Sandwich-type aptamer probe set for detecting antibacterial peptide LL37, kit and preparation method and application thereof

Abstract

The application discloses a sandwich type aptamer probe set for detecting antibacterial peptide LL37, a kit and a preparation method and application thereof. Based on the secondary structure characteristics of a single specific aptamer LL37-01, the application splits the aptamer into two parts, which are used as a capture probe and a signal probe. The 5' end of the capture probe is modified with a thiol group and is fixed to the bottom of a maleimide activated plate through a Michael addition reaction, while the 3' end of the signal probe is modified with biotin for connecting a streptavidin labeled horseradish peroxidase, so as to participate in the subsequent enzymatic oxidation-reduction reaction with a TMB substrate. The probe set and the kit have excellent specificity and sensitivity, and can rapidly complete the detection of LL37 antibacterial peptide in a wound drainage sample within 55 minutes. The kit has a good linear relationship in the target range of 5 ng / mL to 8 mu g / mL, and the detection limit is 3.6 ng / mL.

Description

Technical Field

[0001] This application relates to sandwich-type aptamer probe sets, kits, preparation methods, and applications for detecting the antimicrobial peptide LL37, and belongs to the field of biomedical detection technology. Background Technology

[0002] Cationic antimicrobial peptide LL37 (also known as human antimicrobial peptide LL-37) is currently the only antimicrobial peptide belonging to the cathelicidin family found in the human body. It is formed by the release of the precursor protein hCAP18 after protease cleavage, contains 37 amino acid residues, and possesses cationic properties and a typical amphiphilic α-helix structure. LL37 is widely distributed in neutrophils, epithelial cells, and various body fluids, with significantly increased expression, especially during infection, inflammation, and tissue damage. As an important effector molecule of the innate immune system, LL37 not only has broad-spectrum antimicrobial activity but also participates in various physiological processes such as regulating inflammatory responses, promoting angiogenesis, inducing cell migration, and accelerating wound healing. Recent studies have shown that abnormal expression of LL37 is closely related to autoimmune diseases such as psoriasis, atopic dermatitis, and systemic lupus erythematosus, as well as various infectious diseases. Therefore, changes in its content in biological samples can serve as important indicators for disease diagnosis and disease monitoring.

[0003] Currently, the detection of LL37 mainly relies on immunoassay methods based on antigen-antibody specific recognition, especially enzyme-linked immunosorbent assay (ELISA) kits. This method has advantages such as relatively simple operation and high throughput, and has been widely used in research and clinical testing. However, traditional antibody-based ELISA kits typically require a pair of capture and detection antibodies that can recognize different epitopes of the target. Their preparation process is complex and costly, and the antibodies are sensitive to environmental conditions, easily affected by temperature, pH, and storage conditions, resulting in poor stability and batch-to-batch consistency. Furthermore, for target molecules with small molecular weights, such as the LL37 antimicrobial peptide, their relatively simple structure and limited exposed epitopes make it difficult to simultaneously screen for two antibodies or other recognition elements that can bind to different sites without interference. This limits the application of traditional sandwich-type detection methods in the detection of this type of small molecule target.

[0004] Nucleic acid aptamers are a class of single-stranded DNA or RNA molecules obtained through in vitro screening techniques. They can bind to target molecules with high affinity and high specificity through specific spatial conformations. Compared with traditional antibodies, aptamers have advantages such as simple preparation process, low cost, good stability, ease of chemical modification, and high batch-to-batch consistency, and have shown broad application prospects in the field of biological detection in recent years. However, for antimicrobial peptides like LL37, which have small molecular weights and compact structures, conventional screening strategies are also difficult to obtain two aptamers that can recognize different binding sites, thus limiting the construction of sandwich detection systems based on dual aptamers.

[0005] To address the aforementioned issues, this application, based on a previously obtained, undisclosed, highly specific LL37 aptamer sequence, utilizes its secondary structure characteristics to split and truncate the single aptamer at different sites, optimizing it into two functional fragments, used as a capture probe and a signal probe, respectively. This strategy retains the original aptamer's specific recognition ability for LL37 while achieving synergistic recognition of the same target through structural partitioning, thus successfully constructing a sandwich-type detection system derived from a single aptamer. This method effectively overcomes the technical bottleneck of constructing sandwich detection systems for small molecule antimicrobial peptides, providing a new solution for the highly sensitive and specific detection of LL37, and offering a referable approach for the detection of other small molecules or structurally simple targets. Summary of the Invention

[0006] The purpose of this invention is to address the problems existing in the prior art by providing a sandwich-type aptamer probe kit, a reagent kit, and its preparation method and application for detecting the antimicrobial peptide LL37, aiming to provide a rapid, convenient, and efficient detection method that differs from commercial ELISA antibody kits.

[0007] To achieve the above objectives, this application adopts the following technical solution:

[0008] This application provides a sandwich-type aptamer probe set, which is a pair of split aptamers S1 and S2 that specifically recognize the antimicrobial peptide LL37. It is obtained by splitting an intact single aptamer LL37-01 at a specific site. The sequence of the aptamer LL37-01 is shown in SEQ ID NO: 1.

[0009] Furthermore, the sequence of aptamer S1 is selected from any one of SEQ ID NO: 2-5, and the sequence of aptamer S2 is shown in SEQ ID NO: 6.

[0010] Furthermore, the sequence of aptamer S1 is shown in SEQ ID NO: 4, and the sequence of aptamer S2 is shown in SEQ ID NO: 6.

[0011] Furthermore, the 5' end of the aptamer S1 is modified with a thiol group as a capture probe; the 3' end of the aptamer S2 is modified with biotin as a signal probe.

[0012] In this process, the capture probe is immobilized on the maleimide-activated plate bottom via a Michael addition reaction when the antimicrobial peptide LL37 is actually detected.

[0013] The signal probe Apt-B is modified with biotin at its 3' end to connect to streptavidin-labeled horseradish peroxidase, thereby participating in the subsequent enzymatic redox reaction with the TMB substrate during the detection of the antimicrobial peptide LL37.

[0014] Only when the antimicrobial peptide LL37 is present can the capture probe (Apt-A) and signal probe (Apt-B) bind to different sites of LL37 to form a sandwich structure similar to a three-leaf hairpin.

[0015] Secondly, this application provides the use of the sandwich-type aptamer probe set as described in the first aspect in the preparation of products for detecting the antimicrobial peptide LL37.

[0016] Furthermore, the product includes reagents, reagent kits, or detection chips.

[0017] Thirdly, this application provides a kit for detecting the antimicrobial peptide LL37, the kit comprising at least the sandwich aptamer probe set as described in the first aspect.

[0018] Fourthly, this application provides the use of sandwich-type aptamer probe kits as described in the first aspect or kits as described in the third aspect in detecting the antimicrobial peptide LL37 in in vitro samples.

[0019] Compared with the prior art, this application has the following beneficial effects: This application optimizes the secondary structure of the single nucleic acid aptamer LL37-01 by splitting and truncating it at different sites, constructing a capture probe Apt-A (LL0138) and a signal probe Apt-B (LL0127). While retaining the high affinity and specificity of the original aptamer, this method achieves sandwich-type detection of the small-molecule antimicrobial peptide LL37, effectively overcoming the technical bottleneck of traditional methods that struggle to obtain two molecular probes recognizing different sites. By immobilizing the capture probe on the bottom of a maleimide-activated plate and combining it with a signal amplification system between a biotinylated signal probe and streptavidin-modified HRP, a stable and reliable enzymatic colorimetric detection platform is constructed, improving the sensitivity and specificity of the detection system. This method requires no complex sample pretreatment and can rapidly detect the LL37 antimicrobial peptide in wound drainage samples within 55 minutes, exhibiting good linearity in the range of 5 ng / mL–8 μg / mL, with a detection limit as low as 3.6 ng / mL. Therefore, this invention fills the technical gap in the current difficulty of constructing aptamer sandwich detection systems for small molecule antimicrobial peptides, and provides a new technical means for the rapid and accurate detection of LL37 in complex clinical wound drainage fluid samples, which is of great significance for promoting its application in infectious disease monitoring, wound assessment and related disease diagnosis and treatment. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the detection principle of the sandwich-type aptamer probe set or kit provided in the embodiments of this application.

[0021] Figure 2 These are the test results of different combinations of truncated sequences provided in the embodiments of this application.

[0022] Figure 3 The results are the conformational analysis results of the binding of the capture probe LL0138 to the antimicrobial peptide LL37 by circular dichroism chromatography provided in the embodiments of this application.

[0023] Figure 4 The results are the conformational analysis results of the binding of the signal probe LL0127 and the antimicrobial peptide LL37 by circular dichroism spectroscopy provided in the embodiments of this application.

[0024] Figure 5 The results are the kinetic analysis results of the capture probes LL0138 and LL37 antimicrobial peptides provided in the embodiments of this application.

[0025] Figure 6 The results are the kinetic analysis results of the signal probes LL0127 and LL37 antimicrobial peptides provided in the embodiments of this application.

[0026] Figure 7 This application describes the specificity analysis of the single aptamer sandwich reagent kit provided in the embodiments of this application. Figure 8This is the signal response-target concentration relationship curve of the single aptamer sandwich reagent kit provided in the embodiments of this application.

[0027] Figure 9 The results of the detection of LL37 antimicrobial peptide in clinical drainage fluid samples using the single aptamer sandwich kit provided in this application embodiment are shown. Detailed Implementation

[0028] To make the technical solution of this application clearer and easier to understand, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0029] Unless otherwise specified, the experimental or testing methods described in the following examples are conventional methods; the reagents and materials described are obtained from conventional commercial sources unless otherwise specified.

[0030] Example 1: Selection of capture probe and co-location probe (1) Based on the secondary structure characteristics of the single specific aptamer LL37-01, structural truncation optimization was performed, and the truncated sequence is shown in the table below. LL0165, LL0155, LL0138 and LL0128 were selected as candidate capture probes, and thiol groups were modified at their 5' ends. LL0127 was selected as a signal probe, and biotin was modified at its 3' end.

[0031]

[0032] (2) Dissolve the thiol-modified capture probes thoroughly in sterile water to form a 100 μM storage solution. To avoid repeated freeze-thaw cycles, the solution can be aliquoted into smaller portions. Take out the pre-allotted 100 μM candidate capture probe solutions (LL0165, LL0155, LL0138, and LL0128), bring them to room temperature, and centrifuge them at high speed. Add an equal volume of 20 mM TCEP solution (i.e., the molar ratio of aptamer to TCEP is 1:200) to the candidate capture probe solutions. After the reduction reaction is carried out at room temperature for 2 hours, dilute the candidate capture probe solutions to a working concentration of 500 nM with 1×PBS solution. Subsequently, the capture probes were immobilized by adding 100 μL of capture probe solution to each well, sealing it with adhesive tape, and incubating it at 37°C and 100 rpm for 1 hour. After incubation, the liquid in the wells was discarded, and 200 μL of PBST washing buffer (1×PBS, 0.05% Tween-20, pH=7.45) was added to each well. The wells were then washed four times on a horizontal constant-temperature shaker for 2 minutes each time, and then patted dry on clean absorbent paper.

[0033] (3) Add 100 μL of 5% BSA solution to each well in the microplate with the immobilized capture probe, seal with adhesive tape, and then incubate at 37°C and 100 rpm for 2 hours. After sealing, discard the liquid in the well and repeat the washing steps in (1).

[0034] (4) The wound drainage fluid collected clinically was filtered using a 30 kDa ultrafiltration tube. The recovered retentate was filtered twice to ensure that the original LL37 in the sample was removed. The LL37 antimicrobial peptide standard with a storage concentration of 10 mg / mL was diluted to a final concentration of 5 μg / mL using the pretreated wound drainage fluid. 100 μL of LL37 antimicrobial peptide solution was added to each well of the ELISA plate, sealed with adhesive tape, and incubated at 37°C and 100 rpm for 1 hour. After incubation, the liquid in the well was discarded, and the washing steps in (1) were repeated.

[0035] (5) Dissolve the biotin-modified signal probe LL0127 thoroughly in sterile water to prepare a storage solution with a concentration of 100 μM. To avoid repeated freeze-thaw cycles, it can be aliquoted into smaller portions. Dilute the biotin-labeled signal probe LL0127 to a working concentration of 200 nM with 1×PBS buffer containing 1 mM MgCl2. Then add 100 μL of the signal probe solution to each well, seal with adhesive tape, and incubate at 37°C and 100 rpm for 1 hour. After incubation, discard the liquid in the well and repeat the washing steps in (1).

[0036] (6) Add 100 μL of horseradish peroxidase conjugate modified with streptavidin (SA-HPR) to each well, seal with adhesive tape, and then incubate at 37°C and 100 rpm for 0.5 hours. After incubation, discard the liquid in the well and repeat the washing steps in (1).

[0037] (7) Add 100 μL of TMB colorimetric reagent solution to each well, and then develop the color at 37°C in the dark for 10 minutes.

[0038] (8) Add 50 μL of stop solution (2 M sulfuric acid) to each well, and within 5 minutes after the reaction is terminated, use an ELISA reader to detect the absorbance value of each well at 450 nm (OD450 nm).

[0039] The results show that: Figure 2 As shown, four candidate capture probes were tested in combination with the signal probe LL0127. All four combinations were able to specifically identify the LL37 standard in complex drainage fluid systems. Among them, the test results were the best when LL0138 was used as the capture probe and LL0127 as the signal probe.

[0040] Example 2: Circular dichroism spectroscopy to investigate the binding mode of the capture probe, signal probe and LL37. (1) The capture probe LL0138 and the signal probe LL0127 were diluted to 20 μM with ddH2O, denatured at 95℃ for 30s, and then rapidly cooled to 25℃. (2) Then use a solution containing 1 mM Mg 2+ The annealed and denatured nucleic acid aptamers were diluted to 5 μM with 1×PBS buffer, and then LL37 antimicrobial peptide standard was added to bring the final concentration to 100 μg / mL. The mixture was then incubated at room temperature for 1 hour. (3) The detection was performed using a circular dichroism chromatograph. The temperature was set to 26℃, the scanning wavelength was set to 200-320 nm, the scanning speed was 100 nm / min, and the reaction time was 1 s.

[0041] The results showed that in the presence of 1 mM Mg 2+ In 1×PBS buffer, the positive bands of the CD patterns of capture probe LL0138 and signal probe LL0127 both appeared around 280 nm, and the negative bands both appeared around 240 nm, intersecting the baseline axis around 255 nm, consistent with the CD pattern of standard B-DNA. When capture probe LL0138 and signal probe LL0127 bound to target LL37, the intensity of the positive and negative bands of their CD curves changed dramatically. This was induced by stronger base stacking and protein binding, amplifying the CD signals of capture probe LL0138 and signal probe LL0127. This indicates that the binding of capture probe LL0138 and signal probe LL0127 to target LL37 is mainly through conformational changes rather than electrostatic interactions (see [link to relevant documentation]). Figure 4 ).

[0042] Example 3: Determination of the binding kinetics between the capture probe, signal probe and LL37 using square wave voltammetry (1) Pour an appropriate amount of 1 μm alumina powder onto a polishing cloth, add an appropriate amount of deionized water to moisten the aluminum powder, place each gold disk electrode vertically on the aluminum powder solution and carefully polish it in a figure-eight pattern 100 times. Then rinse the electrode surface with deionized water and place the electrode in an ultrasonic cleaner for 5 minutes. Next, pour an appropriate amount of 0.05 μm alumina powder onto another polishing cloth, add deionized water to moisten the aluminum powder, and perform the same operation as in the previous step, polishing each electrode in a figure-eight pattern. Then rinse the electrode surface with deionized water and place the electrode in an ultrasonic cleaner for 5 minutes. Finally, wipe away excess water from the edges of the gold disk electrodes after ultrasonication with clean, lint-free paper, and wait for the electrochemical cleaning step.

[0043] (2) The gold disk electrode after the physical polishing step is placed in a 0.5 M sodium hydroxide solution. A three-electrode system is used, and cyclic voltammetry is employed to scan 10 times in a voltage range of 0-1.5 V with a step size of 0.1 V / s. This step is mainly used to remove sulfur-containing contaminants.

[0044] (3) Rinse the gold disk electrode with deionized water, then immerse the electrode in a 0.5 M sulfuric acid solution. Select the chronoamperometry method and set the test parameters as Einitial = 0.0 V, Efinal = 2.0 V, pulse duration of 0.02 s, and 320 steps. Begin the test. This step is mainly used to reduce the formed gold oxide. Note that O2 bubbles are generated on the surface of the gold disk electrode in this step. To ensure that the electrode surface can be completely cleaned, the surface bubbles need to be removed by vibrating the electrode before proceeding to the next step.

[0045] (4) Next, select cyclic voltammetry, set the test parameters as Einitial=-0.35 V, Ehigh=1.5 V, and scan rate=4 V / s before starting the test. This step is mainly used to further oxidize and reduce contaminants on the electrode surface, and at the same time form a layer of reduced gold nano-oxide.

[0046] (5) Rinse the gold disk electrode with deionized water, transfer the electrode to a 0.05 M sulfuric acid solution, select cyclic voltammetry, and use cyclic voltammetry to test the redox peak of gold in a voltage range of -0.35-1.5 V with a step size of 0.1 V / s. Read the integrated area of ​​the gold redox peak and divide the obtained area value by 422 μC / cm. 2 This allows us to determine the effective electroactive area of ​​each gold electrode. The effective surface area of ​​a freshly cleaned gold disk electrode is approximately 0.03 cm². 2 Approximately (±30%).

[0047] (6) Take out the pre-amplified 100 μM aptamer LL0138 solution, restore it to room temperature, and centrifuge it at high speed. Add an equal volume of 20 mM TCEP solution to the aptamer LL0138 solution (i.e., the molar ratio of aptamer to TCEP is 1:200). After the reduction reaction is carried out at room temperature for 1 hour, the aptamer LL0138 solution is diluted with 1×PBS solution to a working concentration of 5 μM. Rinse the pretreated gold disk electrode with deionized water and wipe off excess water from the electrode edges with clean, lint-free paper. Then immerse the electrode in the reduced 5 μM aptamer LL0138 solution and allow it to self-assemble at room temperature for 2 hours, so that Au-S bonds are formed between the aptamer LL0138 and the surface of the gold disk electrode, thereby anchoring it to the gold disk electrode. Next, the assembled aptamer LL0138 electrode was rinsed with 1×PBS solution, dried with nitrogen, and then placed in 20 mM 6-mercapto-1-hexanol solution (diluted with 1×PBS buffer) and incubated overnight at 4°C (≥8 hours).

[0048] (7) The wound drainage fluid collected clinically was filtered using a 30 kDa ultrafiltration tube. The recovered retentate was filtered twice to ensure the removal of the original LL37 from the sample. Subsequently, the electrodes with the assembled capture probe and signal probe were placed in a conductivity cell containing 8 mL of pretreated drainage fluid, connected to the various pathways of the electrochemical workstation, and 15 pre-scans were performed using the SWV method to ensure that the electrical signal was stable. Then, 10 μg / mL of LL37 standard was added to the conductivity cell for kinetic testing at a frequency of 300 Hz.

[0049] The results show that: Figure 5 and Figure 6 As shown, in the wound drainage fluid testing system, both the capture probe and the signal probe can rapidly bind to the LL37 antimicrobial peptide, with binding equilibrium times on the order of minutes, far faster than traditional antigen-antibody binding kinetics. Specifically, the kinetic time constant τ between the capture probe LL0138 and the LL37 antimicrobial peptide is 1.41 ± 0.08 minutes, reaching equilibrium within 5 minutes, while the kinetic time constant τ between the signal probe LL0127 and the LL37 antimicrobial peptide is 2.26 ± 0.10 minutes, reaching equilibrium within 10 minutes.

[0050] Example 4: Specificity and sensitivity evaluation of sandwich aptamer kit The pre-loaded ELISA plate with the capture probe LL0138 immobilized on the surface, prepared based on steps (1-3) in Example 1, can be stored at 4°C for up to 6 months in a sealed bag.

[0051] (1) Specificity: The prepared pre-loaded ELISA plates were used to detect different biomolecules (negative control: non-target substances: lysozyme, NSG2 neuronal protein, thrombin; blank control: BSA; positive group: LL37 antimicrobial peptide. The concentrations were kept consistent) and mixtures of the above biomolecules. The incubation time was 5 minutes for all samples. The remaining operation steps were the same as in Example 1, except that the incubation time of the signal probe LL0138 was 10 minutes.

[0052] (2) Sensitivity: LL37 standard was added to the pretreated drainage solution to achieve final concentrations of 0.001, 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 5.0, and 8.0 μg / mL. Subsequently, 100 μL was added to each well of the prepared pre-loaded ELISA plate, and the incubation time was 5 minutes. The remaining operation steps were the same as in Example 1, except that the incubation time of the signal probe LL0138 was 10 minutes. Finally, a standard curve of signal response-concentration was obtained.

[0053] The results show that: Figure 7 It can be seen that the test kit can identify LL37 standard and mixtures containing LL37 with high specificity, but cannot identify other non-target biomolecules (including high isoelectric point lysozyme and NSG2 neuron protein). Figure 8 The standard curve of signal response-concentration shows that the test kit exhibits good linearity within the concentration range of 5 ng / mL to 8 μg / mL. For the criterion for positive results in immunoassay biological experiments, a detection signal at least 2.1 times stronger than the blank background signal is considered biologically significant. Therefore, the estimated LOD value of this test kit is 3.6 ng / mL.

[0054] Example 5: Actual testing of a sandwich-type aptamer kit on clinical drainage fluid samples. To test whether the constructed sandwich aptamer detection kit could specifically identify LL37 antimicrobial peptide in clinical drainage fluid samples, drainage fluid samples from the wounds of sixteen scalded patients were collected from the Department of Burns at Shanghai Ninth People's Hospital. The operation steps were the same as above (see Specificity in Example 4).

[0055] The results showed that the detection kit described in this application can specifically identify LL37 antimicrobial peptide in clinical drainage fluid samples. Figure 9 The concentrations of LL37 antimicrobial peptide in the 16 samples were finally calculated by back-calculating using the standard curve of signal response-concentration (see table below).

[0056]

[0057] The above description is merely a preferred embodiment of this application and is not intended to limit this application in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from this application, and these improvements and additions should also be considered within the scope of protection of this application.

Claims

1. A sandwich-type aptamer probe assembly, characterized in that, The probe set consists of a pair of splitting aptamers S1 and S2 that specifically recognize the antimicrobial peptide LL37. They are obtained by splitting an intact single aptamer LL37-01 at a specific site. The sequence of the aptamer LL37-01 is shown in SEQ ID NO:

1.

2. The sandwich-type aptamer probe assembly according to claim 1, characterized in that, The sequence of aptamer S1 is selected from any one of SEQ ID NO: 2-5, and the sequence of aptamer S2 is shown in SEQ ID NO:

6.

3. The sandwich-type aptamer probe assembly according to claim 1, characterized in that, The sequence of aptamer S1 is shown in SEQ ID NO: 4, and the sequence of aptamer S2 is shown in SEQ ID NO:

6.

4. The sandwich-type aptamer probe assembly according to claim 1, characterized in that, The 5' end of aptamer S1 is modified with a thiol group to serve as a capture probe; the 3' end of aptamer S2 is modified with biotin to serve as a signal probe.

5. The use of the sandwich-type aptamer probe assembly according to any one of claims 1-4 in the preparation of products for detecting the antimicrobial peptide LL37.

6. The application according to claim 5, characterized in that, The products include reagents, reagent kits, or detection chips.

7. A kit for detecting the antimicrobial peptide LL37, characterized in that, The kit includes at least the sandwich-type aptamer probe set as described in any one of claims 1-3.

8. The use of the sandwich-type aptamer probe kit according to any one of claims 1-4 or the kit according to claim 7 in detecting the antimicrobial peptide LL37 in in vitro samples.

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