A diagnostic kit for prostate cancer based on a biosensor chip

Abstract

The present invention discloses a diagnostic kit for prostate cancer based on a biosensor chip, relating to the technical field of biosensor chips. The diagnostic kit comprises the following components: a biosensor chip, streptavidin-modified gold nanoparticles, tetrahedral DNA, a miRNA-helper, an H1 chain, and an H2 chain; the nucleotide sequences of the miRNA-helper, H1 chain, and H2 chain are shown in SEQ ID NOs. 13-15, respectively. The biosensor chip-based diagnostic kit for prostate cancer developed by the present invention can detect the miRNA-21 content in urine samples, with the advantages of strong specificity and high sensitivity. Compared with traditional PSA testing, the diagnostic specificity of the kit of the present invention is as high as over 90%, significantly superior to traditional PSA testing.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensor chips, and in particular to a diagnostic kit for prostate cancer based on a biosensor chip. Background Art

[0002] Prostate cancer is a malignant tumor that affects men's health. Urine specimens offer advantages such as ease of collection and non-invasiveness. Therefore, the development of detection methods based on tumor-derived markers in urine is expected to provide strong technical support for the early, non-invasive diagnosis of prostate cancer. However, in conventional clinical settings, marker detection suffers from issues such as insufficient sensitivity and throughput, as well as high costs for detection equipment and analysis (e.g., sequencing). Therefore, there is an urgent need to develop high-throughput marker analysis products that are highly sensitive, convenient, and clinically suitable.

[0003] Prostate-specific antigen (PSA) is one of the few molecular markers routinely used for the detection, risk stratification, and monitoring of common cancers. Currently, traditional prostate cancer screening relies primarily on serum PSA testing. Enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, polymerase chain reaction (PCR), and radioimmunoassay have been proposed for PSA testing. Despite this, these methods still have disadvantages such as being time-consuming, expensive, inconvenient, and requiring complex instrumentation. Therefore, the development of novel and effective precision diagnostic products for prostate cancer is crucial for the early diagnosis and monitoring of patients. Summary of the Invention

[0004] The purpose of the present invention is to provide a diagnostic kit for prostate cancer based on a biosensor chip to solve the problems of the above-mentioned prior art. The diagnostic kit can detect the miRNA-21 content in urine samples and has the advantages of high specificity and high sensitivity.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a diagnostic kit for prostate cancer based on a biosensor chip, comprising the following components: a biosensor chip, streptavidin-modified gold nanoparticles, tetrahedral DNA, miRNA-helper, H1 chain and H2 chain;

[0007] The nucleotide sequences of the miRNA-helper, the H1 chain and the H2 chain are shown in SEQ ID NOs. 13-15, respectively.

[0008] Furthermore, the tetrahedral DNA is composed of a DNA oligonucleotide chain described in any one of the following (1) to (3):

[0009] (1) S13-1 with a nucleotide sequence as shown in SEQ ID NO.1, S13-2 with a nucleotide sequence as shown in SEQ ID NO.2, S13-3 with a nucleotide sequence as shown in SEQ ID NO.3, and S13-4 with a nucleotide sequence as shown in SEQ ID NO.4;

[0010] (2) S26-1 with a nucleotide sequence as shown in SEQ ID NO.5, S26-2 with a nucleotide sequence as shown in SEQ ID NO.6, S26-3 with a nucleotide sequence as shown in SEQ ID NO.7, and S26-4 with a nucleotide sequence as shown in SEQ ID NO.8;

[0011] (3) S17-1 with a nucleotide sequence as shown in SEQ ID NO.9, S17-2 with a nucleotide sequence as shown in SEQ ID NO.10, S17-3 with a nucleotide sequence as shown in SEQ ID NO.11, and S17-4 with a nucleotide sequence as shown in SEQ ID NO.12;

[0012] The 5' ends of the S13-1, the S13-2, the S13-3, the S26-1, the S26-2, the S26-3, the S17-1, the S17-2 and the S17-3 are all modified with SH-(CH2)6 groups.

[0013] Preferably, the tetrahedral DNA consists of the S17-1, the S17-2, the S17-3 and the S17-4.

[0014] Furthermore, the tetrahedral DNA is obtained by incubating the S17-1, the S17-2, the S17-3 and the S17-4 with PCR.

[0015] Furthermore, the PCR incubation reaction temperature is 95° C. and the time is 10 min.

[0016] Furthermore, the biosensor chip is a LifeDisc™ MetaSPR biosensor chip.

[0017] Furthermore, the streptavidin-modified gold nanoparticles are obtained by mixing streptavidin and gold nanoparticles.

[0018] Furthermore, the temperature of the mixing reaction is 20-30° C., and the time is 5-15 min.

[0019] Furthermore, the gold nanoparticles are 10-30 nm gold nanoparticles.

[0020] Preferably, the gold nanoparticles are 16 nm gold nanoparticles.

[0021] The present invention discloses the following technical effects:

[0022] The present invention has developed a diagnostic kit for prostate cancer based on a biosensor chip, which can detect the miRNA-21 content in urine samples. It has the advantages of strong specificity and high sensitivity. Compared with traditional PSA tests, the specificity of diagnosis using the kit of the present invention is as high as over 90%, which is significantly better than traditional PSA tests.

[0023] To improve detection performance and adapt to diverse application scenarios, the present invention adopts multiple strategies. First, to enhance detection accuracy and sensitivity, the present invention introduces hybridization chain reaction (HCR) amplification technology and surface plasmon resonance (SPR) chip-enhanced signal amplification technology. HCR amplification technology significantly amplifies the detection signal by initiating cyclic hybridization reactions of the chain to form long-chain DNA nanostructures; while the SPR chip utilizes the resonance phenomenon generated by the interaction of surface plasmon waves with the substance to be detected, achieving further signal enhancement by optimizing chip design and detection conditions. This invention provides reliable technical support for the early monitoring, prognosis, and efficacy evaluation of clinical prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1TDN electrophoresis diagram; A is TDN-13 electrophoresis diagram, 1: S13-1, 2: S13-2, 3: S13-3, 4: S13-4, 5: S13-1+S13-2, 6: S13-1+S13-2+S13-3, 7: S13-2+S13-3+S13-4, 8: S13-1+S13-2+S13-3+S13-4, 9: DL5000 DNA Marker; B is the electrophoresis diagram of TDN-17, 1: S17-1, 2: S17-2, 3: S17-3, 4: S17-4, 5: S17-1+S17-2, 6: S17-1+S17-2+S17-3, 7: S17-2+S17-3+S17-4, 8: DL1000 DNA Marker; C is the electrophoresis diagram of TDN-26, 1: S26-1, 2: S26-2, 3: S26-3, 4: S26-4, 5: S26-1+S26-2, 6: S26-1+S26-2+S26-3, 7: S26-2+S26-3+S26-4, 8: S26-1+S26-2+S26-3+S26-4, 9: DL1000 DNA Marker;

[0026] Figure 2 Figure 2 is the electrophoresis diagram of HCR; A is the electrophoresis diagram of TDN-13-HCR, 1: DL1000 DNA Maker, 2: 1.0 μM H1, 3: 1.0 μM H2, 4-9: 1 μM H1 and H2 mixture containing 0.0, 1.5, 1.0, 0.50, 0.20 and 0.10 μM initiator (miRNA-21); B is the electrophoresis diagram of TDN-17-HCR, 1: 1.0 μM H1, 2: 1.0 μM H2, 3-7: 1 μM H1 and H2 mixture containing 0.0, 1.0, 0.50, 0.20 and 0.10 μM initiator (miRNA-21), 8: DL1000 DNA Maker; C is the electrophoresis diagram of TDN-26-HCR, 1: DL1000 DNA Maker, 2: 1.0 μM H1, 3: 1.0 μM H2, 4-9: 0.0, 1.5, 1.0, 0.50, 0.20, and 0.10 μM of initiator (miRNA-21) in a 1 μM mixture of H1 and H2;

[0027] Figure 3 Figure 2 is the characterization result of AuNPs nanoparticles; A, B and C are TEM images of 10 nm, 16 nm, 33 nm AuNPs and AuNPs-SA, respectively; D, E and F are the results of particle size analysis and statistics of TEM images of A, B and C using ImageJ; scale bars in AC are all 100 nm;

[0028] Figure 4 Optimization results of different Mg 2+ Optimization results of different Mg 2+ Optimization results of different Mg

[0029] Figure 5 Optimization results of different reaction temperature

[0030] Figure 6 Optimization results of different TDN

[0031] Figure 7 Optimization results of different AuNPs

[0032] Figure 8 Specificity of biosensor ** P<0.01, *** P<0.001;

[0033] Figure 9 Sensitivity of biosensor

[0034] Figure 10 Signal change of miRNA-21 in clinical sample

[0035] Figure 11Heat map of miRNA-21 levels in 10 healthy donors (HD), 10 benign prostatic hyperplasia patients (BPH), and 15 prostate cancer patients (PC);

[0036] Figure 12 Statistical graph of miRNA-21 levels in healthy donors (HD), benign prostatic hyperplasia patients (BPH), and prostate cancer patients (PC);

[0037] Figure 13 Receiver operating characteristic curve for healthy donors (HD) and prostate cancer patients (PC);

[0038] Figure 14 Receiver operating characteristic curve for benign prostatic hyperplasia patients (BPH) and prostate cancer patients (PC). DETAILED DESCRIPTION

[0039] Various illustrative embodiments of the present application are now described in detail below. The description made herein is not to be construed as limiting the present application, but rather merely as describing certain aspects, features, and embodiments of the present application.

[0040] It should be understood that the terms used herein are merely descriptive, but that the application should not be construed as being limited thereto. In addition, with respect to numerical ranges in the present application, it is to be understood that every intervening value, to the upper and lower limits, is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is also specifically encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art, but rather that the reference is part of the technical literature that is relevant to A person of ordinary skill in the art.

[0042] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof, and it is to be understood that all such modifications and variations warrant the patentable subject matter under the patent laws. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is to be understood that the application is not to be limited to the specific details given.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] The sequence information involved in the present invention is shown in Table 1.

[0045] Table 1 Sequence information

[0046]

[0047] Example 1

[0048] 1. Tetrahedral DNA (TDN) Synthesis and Identification

[0049] 1. Synthesis of TDN

[0050] DNA oligonucleotide chains S13-1-4, S17-1-4, and S26-1-4 (see Table 1) were purchased from Shanghai Sangon Biotechnology Co., Ltd. TDN was prepared using DNA oligonucleotides S13-1-4, S17-1-4, and S26-1-4, respectively, as follows:

[0051] Transfer 2 μL of each of the four DNA oligonucleotides (100 μM stock solution) to a centrifuge tube. Add 92 μL of TM buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 7.0-8.0) to the tube. Mix thoroughly by vortexing quickly and centrifuge briefly (12,000 rpm, 5 seconds) to allow the liquid to settle to the bottom of the tube. Place the tube in a PCR machine and incubate at 95°C for 10 minutes, then slowly cool to 25°C. This results in a final concentration of 1 μM TDN solution, which should be stored at -20°C.

[0052] 2. Identification of TDN

[0053] 10 μL of the synthesized TDN solution was loaded onto a 10% polyacrylamide gel (PAGE) and electrophoresed in TBE buffer (89 mM Tris-boric acid, 2 mM ethylenediaminetetraacetic acid) (110 V, 50 min). The temperature was controlled below 10 °C throughout the electrophoresis. After the electrophoresis, the gel was removed and placed in Gelred stain (diluted 1:5000) at 25 °C for 15 min. The results were analyzed by ChemiDoc. TM Imaging System (Bio-Rad) instrument scanning imaging, the results are shown in Figure 1 The results showed that the present invention successfully synthesized three types of TDN.

[0054] 2. Hybridization Chain Reaction (HCR) Verification

[0055] H1, H2, miRNA-helper and miRNA-21 sequences (see Table 1) were purchased from Shanghai Biotech Bioengineering Co., Ltd. according to the primer sequence synthesis instruction, and prepared into 100 mM storage solution for standby.

[0056] Before HCR, S4, H1, H2 and miRNA-helper were placed in a PCR instrument at 95 °C for 5 min, and then cooled to 25 °C at a rate of 6 °C / min. 2 μL of S4, H1, H2, miRNA-helper and miRNA-21 (100 μM) oligonucleotides were added to a centrifuge tube, and 90 μL of TM buffer was added to the tube. Then vortexed quickly and uniformly, and then point off, and the centrifuge tube was placed at 25 °C for 2 h to obtain the HCR reaction solution. 5 μL of the synthesized HCR reaction solution was mixed with 1 μL of 5× DNA loading buffer (Takara) uniformly, and then loaded into a 2% agarose gel, and electrophoresis was carried out in TBE buffer (89 mM Tris-boric acid, 2 mM ethylenediaminetetraacetic acid) (110 V, 30 min). The results are shown in Figure 1. TM Imaging System (Bio-Rad) instrument scanning imaging, the results are shown in Figure 1. Figure 2 .

[0057] III. Synthesis of gold nanoparticles (AuNPs) of different sizes, streptavidin modification and characterization

[0058] 1. Synthesis of AuNPs of different sizes

[0059] (1) All glassware in the experiment was pre-soaked in freshly prepared aqua regia for 30 min, and then rinsed several times with Milli-Q ultrapure water (DI, >18.25 ) until the container was clean and no aqua regia was left. Put in the oven and dry for standby.

[0060] (2) Preparation of 16 nm AuNPs: In a three-necked flask, prepare 100 mL of 0.01% HAuCl4 aqueous solution and heat and stir to boiling. Then quickly add 3 mL of 1% trisodium citrate solution to the three-necked flask. Observe the color of the solution from gray to red to wine red and keep boiling, count 15 min, slowly cool to 25 °C, filter the AuNPs with a 0.22 μm filter and store at 4 °C. After stabilization, gold nanoparticles with an average diameter of 16.1 ± 0.66 nm are obtained.

[0061] (3) Preparation of 30 nm AuNPs: 30 nm AuNPs were synthesized by hydroquinone reduction. The specific experimental steps are as follows: 100 μL of HAuCl4 solution (1% w / v) was added to a glass bottle containing 9.378 mL of Milli-Q ultrapure water. Next, 400 μL of 16 nm AuNPs seeds were added, and the solution was rapidly stirred at 25°C. 22 μL of sodium citrate solution (1% w / v) was added, followed by the immediate addition of 100 μL of hydroquinone (0.03 M), and the mixture was rapidly stirred at 25°C for 10 min. Finally, the AuNPs were filtered through a 0.22 μm filter and stored at 4°C.

[0062] (4) Preparation of 10 nm AuNPs: 1.0 mL of HAuCl4 (1.0% by mass) was added to 80 mL of Milli-Q ultrapure water and heated to 60°C in a 250 mL conical flask while stirring. Then, a reducing solution was prepared by adding tribasic sodium citrate, tannic acid, and potassium carbonate according to the amounts shown in Table 2 below.

[0063] Table 2 Reduction solution formula

[0064]

[0065] Heat the prepared reducing solution to 60°C and maintain this temperature for 30 minutes. Then, mix the heated reducing solution with the heated HAuCl₄ aqueous solution. When the solution turns red, the reaction is progressing. After the solution turns red, continue the reaction at 60°C for 30 minutes with vigorous stirring. When the tannic acid concentration is low, the reaction may take up to an hour to form larger particles, at which point the solution will turn red. After the reaction, filter and store the gold nanoparticles at 4°C until further use.

[0066] 2. Preparation of Streptavidin-modified AuNPs (AuNPs-SA)

[0067] First, the AuNPs prepared in the previous step were concentrated 10-fold and redispersed in 20 mM borate buffer (pH 7.5). Streptavidin was prepared in borate buffer at varying concentrations (0–100 μg / mL) and added in 50 μL increments to 0.6 mL of AuNP solutions of varying particle sizes (10 nm, 16 nm, and 30 nm). Vortexing was performed after each addition to homogenize the mixture. The mixture was incubated at 25°C for 10 minutes (the reaction conditions can be extended to 20–30°C for 5–15 minutes) to allow the streptavidin to bind to the AuNP surface. To remove excess streptavidin, the mixture was centrifuged, and the precipitate was then resuspended in borate buffer (pH 7.5) to yield AuNPs-SA.

[0068] 3. Characterization of AuNPs and AuNPs-SA

[0069] Take 100 μL of the prepared AuNPs and AuNPs-SA solutions respectively, dilute with pure water until the solution turns light pink, and then aspirate 10 μL and drop it on a sample-loaded copper grid with a pore size of 2 nm. Let it stand at 25°C overnight and observe and photograph it under an electron microscope. Figure 3 AuNPs can be observed in the AC. The AuNPs-SA nanoparticles are spherical and have sizes of about 10 nm, 16 nm, and 33 nm, respectively. The TEM images were analyzed using ImageJ image analysis software. Figure 3 Medium DE.

[0070] 4. Optimization of reaction conditions

[0071] 1. Different Mg 2+ Concentration effect

[0072] (1) Synthesis of TDN: Take 2 µL of each of S17-1, S17-2, S17-3, and S17-4 (100 µM each) and add them to a centrifuge tube. Then add 82 µL of TM buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 8.0). Vortex quickly to mix evenly and centrifuge. Place the centrifuge tube in a PCR instrument, incubate at 95 °C for 10 min, and then slowly reduce the temperature to 25 °C. The final concentration is 1 µM TDN. Add 10 µL of tris(2-carbonylethyl)phosphine hydrochloride (TECP) (30 mM) solution, mix evenly, and incubate at 25 °C for 30 min. Use different MgCl2 concentrations. 2+ Dilute it to 0.2 µM TDN with TM buffer of different concentrations (2 mM, 10 mM, 50 mM) for later use.

[0073] (2) Chip pretreatment: Take the LifeDisc™ MetaSPR biosensor chip (purchased from Liangzhun (Wuhan) Life Science Technology Co., Ltd.), add 50 μL PBS buffer to each well, and detect the OD value at 500-750 nm.

[0074] (3) TDN fixation: Take 50 µL / well of 0.2 µM TDN solution, seal the plate with film, and incubate at 25°C overnight (12-16 hours).

[0075] (4) Washing: Discard the above liquid, wash with PBS three times, add 50 μL / well of PBS buffer again, and detect the OD value at 500-750 nm.

[0076] (5) 6-Mercapto-1-hexanol (MCH) treatment: Add 50 μL / well of 50 mM MCH solution, seal the plate with film, and incubate at 25°C for 30 min. Discard the MCH solution, wash three times with PBS, and add 50 μL / well of PBS buffer again. Detect the OD value at 500-750 nm.

[0077] (6) miRNA-helper treatment: using different Mg 2+ Dilute miRNA-helper (100 µM) to 0.2 µM with TM buffer at various concentrations (2 mM, 10 mM, and 50 mM). Dispense 50 µL / well and incubate at 25°C for 2 hours. Discard the solution, wash three times with PBS, and then add 50 µL / well of PBS buffer. Measure the OD value at 500-750 nm.

[0078] (7) miRNA-21 treatment: using different Mg 2+ MiRNA-21 (100 µM) was diluted to 200 nM, 100 nM, 40 nM, 20 nM, and 10 nM using TM buffer at different concentrations (2 mM, 10 mM, and 50 mM). Three replicate wells were prepared for each concentration, with 50 µL / well dispensed. The cells were incubated at 25°C for 2 hours. The solution was discarded, and the cells were washed three times with PBS. 50 µL / well of PBS was added again, and the OD values ​​were measured at 500-750 nm.

[0079] (8) H1 and H2 treatment: using different Mg 2+ Dilute H1 and H2 (100 µM) to a final concentration of 0.2 µM with TM buffer at various concentrations (2 mM, 10 mM, and 50 mM). Dispense 50 µL / well and incubate at 25°C for 2 hours. Discard the solution, wash three times with PBS, and then add 50 µL / well of PBS buffer. Measure the OD value at 500-750 nm.

[0080] (9) AuNPs-SA treatment: Add 16 nm AuNPs-SA and incubate at 25°C for 1 hour. Discard the liquid, wash with PBS buffer three times, add 50 μL / well of PBS buffer again, and measure the OD value at 500-750 nm.

[0081] (10) Data processing: Statistical OD value difference result = OD (AuNPs-SA) -OD (miRNA-helper) .

[0082] Figure 4 The results showed that when Mg 2+ The signal response was strongest at a concentration of 10 mM.

[0083] 2. Effect of different temperatures

[0084] (1) Synthesis of TDN: Take 2 µL of each of S17-1, S17-2, S17-3, and S17-4 (100 µM each) and add them to a centrifuge tube. Then add 82 µL of TM buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 8.0). Vortex quickly to mix thoroughly and centrifuge. Place the centrifuge tube in a PCR instrument and incubate at 95 °C for 10 min, then slowly reduce the temperature to 25 °C. The final concentration is 1 µM TDN. Add 10 µL of TECP (30 mM) solution, mix thoroughly, and incubate at 25 °C for 30 min. Dilute it to 0.2 µM TDN using TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0) for later use.

[0085] (2) Chip pretreatment: Take the LifeDisc™ MetaSPR biosensor chip, add 50 μL PBS buffer to each well, and detect the OD value at 500-750 nm.

[0086] (3) TDN fixation: Take 50 µL / well of 0.2 µM TDN solution, seal the plate with film, and incubate at 4°C, 25°C, and 37°C overnight (12-16 hours).

[0087] (4) Washing: Discard the above liquid, wash with PBS three times, add 50 μL / well of PBS buffer again, and detect the OD value at 500-750 nm.

[0088] (5) MCH treatment: Add 50 μL / well of 50 mM MCH solution, seal the plate with film, and incubate at 25°C for 30 min. Discard the MCH solution, wash three times with PBS buffer, add 50 μL / well of PBS buffer again, and measure the OD value at 500-750 nm.

[0089] (6) miRNA-helper treatment: miRNA-helper (100 µM) was diluted to 0.2 µM with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0), 50 µL / well was taken and incubated at 4°C, 25°C and 37°C for 2 hours respectively. The above liquid was discarded, washed with PBS buffer for 3 times, and then 50 µL / well of PBS buffer was added again, and the OD value at 500-750 nm was detected.

[0090] (7) miRNA-21 treatment: miRNA-21 (100 µM) was diluted to 200 nM, 100 nM, 40 nM, 20 nM and 10 nM respectively with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0), and 3 replicates were set for each concentration, 50 µL / well was taken and incubated at 4°C, 25°C and 37°C for 2 hours respectively. The remaining liquid was discarded, washed with PBS buffer for 3 times, and then 50 µL / well of PBS buffer was added again, and the OD value at 500-750 nm was detected.

[0091] (8) H1 and H2 treatment: H1 and H2 (100 µM) were diluted to a final concentration of 0.2 µM with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0), 50 µL / well was taken and incubated at 4°C, 25°C and 37°C for 2 hours respectively. The above liquid was discarded, washed with PBS buffer for 3 times, and then 50 µL / well of PBS buffer was added again, and the OD value at 500-750 nm was detected.

[0092] (9) AuNPs-SA treatment: 16 nm AuNPs-SA was added and incubated at 4°C, 25°C and 37°C for 2 hours respectively. The remaining liquid was discarded, washed with PBS buffer for 3 times, and then 50 µL / well of PBS buffer was added again, and the OD value at 500-750 nm was detected.

[0093] (10) Data processing: the difference of OD value = OD (AuNPs-SA) - OD (miRNA-helper) .

[0094] Figure 5 The results showed that when the reaction temperature was 25°C, the signal response was the strongest.

[0095] 3. Influence of different sizes of TDN

[0096] (1) Synthesis of TDN-13, TDN-17, and TDN-26: TDN-13: 2 μL of each of S13-1, S13-2, S13-3, and S13-4 (100 μM each) were added to a centrifuge tube, and then 82 μL of TM buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 8.0) was added. TDN-17: 2 μL of each of S17-1, S17-2, S17-3, and S17-4 (100 μM each) were added to a centrifuge tube, and then 82 μL of TM buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 8.0) was added. TDN-26: 2 μL of each of S26-1, S26-2, S26-3, and S26-4 (100 μM each) were added to a centrifuge tube, and then 82 μL of TM buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 8.0) was added. Mix thoroughly with 100 mM Tris-HCl, 50 mM MgCl2, pH 8.0. Vortex quickly to mix thoroughly and then centrifuge. Place the tube in a PCR instrument and incubate at 95°C for 10 minutes. Then slowly reduce the temperature to 25°C to prepare a final concentration of 1 µM for TDN-13, TDN-17, and TDN-26. Add 10 µL of 30 mM TECP solution, mix thoroughly, and incubate at 25°C for 30 minutes. Dilute to 0.2 µM TDN using TM buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 8.0) before use.

[0097] (2) Chip pretreatment: Take the LifeDisc™ MetaSPR biosensor chip, add 50 μL PBS buffer to each well, and detect the OD value at 500-750 nm.

[0098] (3) TDN fixation: Take 50 µL / well of 0.2 µM TDN-13, TDN-17, or TDN-26 solution, seal the plate with film, and incubate at 25°C overnight (12-16 hours).

[0099] (4) Washing: Discard the above liquid, wash with PBS buffer three times, add 50 μL / well of PBS buffer again, and detect the OD value at 500-750 nm.

[0100] (5) MCH treatment: Add 50 μL / well of 50 mM MCH solution, seal the plate with film, and incubate at 25°C for 30 min. Discard the MCH solution, wash three times with PBS buffer, add 50 μL / well of PBS buffer again, and measure the OD value at 500-750 nm.

[0101] (6) Treatment with miRNA-helper: Dilute miRNA-helper (100 µM) to 0.2 µM with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0), add 50 µL / well, and incubate at 25°C for 2 h. Discard the above liquid, wash three times with PBS buffer, add 50 µL / well of PBS buffer again, and measure the OD value at 500-750 nm.

[0102] (7) Treatment with miRNA-21: miRNA-21 (100 µM) was diluted to 200 nM, 100 nM, 40 nM, 20 nM, and 10 nM using TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0), with 50 µL / well added. The cells were incubated at 25°C for 2 h. The remaining liquid was discarded, and the cells were washed three times with PBS buffer. 50 µL / well of PBS buffer was added, and the OD value at 500-750 nm was measured.

[0103] (8) H1 and H2 treatment: H1 (100 µM) and H2 (100 µM) were diluted to a final concentration of 0.2 µM using TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0). 50 µL / well of each concentration was added in triplicate and incubated at 25°C for 2 h. The cells were discarded and washed three times with PBS buffer. 50 µL / well of PBS buffer was added again, and the OD values ​​at 500-750 nm were measured.

[0104] (9) AuNPs-SA treatment: Add 16 nm AuNPs-SA and incubate at 25°C for 1 hour. Discard the remaining liquid, wash with PBS buffer three times, add 50 μL / well of PBS buffer again, and measure the OD value at 500-750 nm.

[0105] (10) Data processing: Calculate the difference in OD values ​​= OD (AuNPs-SA) -OD (miRNA-helper) .

[0106] Figure 6 The results show that when the TDN size is 17, the signal response is the strongest.

[0107] 4. Effect of different sizes of AuNPs (10 nm, 16 nm, 30 nm)

[0108] (1) Synthesis of TDN: Take 2 μL of each of S17-1, S17-2, S17-3, S17-4 (100 μM each) and add to a centrifuge tube, then add 82 μL of TM buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 8.0). Mix well by vortexing quickly and then spot. Place the centrifuge tube in a PCR machine and incubate at 95 °C for 10 min, then slowly reduce to 25 °C. The final concentration is 1 μM TDN. Add 10 μL of TECP (30 mM) solution and mix well, then incubate at 25 °C for 30 min. Dilute to 0.2 μM TDN with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0) and store for later use.

[0109] (2) Chip pretreatment: Take LifeDisc™ MetaSPR biosensor chip and add 50 μL of PBS buffer to each well, then detect the OD value at 500-750 nm.

[0110] (3) TDN immobilization: Take 50 μL of 0.2 μM TDN-17 solution per well, seal with a plate membrane, and incubate at 25 °C overnight (12-16 hours).

[0111] (4) Washing: Discard the above liquid and wash with PBS buffer 3 times, then add 50 μL of PBS buffer per well again, and detect the OD value at 500-750 nm.

[0112] (5) MCH treatment: Add 50 μL of 50 mM MCH solution per well, seal with a plate membrane, and incubate at 25 °C for 30 min. Discard the MCH solution and wash with PBS buffer 3 times, then add 50 μL of PBS buffer per well again, and detect the OD value at 500-750 nm.

[0113] (6) miRNA-helper treatment: Dilute the miRNA-helper (100 μM) to 0.2 μM with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0), take 50 μL per well, and incubate at 25 °C for 2 hours. Discard the above liquid and wash with PBS buffer 3 times, then add 50 μL of PBS buffer per well again, and detect the OD value at 500-750 nm.

[0114] (7) miRNA-21 treatment: miRNA-21 (100 µM) was diluted to 200 nM, 100 nM, 40 nM, 20 nM, 10 nM, respectively, with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0), 50 µL / well for each concentration, 3 replicates, incubated at 25°C for 2 hours. Discard the remaining liquid, wash 3 times with PBS buffer, add 50 µL / well of PBS buffer, and detect the OD value at 500-750 nm.

[0115] (8) H1, H2 treatment: H1 (100 µM) and H2 (100 µM) were diluted to a final concentration of 0.2 µM with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0), 50 µL / well, incubated at 25°C for 2 hours. Discard the above liquid, wash 3 times with PBS buffer, add 50 µL / well of PBS buffer again, and detect the OD value at 500-750 nm.

[0116] (9) AuNPs-SA treatment: 10 nm, 16 nm, 33 nm AuNPs-SA were added, incubated at 25°C for 1 hour. Discard the remaining liquid, wash 3 times with PBS buffer, add 50 µL / well of PBS buffer again, and detect the OD value at 500-750 nm.

[0117] (10) Data processing: calculate the difference in OD value = OD (AuNPs-SA) - OD (miRNA-helper) .

[0118] Figure 7 The results show that the signal of 16 nm AuNPs-SA is the strongest, so 16 nm AuNPs-SA is selected for subsequent experiments.

[0119] Five, detection specificity verification

[0120] (1) Synthesis of TDN: Take 2 µL of each of S17-1, S17-2, S17-3, and S17-4 (100 µM each) and add them to a centrifuge tube. Then add 82 µL of TM buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 8.0). Vortex quickly to mix thoroughly and centrifuge. Place the centrifuge tube in a PCR instrument and incubate at 95 °C for 10 min, then slowly reduce the temperature to 25 °C. The final concentration is 1 µM TDN. Add 10 µL of TECP (30 mM) solution, mix thoroughly, and incubate at 25 °C for 30 min. Dilute it to 0.2 µM TDN using TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0) for later use.

[0121] (2) Chip pretreatment: Take the LifeDisc™ MetaSPR biosensor chip, add 50 μL PBS buffer to each well, and detect the OD value at 500-750 nm.

[0122] (3) TDN fixation: Take 50 µL / well of 0.2 µM TDN-17 solution, seal the plate with film, and incubate at 25°C overnight (12-16 hours).

[0123] (4) Washing: Discard the above liquid, wash with PBS buffer three times, add 50 μL / well of PBS buffer again, and detect the OD value at 500-750 nm.

[0124] (5) MCH treatment: Add 50 μL / well of 50 mM MCH solution, seal the plate with film, and incubate at 25°C for 30 min. Discard the MCH solution, wash three times with PBS buffer, add 50 μL / well of PBS buffer again, and measure the OD value at 500-750 nm.

[0125] (6) Treatment with miRNA-helper: Dilute miRNA-helper (100 µM) to 0.2 µM with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0), add 50 µL / well, and incubate at 25°C for 2 h. Discard the above liquid, wash three times with PBS buffer, add 50 µL / well of PBS buffer again, and measure the OD value at 500-750 nm.

[0126] (7) miRNA-21, miRNA-21(M)1, miRNA-21(M)2, miRNA-21(M)3, miRNA-122, miRNA-NC treatment: miRNA-21, miRNA-21(M)1, miRNA-21(M)2, miRNA-21(M)3, miRNA-122, miRNA-NC (100 µM) were diluted to 2 nM with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0), 3 replicates were set for each concentration, 50 µL / well, incubated at 25°C for 2 hours. Discard the remaining liquid, wash 3 times with PBS buffer, add 50 µL / well of PBS buffer, and detect the OD value at 500-750 nm.

[0127] (8) H1, H2 treatment: H1 (100 µM) and H2 (100 µM) were diluted to a final concentration of 0.2 µM with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0), 50 µL / well, incubated at 25°C for 2 hours. Discard the above liquid, wash 3 times with PBS buffer, add 50 µL / well of PBS buffer again, and detect the OD value at 500-750 nm.

[0128] (9) AuNPs-SA treatment: 16 nm AuNPs-SA was added, incubated at 25°C for 1 hour. Discard the remaining liquid, wash 3 times with PBS buffer, add 50 µL / well of PBS buffer again, and detect the OD value at 500-750 nm.

[0129] (10) Data processing: the difference result of OD value = OD (AuNPs-SA) - OD (miRNA-helper) .

[0130] Figure 8 The results show that the biosensor can specifically bind miRNA-21 compared with the control group.

[0131] Six, detection sensitivity verification

[0132] (1) Synthesis of TDN: Take 2 μΐ of each of S17-1, S17-2, S17-3, S17-4 (100 μΜ each) and add to a centrifuge tube, then add 82 μΐ of TM buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 8.0). Mix well by vortexing quickly and then spot off. Place the centrifuge tube in a PCR instrument and incubate at 95 °C for 10 min, then slowly reduce to 25 °C. The final concentration is 1 μΜ TDN. Add 10 μΐ of TECP (30 mM) solution and mix well, then incubate at 25 °C for 30 min. Dilute to 0.2 μΜ TDN with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0) and store for later use.

[0133] (2) Chip pretreatment: Take LifeDisc™ MetaSPR biosensor chip and add 50 μΐ of PBS to each well, then detect the OD value at 500-750 nm.

[0134] (3) TDN immobilization: Take 50 μΐ of 0.2 μΜ TDN-17 solution per well, seal with a plate film, and incubate at 25 °C overnight (12-16 hours).

[0135] (4) Washing: Discard the above liquid and wash 3 times with PBS buffer, then add 50 μΐ of PBS buffer per well again, and detect the OD value at 500-750 nm.

[0136] (5) MCH treatment: Add 50 μΐ of 50 mM MCH solution per well, seal with a plate film, and incubate at 25 °C for 30 min. Discard the MCH solution and wash 3 times with PBS buffer, then add 50 μΐ of PBS buffer per well again, and detect the OD value at 500-750 nm.

[0137] (6) miRNA-helper treatment: Dilute the miRNA-helper (100 μΜ) to 0.2 μΜ with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0) and take 50 μΐ per well, then incubate at 25 °C for 2 hours. Discard the above liquid and wash 3 times with PBS buffer, then add 50 μΐ of PBS buffer per well again, and detect the OD value at 500-750 nm.

[0138] (7) miRNA-21 treatment: miRNA-21 (100 µM) was diluted to 2nM, 200 pM, 20 pM, 2 pM, 200 fM, 20 fM, respectively, with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0), 50 µL / well was taken, and incubated at 25°C for 2 hours. Discard the remaining liquid, wash with PBS buffer for 3 times, add PBS 50 µL / well, and detect the OD value at 500-750 nm.

[0139] (8) H1, H2 treatment: H1 (100 µM) and H2 (100 µM) were diluted to a final concentration of 0.2 µM with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0), 50 µL / well was taken, and incubated at 25°C for 2 hours. Discard the above liquid, wash with PBS buffer for 3 times, add PBS buffer 50 µL / well again, and detect the OD value at 500-750 nm.

[0140] (9) AuNPs-SA treatment: 16 nm AuNPs-SA was added, and incubated at 25°C for 1 hour. Discard the remaining liquid, wash with PBS buffer for 3 times, add PBS buffer 50 µL / well again, and detect the OD value at 500-750 nm.

[0141] (10) Data processing: the difference result of OD value = OD (AuNPs-SA) - OD (miRNA-helper) .

[0142] Figure 9 The results show that there is a linear relationship between the signal response and the concentration.

[0143] Example 2

[0144] A diagnostic kit for prostate cancer based on a biosensor chip, comprising the following components:

[0145] a biosensor chip, such as LifeDisc™ MetaSPR biosensor chip;

[0146] tetrahedral DNA, such as TDN-17 (see Example 1 for preparation method);

[0147] miRNA-helper, see Table 1 for specific sequence;

[0148] H1 chain and H2 chain, see Table 1 for specific sequence;

[0149] AuNPs-SA, such as 16 nm AuNPs-SA (see Example 1 for preparation method).

[0150] Example 3

[0151] Clinical specimen detection was performed using the diagnostic kit of Example 2:

[0152] 1. RNA Extraction from Urine Sediment

[0153] (1) Collect 50 mL of morning urine from patients with prostate cancer, prostate hyperplasia, and healthy patients, centrifuge at 1500 g for 5 minutes, discard the supernatant, collect the urine sediment, add 500 μL of Trizol, vortex and mix, and let it stand on ice for 5 minutes;

[0154] (2) Add 1 / 5 volume of chloroform (100 μL) to each tube, vortex thoroughly for 15 seconds, precipitate on ice for 5 minutes, and centrifuge at 12,000 rpm at 4°C for 5 minutes.

[0155] (3) Prepare a new 1.5 mL EP tube and mark it. Take 250 μL of the supernatant and transfer it to the corresponding tube. Add an equal volume of isopropanol, invert and mix 10 times, and let it settle on ice for 15 min.

[0156] (4) Centrifuge at 12,000 rpm for 15 min at 4°C and discard the supernatant.

[0157] (5) Prepare 75% ethanol in advance and pre-cool it on ice. Add 1 mL of 75% ethanol to each tube to rinse the RNA precipitate. Centrifuge at 12,000 rpm at 4°C for 5 min, discard the supernatant, then empty the tube for 1 min, discard the remaining ethanol, and place the precipitate at 42°C to dry.

[0158] (6) Add 15 μL of DEPC water to dissolve the RNA precipitate and store at -30°C.

[0159] 2. Detection of miRNA-21 content in clinical specimens using biosensors

[0160] (1) Dilute TDN-17 to 0.2 μM using TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0).

[0161] (2) Chip preprocessing: Get LifeDisc TM MetaSPR biosensor chip, add 50 μL PBS to each well, and detect the OD value at 500-750 nm.

[0162] (3) TDN fixation: Take 50 µL / well of 0.2 µM TDN-17 solution, seal the plate with film, and incubate at 25°C overnight (12-16 hours).

[0163] (4) Washing: Discard the above liquid, wash with PBS buffer three times, add 50 μL / well of PBS buffer again, and detect the OD value at 500-750 nm.

[0164] (5) MCH treatment: Add 50 μL / well of 50 mM MCH solution, seal the plate with film, and incubate at 25°C for 30 min. Discard the MCH solution, wash three times with PBS buffer, add 50 μL / well of PBS buffer again, and measure the OD value at 500-750 nm.

[0165] (6) Treatment with miRNA-helper: Dilute miRNA-helper (100 µM) to 0.2 µM with TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0), add 50 µL / well, and incubate at 25°C for 2 h. Discard the above liquid, wash three times with PBS buffer, add 50 µL / well of PBS buffer again, and measure the OD value at 500-750 nm.

[0166] (7) Treatment of clinical specimen RNA: Dilute the clinical specimen RNA to 50 ng / µL using TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0). Set up three replicate wells for each specimen, take 50 µL / well, and incubate at 25°C for 2 hours. Discard the remaining liquid, wash three times with PBS buffer, add 50 µL / well of PBS buffer, and measure the OD value at 500-750 nm.

[0167] (8) H1 and H2 treatment: H1 (100 µM) and H2 (100 µM) were diluted to a final concentration of 0.2 µM using TM buffer (20 mM Tris-HCl, 10 mM MgCl2, pH 8.0). 50 µL / well was added and incubated at 25°C for 2 h. The above liquid was discarded, and the cells were washed three times with PBS buffer. 50 µL / well of PBS buffer was added again, and the OD value at 500-750 nm was measured.

[0168] (9) AuNPs-SA treatment: Add 16 nm AuNPs-SA and incubate at 25°C for 1 hour. Discard the remaining liquid, wash with PBS buffer three times, add 50 μL / well of PBS buffer again, and measure the OD value at 500-750 nm.

[0169] (10) Data processing: Difference in OD value = OD (AuNPs-SA) -OD (miRNA-helper) .

[0170] Figure 10- Figure 14The results show that the miRNA-21 content in urine of the prostate patients is obviously higher than that of the healthy control group.

[0171] The above-described embodiments are merely intended to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application defined by the claims.

Claims

1. A diagnostic kit for prostate cancer based on a biosensor chip, characterized in that: The invention comprises the following components: a biosensor chip, streptavidin-modified gold nanoparticles, tetrahedral DNA, miRNA-helper, H1 chain and H2 chain; The nucleotide sequences of the miRNA-helper, the H1 chain, and the H2 chain are shown in SEQ ID NOs. 13-15, respectively; The tetrahedral DNA is composed of the following DNA oligonucleotide chains: S17-1 having a nucleotide sequence as shown in SEQ ID NO.9, S17-2 having a nucleotide sequence as shown in SEQ ID NO.10, S17-3 having a nucleotide sequence as shown in SEQ ID NO.11, and S17-4 having a nucleotide sequence as shown in SEQ ID NO.12; The 5' ends of the S17-1, S17-2, and S17-3 are all modified with SH-(CH2)6 groups; The gold nanoparticles are 16 nm gold nanoparticles.

2. The diagnostic kit according to claim 1, characterized in that The tetrahedral DNA is obtained by subjecting the S17-1, the S17-2, the S17-3 and the S17-4 to a PCR incubation reaction.

3. The diagnostic kit according to claim 2, characterized in that The PCR incubation reaction temperature was 95° C. and the time was 10 min.

4. The diagnostic kit according to claim 1, characterized in that The biosensor chip is a LifeDisc™ MetaSPR biosensor chip.

5. The diagnostic kit according to claim 1, characterized in that The streptavidin-modified gold nanoparticles are obtained by mixing streptavidin and gold nanoparticles.

6. The diagnostic kit according to claim 5, characterized in that The temperature of the mixing reaction is 20-30° C., and the time is 5-15 minutes.

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