Kit for detecting alpha-synapsin oligomer and application thereof

Abstract

The invention relates to a kit for detecting alpha-synapsin oligomer and application of the kit, and belongs to the technical field of protein detection. Wherein a combination pad of the lateral flow test paper contains gold nanoparticles coupled with a first split aptamer and first DNA for quality control, and a detection line of the lateral flow test paper is loaded with a biotinylated second split aptamer combined with streptavidin; a biotinylation second quality control DNA combined with streptavidin is loaded on a quality control line of the lateral flow test paper; the base sequence of the first split aptamer is as shown in SEQ ID No. 1, and the base sequence of the second split aptamer is as shown in SEQ ID No. 2; the first DNA for quality control and the second DNA for quality control comprise complementary base sequences. According to the invention, the simple, portable, economical and efficient lateral chromatography test paper is established by utilizing the characteristic of the split aptamer (two chains can form a correct secondary structure through hybridization and develop color only in the presence of a target), and early diagnosis and visual analysis of a PD patient are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein detection, and in particular relates to a kit for detecting alpha-synaptophysin oligomers and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Parkinson's disease (PD) is the second most common neurodegenerative disorder. Currently, the diagnosis of PD relies primarily on typical motor symptoms and imaging findings. However, when PD patients develop typical clinical symptoms, it indicates that the disease has entered the mid-to-late stage. At this stage, 50%-70% of dopaminergic neurons in the patient's substantia nigra have degenerated, significantly reducing the effectiveness of drug treatment. Early and accurate diagnosis can help identify PD patients in the prodromal or preclinical stages of the disease, enabling them to receive neuroprotective treatment as early as possible, thereby improving treatment outcomes and prognosis for PD patients.

[0004] The neuropathological hallmarks of Parkinson's disease (PD) include the formation of Lewy bodies (LBs) and degeneration and loss of dopaminergic neurons in the substantia nigra, striatum, and other brain regions. LBs are primarily composed of abnormally aggregated and misfolded α-synuclein (α-syn), also known as α-syn oligomers. As a misfolded form of the protein, α-syn oligomers have been shown to play a role in the pathogenesis of PD. They are not only abnormally elevated in various biological fluids, including saliva, blood, and cerebrospinal fluid, but can also be detected in peripheral tissues, including the skin, olfactory bulb, and salivary glands, with levels significantly elevated compared to healthy controls. Because abnormal α-syn oligomer levels often precede the onset of motor symptoms, α-syn oligomers are considered a characteristic biomarker, crucial for the early diagnosis, disease monitoring, and therapeutic efficacy evaluation of PD.

[0005] Saliva, due to its non-invasive nature, can be readily obtained as a test fluid. Prior art uses aptamers in electrochemical sensors to detect α-syn oligomers. The principle is that adsorption of aptamers labeled with FAM (an azide derivative) onto graphene oxide quenches FAM fluorescence; binding of the aptamers to α-syn oligomers restores FAM fluorescence. Previous detection methods required expensive and complex instrumentation and specialized technicians, resulting in time-consuming and costly procedures.

[0006] Lateral flow chromatography (LFC) is a membrane-based sensor, most commonly implemented as a lateral flow test strip. These strips consist of five components: a sample pad, a conjugate pad (also known as a conjugate pad), a nitrocellulose membrane, absorbent paper, and a backing plate. Detection and self-testing of the test strip's performance are achieved by observing changes in the color depth or signal intensity of the test and control lines on the strip, thereby diagnosing the target and performing a self-test. Summary of the Invention

[0007] To address the shortcomings of the existing technology, the present invention provides a kit for detecting α-synuclein oligomers and its application. This kit utilizes split-α-syn oligomer-specific aptamers and gold nanoparticles (AuNPs) as biorecognition elements. The AuNPs and the detection line of a test strip each contain two segments of the split aptamer. In the presence of α-syn oligomers, the gold nanoparticle conjugate, α-syn oligomers, and the split aptamer on the detection line form an "aptamer-α-syn oligomer-aptamer" sandwich structure, trapping the AuNPs on the detection line and coloring the test strip. This color change can be observed directly with the naked eye, enabling visual analysis of PD diagnosis.

[0008] In order to achieve the above object, the technical solution of the present invention is: In a first aspect, a kit for detecting α-synaptophysin oligomers comprises a lateral flow test strip, wherein the conjugate pad of the lateral flow test strip contains gold nanoparticles coupled to a first split aptamer and a first quality control DNA, the detection line of the lateral flow test strip carries a biotinylated second split aptamer bound to streptavidin, and the quality control line of the lateral flow test strip carries a biotinylated second quality control DNA bound to streptavidin; The base sequence of the first split aptamer is shown as SEQ ID No. 1, and the base sequence of the second split aptamer is shown as SEQ ID No. 2; the first quality control DNA and the second quality control DNA include complementary base sequences.

[0009] Optionally, the kit includes a running buffer for maintaining pH stability and ion balance in the reaction environment and a protease inhibitor for preventing degradation of the target protein, wherein the running buffer has a pH of 7.5-8.0 and includes the following components at the following concentrations: 50-60 mM HEPES, 100-120 mM NaCl, 5-20 mM MgCl2, and 1.0-1.5 wt% PVP.

[0010] In a second aspect, a method for preparing the lateral flow test paper in the above-mentioned kit for detecting α-synaptophysin oligomers comprises: S1, mixing the TCEP-treated first split aptamer, the TCEP-treated first quality control aptamer, and gold nanoparticles (AuNPs) in a solution at a molar ratio of (100-120):(100-120):1, and removing the unconjugated aptamer to obtain a gold nanoparticle conjugate; S2, adding a stabilizer of a set concentration to the dispersion containing the gold nanoparticle conjugate and spraying the stabilizer onto the conjugate pad, and drying the conjugate pad for later use; S3. Mixing and incubating the biotinylated second split aptamer with streptavidin, removing unbound streptavidin and biotinylated second split aptamer, obtaining a streptavidin-second split aptamer, and immobilizing it on a nitrocellulose membrane to form a test line; mixing and incubating the biotinylated second quality control aptamer with streptavidin, removing unbound streptavidin and biotinylated second quality control aptamer, obtaining a streptavidin-biotinylated second quality control aptamer, and immobilizing it on the same nitrocellulose membrane to form a quality control line; the test line and the quality control line are separated by a set distance; S4. Assemble the sample pad, the conjugate pad, the nitrocellulose membrane, the absorbent paper and the back plate into a lateral flow test paper.

[0011] Optionally, in the above process, the base sequences of the first split aptamer and the second split aptamer are obtained by screening, and the screening method includes: ITC measuring the thermodynamic curves thereof with α-syn monomer and α-syn oligomer, respectively, and selecting the first split aptamer and the second split aptamer with the smallest dissociation constant.

[0012] Optionally, in S1, the TCEP treatment method includes: mixing TCEP with the first split aptamer or the first quality control aptamer in a molar ratio of (50-60):1 and incubating for 2-3 hours.

[0013] Optionally, in S2, the concentration of the solution containing the gold nanoparticle conjugate is 40 nM, the concentration of the stabilizer is 10-20 wt%, and the spraying amount is 2-5 μL / cm.

[0014] Optionally, in S2, the stabilizer is selected from one or more of sucrose, tris(hydroxymethyl)aminomethane (Tris) and hydrogen chloride (HCl).

[0015] Optionally, in S3, the mixed incubation method includes: incubating at room temperature for 1 to 2 hours.

[0016] Optionally, in S3, the method for removing unbound streptavidin-biotinylated second split aptamer includes: centrifuging the incubated mixture at 12,000-14,000 rpm for 20-40 min, resuspending the precipitate with PBS buffer, and then repeating the operation twice.

[0017] Optionally, in S3, the method for removing unbound streptavidin-biotinylated second quality control adaptor includes: centrifuging the incubated mixture at 12,000 to 14,000 rpm for 20 to 40 minutes, resuspending the precipitate with PBS buffer, and then repeating the operation twice.

[0018] Optionally, in S4, the sample pad preparation method includes: soaking the sample pad material in a glass fiber solution for 5 to 7 minutes, and then drying it in an oven at 30 to 37°C, wherein the glass fiber solution has a pH of 7.5 to 8.5 and includes the following components at the following concentrations: 20 to 30 mM Tris-HCl, 150 to 160 mM NaCl, and 0.25 to 0.35 wt% Triton X-100.

[0019] In a third aspect, the use of the above-mentioned kit for detecting α-synaptophysin oligomers in detecting PD includes: dissolving the freeze-dried saliva sample with a running buffer, dripping it onto the lateral flow test paper, and observing and analyzing the color development results of the test line and the quality control line.

[0020] Optionally, the method for preparing the saliva sample includes: adding protease inhibitors to the saliva collected from the subject, centrifuging to retain the supernatant, and freeze-drying.

[0021] Optionally, the method of centrifuging and retaining the supernatant includes: centrifuging at a speed of 3500-4000 rpm for 10-20 minutes to remove impurities and retain the supernatant, and then centrifuging at a speed of 15000-17000 rpm for 20-40 minutes to remove bacteria contained in the saliva.

[0022] One or more of the above technical solutions have the following beneficial effects: 1. This invention combines split aptamers with lateral flow chromatography technology to create a simple, portable, cost-effective lateral flow test strip. This method utilizes the properties of split aptamers, splitting the intact aptamer at a site unrelated to binding, forming two nucleic acid strands. Only in the presence of the target can the two strands hybridize to form the correct secondary structure and develop color. In the absence of the target, the two split aptamer segments exist independently and do not develop color. This test strip is used to detect α-syn oligomers in the saliva of patients with Parkinson's disease (PD), enabling early diagnosis and visual analysis of PD.

[0023] 2. The present invention utilizes isothermal calorimetric titration to screen suitable split aptamer sequences, screens the split aptamers used in the test strips, and optimizes the parameters of the constructed test strips, thereby obtaining the test strips with optimal performance.

[0024] 3. This study uses saliva as a biofluid, rather than cerebrospinal fluid or blood. This is because obtaining cerebrospinal fluid is cumbersome and can cause unnecessary damage to the patient. Ruptured red blood cells in blood also contain α-synuclein, and improper blood collection and storage can easily affect test results. Saliva, as a viable biofluid, is easily accessible and less susceptible to influences from other substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0026] Figure 1 This is a schematic diagram of the lateral flow test strip principle and operating process in Example 1 of the present invention; wherein, (A): PD patients collect naturally flowing saliva after rinsing their mouths; (B): The collected saliva is centrifuged to remove impurities and then added dropwise to a lateral flow test strip to detect α-syn in the saliva; (C) shows the operating principle of the lateral flow test strip.

[0027] Figure 2 1-14: 50% IEC Buffer B; (D): ThT monitoring 150 μM During the aggregation process of α-syn, the fluorescence intensity of ThT was measured every two hours (excitation wavelength: 444 nm, emission wavelength: 485 nm), and the experimental data were fitted with a sigmoid curve.

[0028] Figure 3Figure 1 is a thermodynamic diagram of ITC characterization of intact and split aptamers and an isothermal calorimetric titration curve at 25°C in Example 1 of the present invention; wherein, (A): 10 μM intact aptamer and 2.5 μM α-syn oligomer, (B): 10 μM intact aptamer and 2.5 μM α-syn monomer, (C): 10 μM S-Apt 1 and 2.5 μM α-syn oligomer, (D): 10 μM S-Apt 2 and 2.5 μM α-syn oligomer.

[0029] Figure 4 1 is a diagram showing the characterization results of the gold nanoparticle conjugate in Example 1 of the present invention; wherein, (A) is a transmission electron micrograph of the gold nanoparticle conjugate; and (B) is a UV-visible absorption spectrum and optical photograph of the gold nanoparticle conjugate.

[0030] Figure 5 Figures 1 and 2 show the results of optimizing the operating conditions and evaluating the fluidity of the lateral flow test strip in Example 2 of the present invention. (A) Optimization of the MgCl2 concentration in the running buffer shows, from left to right, the color development of the lateral flow test strip under operating conditions of 5 mM MgCl2, 10 mmol / L MgCl2, 15 mmol / L MgCl2, and 20 mmol / L MgCl2. (B) Determination of the relative intensity of the lateral flow test strip under different MgCl2 operating conditions. (C) Optimization of the PVP concentration in the running buffer shows, from left to right, the color development of the lateral flow test strip under operating conditions of 0.5% PVP, 1.0% PVP, 1.5% PVP, and 2.0% PVP. (D) Determination of the relative intensity of the lateral flow test strip under different PVP concentrations. (E): Color development of the lateral flow strip at different times (from left to right): 1, 2, 3, 4, 5, and 6 min. (F): Relative intensity of the lateral flow strip was measured at different times. The concentration of α-syn oligomers was 5 μM. The relative intensity of the lateral flow strip was quantified using Image J software. Relative intensity was quantified as [relative intensity] = [test line intensity] / [control line intensity]. Error bars represent the standard deviation of three independent experiments.

[0031] Figure 6 This figure shows the detection limit results of the lateral flow test strip in Example 1 of the present invention. (A) shows the color development of the lateral flow test strip at different concentrations of α-syn oligomers, and (B) shows the correlation between the relative intensity of the lateral flow test strip and the concentration of α-syn oligomers. The concentrations of α-syn oligomers and thrombin were 0, 0.1 μM, 0.5 μM, 1 μM, 3 μM, 5 μM, 8 μM, and 10 μM, respectively. Error bars represent the standard deviation from three independent experiments.

[0032] Figure 7Figure 1 shows the specificity test results of the lateral flow test strip in Example 1 of the present invention. (A) Specificity test results of the lateral flow test strip, from left to right, showing blank buffer, BSA, IgG, α-syn monomer, α-syn oligomer, and thrombin. (B) Relative intensity of the lateral flow test strip under different test samples. The concentrations of BSA, IgG, α-syn monomer (α-synm), α-syn oligomer (α-syno), and thrombin were all 5 μmol / L. Error bars represent the deviation from three independent experiments.

[0033] Figure 8 1 is a diagram showing the test paper detection results of PD patients and normal subjects in Example 1 of the present invention, wherein (A) shows the test paper color development result of the normal control group, and (B) shows the test paper color development result of the saliva of PD patients. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0035] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0036] Example 1 This example involves the preparation and related detection of a kit for detecting α-synaptophysin oligomers and a lateral flow test paper therein, including the following steps: 1. Expression and purification of recombinant α-syn monomer, including the following steps: Step 1. Refer to the technical document (DOI:10.1074 / jbc.M800747200) for transformation of the pT7-7 plasmid encoding wild-type α-syn. Culture the transformed BL-21 cells in solid culture medium, select a single colony from the solid culture medium and mix it with LB medium containing ampicillin. Incubate with shaking overnight to obtain a preculture. Inoculate the preculture into LB medium at a ratio of 1:20 and incubate at 37°C with stirring until the ultraviolet absorbance at 600 nm reaches 0.6-0.8; add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM to induce α-syn expression, and then continue incubation for 4-6 h (the LB medium used contains 100 μg / mL ampicillin).

[0037] Step 2: Selectively disrupt the cell membrane using the osmotic shock method to obtain α-syn, specifically including: resuspending the cell pellet from 1 L of bacterial culture in 100 mL of osmotic shock buffer (30 mM Tris–HCl, 40 wt% sucrose, 2 mM EDTA, pH = 7.2), incubating at room temperature for 10 minutes, centrifuging at 12,000 rpm for 20 minutes, and removing the supernatant.

[0038] Step 3: Continue to purify α-syn using the acid precipitation method, specifically including: resuspending the α-syn precipitate with 90 mL of cold water, adding 37.5 μL of saturated MgCl2 solution, and incubating on ice for 3 minutes; then centrifuging at 12000 rpm for 20 minutes to obtain a supernatant containing periplasmic proteins; then adjusting the pH of the supernatant to 3.5 with a 1M HCl solution and stirring at room temperature for 10 minutes; then centrifuging at 12000 rpm for 20 minutes, and then adjusting the pH of the supernatant to 7.5 with a 1M NaOH solution to remove impurity proteins that have become precipitates to obtain a supernatant containing α-syn, which was separated and stored at 4°C.

[0039] Step 4: Ion exchange chromatography (IEC) was used to separate and detect α-syn, including: the chromatography buffer and α-syn sample were filtered through a 0.22 μm filter membrane and degassed, the α-syn sample was loaded onto a Q Sepharose Fast Flow anion exchange column, and the column was equilibrated with 3 column volumes (CV) of IEC buffer A (20 mM Tris–HCl, pH 7.5); α-syn was then eluted with 8 CV of 20-50% gradient volume concentration of IEC buffer B (10 mM Tris and 0.75 M NaCl, pH 7.5); finally, unbound proteins were washed with 3 CV of IEC buffer B to remove them. To determine the elution concentration of α-syn, the eluate was collected and subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining using Bis-Tris gel with a gradient volume concentration of 4-20%. The eluate containing the protein band corresponding to the expected molecular weight of α-syn (14.4 kDa) was collected, lyophilized, and stored at -80°C to become lyophilized α-syn monomers. Centrifugation and chromatography were performed at 4°C.

[0040] Figure 2 (A) shows the SDS-PAGE results before and after acid precipitation in step 3. The results indicate that acid precipitation removes most impurities with molecular weights larger than the α-syn monomer. On a 4%-20% SDS-PAGE, α-syn exhibits an apparent molecular mass above 16 kDa, exceeding the theoretical value of 14 kDa. This unusually slow migration may be related to the low affinity of the highly acidic C-terminus of α-syn for SDS.

[0041] Figure 2 (B) and (C) in the figure respectively show the partial SDS-PAGE results of the Buffer B eluate during the IEC process, indicating that the α-syn monomer can be eluted from the chromatography column by 30%-40% IEC Buffer B, while no obvious protein bands are observed under other concentrations of IEC Buffer B. The SDS-PAGE results of the eluate show that the α-syn monomer band is obvious, indicating a high purity and no obvious impurity proteins are observed.

[0042] 2. Preparation and detection of α-syn oligomers, including the following steps: Step 1: Dissolve the freeze-dried α-syn monomer in PBS solution, measure the absorbance of the protein solution at 280 nm and use 5960 M -1 cm -1The α-syn monomer solution was quantified to 150 μM using an extinction coefficient of 1.5; the insoluble protein was removed by centrifugation at 15,000 rpm for 30 min at 4°C; and α-syn oligomers were prepared by incubating the mixture for 7 days at 37°C and 240 rpm using a constant temperature shaker.

[0043] Step 2: Use thioflavin T (ThT) to monitor the formation of α-syn oligomers. Use a microplate reader to measure the fluorescence intensity of ThT. The excitation and emission wavelengths of ThT are 444 nm and 485 nm, respectively.

[0044] Figure 2 (D) shows the changes in ThT fluorescence intensity during α-syn oligomer formation: During the first 48 hours of incubation, α-syn oligomer aggregation proceeded slowly, with no significant change in ThT fluorescence intensity. After 48 hours of incubation, ThT fluorescence intensity increased exponentially, indicating the onset of significant aggregation of α-syn oligomers. Approximately 140 hours of incubation, ThT fluorescence intensity reached its peak, and increasing incubation time did not result in a change in fluorescence intensity, confirming the completion of α-syn aggregation.

[0045] 3. Screening of split aptamers, including the steps of: Step 1: Take the complete aptamer with a base sequence as shown in SEQ ID No. 3, and prepare split aptamers according to the two schemes shown in Table 1: In the first scheme, S-Apt 1-1 is the first split aptamer, which includes the base sequence shown in SEQ ID No. 4, and S-Apt 1-2 is the second split aptamer, which includes the base sequence shown in SEQ ID No. 5. S-Apt 1-1 and S-Apt 1-2 can constitute the aptamer S-Apt 1; In the second scheme, S-Apt 2-1 is the first split aptamer, which includes the base sequence shown in SEQ ID No. 1, and the second split aptamer includes the base sequence shown in SEQ ID No. 2. S-Apt 2-1 and S-Apt 2-2 can constitute the aptamer S-Apt 2; In Table 1, SH refers to thiol modification and Biotin refers to biotin modification.

[0046] Table 1

[0047] Step 2. The isothermal titration calorimetry (ITC) method was used to detect the thermodynamic curves of the set nucleic acid aptamer and α-syn monomer or α-syn oligomer: 1495 mL of α-syn oligomer or α-syn monomer protein solution (2.5 μM) was placed in the sample cell; running buffer (50 mM HEPES, 100 mM NaCl, 15 mM MgCl2, pH 7.5) was used to prepare solutions (10 μM) containing the set nucleic acid aptamer (complete aptamer, S-Apt 1 composed of two split aptamers in the first scheme, and S-Apt 2 composed of two split aptamers in the second scheme); 300 μL of the solution containing the set nucleic acid aptamer was drawn up with a titration needle for 25 injections, with the first injection volume of 5 μL and the remaining injection volumes of 10 μL, and the speed of each injection was 0.5 μL / s; the stirring speed of the sample cell was 307 rpm, the interval between each injection was 180 s, and the entire ITC detection process was carried out at 25 °C.

[0048] Step 3: Use Origin software to analyze and obtain Figure 3 The thermodynamic curve shown, where Figure 3 (A) is 10 μM complete aptamer and 2.5 μM α-syn oligomers. Figure 3 (B) is: 10 μM complete aptamer and 2.5 μM α-syn monomer, Figure 3 (C) is 10 μM S-Apt 1 and 2.5 μM α-syn oligomers. Figure 3 Figure (D) shows the isothermal calorimetric titration curves of 10 μM S-Apt 2 and 2.5 μM α-syn oligomers at 25°C. The dissociation constant (Kd), enthalpy change (ΔH), and entropy change (ΔS) were obtained and fitted to the curves. The results are shown in Table 2.

[0049] Table 2

[0050] It can be seen that the dissociation constant between the complete aptamer and α-syn oligomer is 1.29×10 -8 M -1 The dissociation constant between the complete aptamer and α-syn monomer is 9.98×10 -7 M -1 The dissociation constant between S-Apt 1 and α-syn oligomers is 2.24×10 -8 M -1 The dissociation constant between S-Apt 2 and α-syn oligomers is 1.62×10 -8 M-1 The smaller the dissociation constant, the greater the affinity between the aptamer and the target. Therefore, the affinity of the intact aptamer for α-syn oligomers is much greater than that for α-syn monomers, so the test strip is less affected by α-syn monomers during operation. The dissociation constant of S-Apt 2 is smaller than that of S-Apt 1, so S-Apt 2 has a stronger affinity for α-syn oligomers. The second scheme of the screening step is used to construct a lateral flow test strip.

[0051] The base sequence of the first quality control aptamer is shown in SEQ ID No. 6, i.e., C-AuNPs in Table 1, and the base sequence of the second quality control aptamer is shown in SEQ ID No. 7, i.e., C-capture in Table 1. The first quality control aptamer and the second quality control aptamer are randomly generated complementary sequences. After simulation using nupack software, they do not affect the binding of the test line sequence, meeting the requirements for quality control aptamers.

[0052] 4. Constructing a gold nanoparticle conjugate, comprising the steps of: Step 1: Prepare gold nanoparticles by reducing HAuCl with sodium citrate. Specifically, the following steps were performed: incubating the designated DNA strand (the first split aptamer or the first quality control aptamer) with TCEP (a thiol reducing agent) at a molar ratio of 1:50 for 2 h to reduce the DNA to obtain thiolated DNA; then, mixing the TCEP-treated first split aptamer, the TCEP-treated first quality control aptamer, and gold nanoparticles at a molar ratio of 100:100:1 in 1040 μL of AuNPs solution, followed by incubation at room temperature for 16 h.

[0053] Step 2: Add salt buffer (57 mM Tris-HCl, 2280 mM NaCl, pH 8.0) with a total volume of 100 μL in 10 times, with an interval of 40 minutes each time, to finally reach a salt concentration of 5 mM Tris-HCl and 200 mM NaCl; continue to stir the solution for 24 hours to obtain modified AuNPs.

[0054] Step 3: Transfer the modified AuNPs to a centrifuge tube and centrifuge at 14,000 rpm for 30 minutes. Carefully remove the supernatant and resuspend the red precipitate in 10 mM Tris-HCl (pH 8.0) buffer. Wash three times as described in Step 3 to remove unconjugated DNA strands. Finally, resuspend the washed gold nanoparticle conjugate in 100 μL of 10 mM Tris-HCl solution and store at 4°C. Centrifugation should be performed at 4°C.

[0055] Figure 4(A) in the figure is a gold nanoparticle conjugate characterized by transmission electron microscopy. It can be seen that the gold nanoparticles are well dispersed and have uniform particle size, with an average diameter of 13.1 nm.

[0056] Figure 4 (b) is the UV-visible absorption spectrum measured after dilution. It can be seen that there is a maximum absorption peak at 520 nm. According to 2.7 × 10 8 M -1 •cm -1 The concentration of the gold nanoparticle conjugate was calculated to be 8.07 nM based on the molar extinction coefficient.

[0057] 5. Preparation of lateral flow test paper, comprising a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad, including the following steps: Step 1: Soak the sample pad material in a glass fiber solution (20 mM Tris-HCl, 150 mM NaCl, 0.25 wt% Triton X-100, pH 8.0) for 5 min and then dry it in an oven at 37 °C to obtain a sample pad.

[0058] Step 2: Prepare a 40 nM gold nanoparticle conjugate dispersion using sucrose as a stabilizer, and add sucrose to a final concentration of 10 wt % to obtain a dispersion for preparing a conjugate pad. Spray the dispersion onto the conjugate pad material at a volume of 3 μL / cm and dry to obtain a conjugate pad.

[0059] Step 3: Mix 20 μL of 100 μM biotinylated second split aptamer with 20 μL of 2 mg / mL streptavidin and incubate at room temperature for 1 hour. Then, transfer the incubation solution to a 100 kDa ultrafiltration tube, centrifuge at 12,000 rpm for 30 minutes, and resuspend in 100 μL of PBS solution. Continue centrifugation and resuspending twice (washing 3 times in total) to remove unbound streptavidin and biotinylated second split aptamer. Use a gold spray membrane analyzer to fix the streptavidin-biotinylated second split aptamer to the nitrocellulose membrane at a dosage of 0.5 μL / cm to form the detection line. Mix 20 μL of 100 μM biotinylated second quality control aptamer with 20 μL of 2 mg / mL streptavidin and incubate at room temperature for 1 hour. Then, transfer the incubation solution to a 100 kDa ultrafiltration tube, centrifuge at 12,000 rpm for 30 minutes, and resuspend in 100 μL of PBS solution. Continue centrifugation and resuspending twice (washing three times in total) to remove unbound streptavidin and biotinylated second quality control aptamer. Use a gold spray film streaker to immobilize the streptavidin-biotinylated second quality control aptamer on a nitrocellulose membrane at a volume of 0.5 μL / cm to form a quality control line. The interval between the test line and the quality control line is 2 mm.

[0060] Step 4: Assemble the lateral flow test paper, specifically including: assembling the polyester base plate, nitrocellulose membrane, absorbent paper, conjugate pad, and sample pad in sequence, with the detection line located between the quality control line and the sample pad, and adjacent structures overlapping by 2 mm to ensure the fluidity of the liquid; then cut the assembly into 4 mm strips to obtain the lateral flow test paper, and store it in a dry, dark environment.

[0061] Step 5: Prepare a running buffer with a pH of 7.5 using 50 mM HEPES, 100 mM NaCl, 15 mM MgCl2, and 1.0 wt% PVP to dissolve the saliva sample; in this example, the protease inhibitor selected was P1005 from Beyotime.

[0062] Step 6: Combining running buffer, protease inhibitors and lateral flow test paper to form a kit for detecting α-synaptophysin oligomers.

[0063] 6. Analysis of the detection effect of lateral flow test strips on α-syn oligomers, including the following steps: Step 1. Add the prepared α-syn oligomer sample to the running buffer (50 mM HEPES, 100 mM NaCl, 15 mM MgCl2, 1.0wt% PVP, pH 7.5), mix well to obtain the test liquid, take 80 μL of the test liquid and add it dropwise onto the prepared lateral flow test paper, and observe the color development of the test paper after the set time.

[0064] Step 2: Analyze the color development effect, including: Figure 6 As shown in (A), as the concentration of α-syn oligomers in the sample increases, the color of the test line gradually becomes darker, especially 100 nM α-syn oligomers can be easily identified by the naked eye; Image J software is used to read the color intensity of the test area and the control area of ​​the test strip, and the intensity ratio of the test line and the quality control line (T / C) is plotted against the concentration of α-syn oligomers. The results are shown in Figure 2. Figure 6 As shown in (B), the T / C signal showed a good linear relationship with the concentration of α-syn oligomers, and a linear calibration curve Y (T / C) = 0.162X (μM) + 0.097 was obtained, with a linear correlation coefficient R=0.9916; based on 3σ / k (σ refers to the standard deviation of the blank control, k refers to the slope of the linear equation), the limit of detection (LOD) was calculated to be 88.02 nM.

[0065] Step 3: Perform detection specificity analysis to determine the feasibility of detecting α-syn oligomers in saliva of PD patients without sample separation. Specifically, α-syn monomers or other common proteins, including bovine serum albumin (BSA), immunoglobulin G (IgG), and thrombin, at a concentration of 5 μM are used to replace α-syn oligomers to obtain the test liquid, and the test is performed using a lateral flow test strip according to the method in step 1. The results are as follows: Figure 7 As shown in (A), compared with the negative control group, the test strip detection line will show obvious color change only when the sample contains α-syn oligomers; the color development effect of the test strip was quantitatively analyzed using Image J software, and the results are shown in Figure 2. Figure 7 As shown in (B), the lateral flow test paper used to detect α-syn oligomers in the saliva of PD patients has high selectivity; in particular, compared with α-syn monomers, the test paper shows obvious discrimination, reducing the possibility of false positives in the lateral flow test paper, and verifying the high specificity of the aptamer in recognizing the target.

[0066] Example 2 To ensure the detection performance of the lateral flow test strip, this example optimized the operating conditions of the lateral flow test strip, including the MgCl2 concentration and the polyvinyl pyrrolidone (PVP) content in the running buffer: The color development effect under different reaction conditions was characterized by visual lateral flow analysis, and the color intensity of the test strips was quantified using ImageJ software. Figure 5 As shown in (A), as the concentration of MgCl2 in the reaction solution increases, the color development effect of the test paper detection line gradually increases. Figure 5 As shown in (B), it is a quantitative analysis of the color development effect of the test paper under different MgCl2 concentrations. When the MgCl2 concentration increases to 15mM, the color development effect of the test paper detection line reaches the best signal-to-noise ratio.

[0067] A solution containing α-syn oligomers was used as a sample and dripped onto the sample pad. The PVP content was optimized based on the color development and quantitative results of the test paper. Figure 5 As shown in (C), the color development of the test line and the quality control line changes significantly with the PVP content of the running buffer. Figure 5 (D) in the figure is the quantitative analysis result. When the PVP content increases from 0.5wt% to 1.0wt%, the color development effect of the test line and the quality control line is significantly improved, proving that with the increase of PVP content, the release amount of AuNPs on the binding pad gradually increases. When the PVP concentration continues to increase to 2.0wt%, the color of the test line and the quality control line becomes lighter, indicating that excessively high concentrations of PVP can lead to a decrease in the fluidity of AuNPs and a decrease in the performance of the test paper. In addition, the fluidity of the test paper was verified by adding 80 μL of a sample containing 5 μM α-syn oligomers to the sample pad. Over time, the red color on the binding pad gradually weakened, as shown in Figure 2. Figure 5 As shown in (E), the red color of the test line and the quality control line appears and gradually increases. Figure 5 (F) in the figure shows that the test line and the quality control line can show a visible red color within 1 minute after the sample solution is added. After 5 minutes, the color of the test paper tends to be stable, proving that the constructed lateral flow test paper has good fluidity and a fast color development speed.

[0068] Example 3 This embodiment provides the use of a kit for detecting α-synaptophysin oligomers in detecting PD, comprising the following steps: 1. Saliva collection was performed between 9 and 11 am. Figure 1As shown in (A), specifically, the subjects fasted and abstained from drinking water for at least 1 hour before collection; the subjects rinsed their mouths with water for 1 minute before collection, and after resting for 5 minutes, the subjects sat forward with their heads moderately bent so that their mouths were directly above the opening of the collection tube, and were asked to relax their facial and perioral muscles and maintain natural breathing; at this time, saliva naturally flowed out of the mouth under the action of gravity and fell into the sterile collection tube. The saliva collection time was 20 minutes, or until 5 ml was obtained; protease inhibitors were immediately added to the collected saliva, and the saliva was centrifuged at 3500 rpm at 4°C for 15 minutes to remove impurities in the saliva; the supernatant was retained and centrifuged again at 3500 rpm at 4°C for 15 minutes to remove bacteria, etc. contained in the saliva; the supernatant was retained after centrifugation, freeze-dried, and stored at -80°C.

[0069] 2. If Figure 1 As shown in (B), the lyophilized saliva sample was dissolved in running buffer, mixed, and 80 μL of the mixture was dripped dropwise onto the lateral flow test paper obtained in Example 1. The color development of the test paper was observed after 5 minutes. Figure 8 (A) and (B) in the figure show the test paper colorimetric results of saliva from normal subjects and PD patients, respectively: the saliva colorimetric results of the normal control group subjects were negative, that is, no α-syn oligomers were detected; in the PD patient group, the test paper colorimetric effect was analyzed using Image J software, showing that the test results of 7 PD patients were positive, indicating that the collected saliva contained α-syn oligomers.

[0070] The changes that occur in the lateral flow test paper during the process are as follows Figure 1 As shown in (C), the sample flows in a set direction under capillary action: as the sample flows through the conjugate pad, the gold nanoparticle conjugate follows. When the sample reaches the nitrocellulose membrane, if α-syn oligomers are present, the aptamer sequence on the gold nanoparticle conjugate reassembles with the aptamer sequence on the nitrocellulose membrane, forming an "aptamer-α-syn oligomer-aptamer" sandwich structure, thereby capturing the gold nanoparticle conjugate at the test line. Because the gold nanoparticle conjugate is red, its aggregation causes a visible red band on the test line. Simultaneously, the control line contains a sequence complementary to the gold nanoparticle conjugate, which partially traps the gold nanoparticle conjugate, thus enabling self-testing of the test strip's performance. Therefore, when both the test line and the control line appear red, α-syn oligomers are present in the sample. If only the control line changes color while the test line remains unchanged, α-syn oligomers are not detected in the sample.

[0071] The above demonstrates that lateral flow test strips can be used for clinical detection of α-syn oligomers in the saliva of PD patients.

[0072] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A kit for detecting α-synaptophysin oligomers, characterized in that: The method comprises a lateral flow test paper, wherein the conjugate pad of the lateral flow test paper contains gold nanoparticles coupled to a first split aptamer and a first quality control DNA, the detection line of the lateral flow test paper carries a biotinylated second split aptamer bound to streptavidin, and the quality control line of the lateral flow test paper carries a biotinylated second quality control DNA bound to streptavidin; The base sequence of the first split aptamer is shown as SEQ ID No. 1, and the base sequence of the second split aptamer is shown as SEQ ID No. 2; the first quality control DNA and the second quality control DNA include complementary base sequences.

2. The kit for detecting α-synaptophysin oligomers according to claim 1, wherein The kit includes a running buffer and a protease inhibitor. The running buffer has a pH of 7.5-8.0 and includes the following components at the following concentrations: 50-60 mM HEPES, 100-120 mM NaCl, 5-20 mM MgCl2, and 1.0-1.5 wt% PVP.

3. A method for preparing a lateral flow test paper in a kit for detecting α-synaptophysin oligomers according to any one of claims 1 to 2, characterized in that: Including steps: S1, mixing the TCEP-treated first split aptamer, the TCEP-treated first quality control aptamer, and gold nanoparticles in a solution at a molar ratio of (100-120):(100-120):1, and removing the unconjugated aptamer to obtain a gold nanoparticle conjugate; S2, adding a stabilizer of a set concentration to the dispersion containing the gold nanoparticle conjugate and spraying the stabilizer onto the conjugate pad, and drying the conjugate pad for later use; S3. Mixing and incubating the biotinylated second split aptamer with streptavidin, removing unbound streptavidin and biotinylated second split aptamer, obtaining a streptavidin-second split aptamer, and immobilizing it on a nitrocellulose membrane to form a test line; mixing and incubating the biotinylated second quality control aptamer with streptavidin, removing unbound streptavidin and biotinylated second quality control aptamer, obtaining a streptavidin-biotinylated second quality control aptamer, and immobilizing it on the same nitrocellulose membrane to form a quality control line; the test line and the quality control line are separated by a set distance; S4. Assemble the sample pad, the conjugate pad, the nitrocellulose membrane, the absorbent paper and the back plate into a lateral flow test paper.

4. The preparation method according to claim 3, wherein The base sequences of the first split aptamer and the second split aptamer are obtained by screening. The screening method includes: measuring the thermodynamic curves of the first split aptamer and the second split aptamer with the smallest dissociation constant by ITC.

5. The preparation method according to claim 3, wherein In S1, the TCEP treatment method includes: mixing TCEP and the first split aptamer or the first quality control aptamer at a molar ratio of (50-60):1 and incubating for 2-3 hours.

6. The preparation method according to claim 3, wherein In S2, the concentration of the solution containing the gold nanoparticle conjugate is 40 nM, the concentration of the stabilizer is 10-20 wt%, and the spraying amount is 2-5 μL / cm; Or, in S2, the stabilizer is selected from one or more of sucrose, tris(hydroxymethyl)aminomethane and hydrogen chloride.

7. The preparation method according to claim 3, wherein In S3, the mixed incubation method includes: incubating at room temperature for 1 to 2 hours; Alternatively, in S3, the method for removing unbound streptavidin-biotinylated second split aptamer comprises: centrifuging the incubated mixture at 12,000 to 14,000 rpm for 20 to 40 minutes, resuspending the precipitate with PBS buffer, and then repeating the operation twice; Alternatively, in S3, the method for removing unbound streptavidin-biotinylated second quality control adaptor comprises: centrifuging the incubated mixture at 12,000 to 14,000 rpm for 20 to 40 minutes, resuspending the precipitate with PBS buffer, and then repeating the operation twice.

8. The preparation method according to claim 3, wherein The preparation method of the sample pad in S4 includes: soaking the sample pad material in a glass fiber solution for 5 to 7 minutes, and then drying it in an oven at 30 to 37°C. The glass fiber solution has a pH of 7.5 to 8.5 and includes the following components at the following concentrations: 20 to 30 mM Tris-HCl, 150 to 160 mM NaCl, and 0.25 to 0.35 wt% Triton X-100.

9. Use of a kit for detecting α-synaptophysin oligomers according to any one of claims 1 to 2, characterized in that: include: The freeze-dried saliva sample was dissolved in running buffer, dropped onto the lateral flow test paper, and the color development results of the test line and the quality control line were observed and analyzed.

10. The use according to claim 9, characterized in that The method for preparing the saliva sample comprises: adding a protease inhibitor to the saliva collected from the subject, centrifuging to retain the supernatant, and freeze-drying; Alternatively, the method of centrifuging and retaining the supernatant comprises: centrifuging at a speed of 3500-4000 rpm for 10-20 minutes to remove impurities and retain the supernatant, and then centrifuging at a speed of 15000-17000 rpm for 20-40 minutes to remove bacteria contained in the saliva.

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