthocyanin dye 3. The concentrations of the ADNA and CDNA solutions are 2-12 mM.

2. The method for preparing a metal nanoparticle reagent kit as described in claim 1, characterized in that, In S1, the concentration of sodium citrate solution is 1 wt% and the volume is 1-3 mL, and the concentration of chloroauric acid aqueous solution is 0.01 wt% and the volume is 80-120 mL.

3. The method for preparing a metal nanoparticle reagent kit as described in claim 1, characterized in that, The concentration of MBN solution in S2 is 1×10⁻⁶. -5 M, volume 20 μL; silver nitrate solution concentration 1 mM, volume 100-150 μL; ascorbic acid solution concentration 0.01 mM, volume 80 μL; stirring time 30 min.

4. The method for preparing a metal nanoparticle reagent kit as described in claim 1, characterized in that, The immersion time in S3 is 6-24 hours.

5. The method for preparing a metal nanoparticle reagent kit as described in claim 1, characterized in that, The shaking speed of the shaker in S4 is 120 rpm, the temperature of the shaker is 37°C, and the incubation time is 12h.

6. A metal nanoparticle kit prepared by the method according to any one of claims 1 to 5.

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