CN117030672B - Fluorescent probe for detecting activity of coagulation factor FXIa inhibitor in complex system and application thereof - Google Patents
Fluorescent probe for detecting activity of coagulation factor FXIa inhibitor in complex system and application thereof Download PDFInfo
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- CN117030672B CN117030672B CN202311009243.7A CN202311009243A CN117030672B CN 117030672 B CN117030672 B CN 117030672B CN 202311009243 A CN202311009243 A CN 202311009243A CN 117030672 B CN117030672 B CN 117030672B
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Abstract
The invention discloses a fluorescent probe for detecting the activity of a coagulation factor FXIa inhibitor in a complex system and application thereof. The fluorescent probe comprises a recognition probe and a capture probe; the recognition probe consists of fluorescent quantum dots, enterokinase and an aptamer which are respectively modified on the fluorescent quantum dots; the capture probe consists of a nano magnetic sphere and FXIa covalently modified on the nano magnetic sphere; the binding affinity of the aptamer to the FXIa active site is lower than the binding affinity of the FXIa inhibitor to the FXIa active site. The development of the method provides a key technical means for the high-sensitivity discovery, high-throughput quantitative analysis and high-connotation activity screening of the active ingredients of the FXIa inhibitor in a complex system, and has a certain application value in the drug research of novel anticoagulant peptides and the screening research of the active ingredients of the FXIa inhibitor in a traditional Chinese medicine complex system.
Description
Technical Field
The invention relates to a fluorescent probe and application thereof, in particular to a fluorescent probe for detecting the activity of a coagulation factor FXIa inhibitor in a complex system and application thereof.
Background
Thrombotic diseases have the characteristics of high morbidity, high disability rate, high mortality rate and the like, and are common diseases endangering human health. The current commonly used antithrombotic drugs have potential bleeding risk due to interference with the delicate balance of coagulation and anticoagulation of the body. Clinical researches show that the blood coagulation factor XIa (FXIa) is an effective and safe target point for developing antithrombotic drugs, and is expected to become a novel, safe and effective antithrombotic drug due to the low bleeding risk of the FXIa inhibitor.
Thrombotic diseases are common diseases which are harmful to human health, and have the characteristics of high morbidity, high disability rate and high mortality rate. More than 43% of deaths per year are associated with cardiovascular disease, based on WHO statistics, currently 1730 tens of thousands, with 2030 being expected to be a number exceeding 2360 tens of thousands. Guidelines established by the american heart association, european heart association, and the like, each apply anti-thrombotic therapy as a therapeutic basis for the prevention and treatment of acute coronary syndromes (Acute coronary syndrome, ACS) and for the recurrence of cardiovascular events in patients undergoing percutaneous coronary intervention (Percutaneous coronary intervention, PCI). The current commonly used antithrombotic drugs (heparin, warfarin and the like) have better curative effects in clinic, but have potential bleeding risks due to interference of the delicate balance of coagulation and anticoagulation of organisms. Clinical data show that patients with congenital deficiency of Factor XI (FXI) have a lower probability of suffering from ischemic stroke and deep venous thrombosis, and generally have no risk of spontaneous hemorrhage. Studies have shown that FXI can increase thrombin generation during thrombosis, and can inhibit limited thrombin generation during hemostasis with reduced risk of bleeding. Therefore, the research and development of anticoagulant drugs based on FXI/FXIa targets becomes a research hot spot of current new drugs. FXIa is a key coagulation factor in the intrinsic coagulation pathway, which activates FIX to FIXa, thereby producing thrombin through the common coagulation pathway. By means of a feedback amplification loop on FXIa, a large amount of thrombin can be further produced, thereby inducing thrombosis. In 2022, 8 months, with the release of the results of PACIFIC-AMI and PACIFIC-STROKE studies in the European cardiology department, the novel oral FXIa inhibitor Asundexian combined with dual antibody therapy (DAPT) did not increase bleeding in patients with acute myocardial infarction intervention. Studies have shown that Asundexian can reduce the risk of recurrence of symptomatic ischemic stroke or transient ischemic attacks without increasing the risk of massive or intracranial bleeding. FXIa inhibitors are prominent in the cardiovascular and cerebrovascular disease field, making FXIa targets a focus of attention for cardiovascular disease again.
At present, a plurality of F XI a inhibitors are in different research and development stages, and the inhibitors are mainly derived from natural products and derivatives thereof, chemically synthesized small molecular compounds, polypeptides, antisense oligonucleotides, monoclonal antibodies and other macromolecular drugs. In 1982, APLYSINELLIDAE et al isolated and extracted 2 novel bioactive bromophenol alkaloids from the extract of sponge Suberea clavata, and found that CLAVATADINE A thereof was able to irreversibly bind to the active site of fci a and exert an inhibitory effect (ic50=1.3 μmol/L). Al-Horani et Al found that sulfated pentaglucopyranoside (SPGG, 3) is a potent allosteric inhibitor of F XI a and exerts an inhibitory effect by recognizing a plurality of anionic allosteric sites on F XI a and binding to these sites. Argade et al, screened a library of molecules containing fragments of natural products for compounds of different backbone structures, found a benzopyran structure-containing monosulfated trimer having an inhibitory effect on F XI a, which exhibited the inhibitory effect on F XI a by inducing conformational changes in the active site of F XI a. David a.donkey et al, using an in vitro iterative selection procedure of exponential enrichment ligand systematic evolution (SELEX) 19, screened for aptamers that bind at or near the FXIa active site by 10 rounds of positive and negative selection, found that aptamer FELIAP bound to the active site of FXIa with a high affinity (KD of 1.8 nM) that was much higher than other candidate aptamers such as Apt10-E (40). The above results indicate that a highly sensitive, rapid, simple and specific method for screening and evaluating activities is needed for the discovery of novel FXIa inhibitors.
Disclosure of Invention
The invention aims to: the invention aims to provide a fluorescent probe which is high in sensitivity, quick, simple, convenient and strong in specificity and is used for detecting the activity of a coagulation factor FXIa inhibitor in a complex system; another object of the invention is to provide a method for detecting the activity of an inhibitor of the coagulation factor FXIa in a complex system; another object of the present invention is to provide a method for screening for the inhibition activity of FXIa in a complex system.
The technical scheme is as follows: the invention discloses a fluorescent probe for detecting the activity of a coagulation factor FXIa inhibitor in a complex system, which comprises a recognition probe and a capture probe; the recognition probe consists of fluorescent quantum dots, enterokinase and an aptamer which are respectively modified on the fluorescent quantum dots; the capture probe consists of a nano magnetic sphere and FXIa covalently modified on the nano magnetic sphere; the binding affinity of the aptamer to the FXIa active site is lower than the binding affinity of the FXIa inhibitor to the FXIa active site.
As a further improvement of the scheme, the recognition probe is AptE-QDs-EK, wherein the enterokinase is recombinant human enterokinase, the fluorescent quantum dots are ZnSe@ZnS quantum dots, and the aptamer is AptE.
As a further improvement of the scheme, the preparation method of AptE-QDs-EK comprises the following steps:
ZnSe@ZnS dispersion, aptE, recombinant human enterokinase, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride in a buffer solution, and incubating for a period of time;
after the incubation, the pellet was collected as AptE-QDs-EK.
As a further improvement of the scheme, the capture probe is MNPs-FXIa, and the preparation method of the MNPs-FXIa comprises the following steps:
MNPs-COOH dispersion, FXIa solution and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride are mixed and then reacted for a period of time at room temperature, and the MNPs-FXIa is obtained after magnetic separation.
As a further improvement of the scheme, the fluorescent probe further comprises a signal probe, wherein the signal probe is hexapolyarginine peptide-CdTe@ZnS QDs complex.
As a further improvement of the scheme, the preparation method of the hexapolyarginine peptide-CdTe@ZnS QDs compound comprises the following steps of:
Fully mixing the hexapolyarginine peptide solution with the CdTe@ZnS QDs solution, and collecting the product, namely the hexapolyarginine peptide-CdTe@ZnS QDs compound; wherein the mass ratio of the hexapolyarginine peptide in the hexapolyarginine peptide solution to CdTe@ZnS in the CdTe@ZnS QDs solution is 1-7:15.
In another aspect, the invention provides a method for detecting the activity of a factor FXIa inhibitor in a complex system, the method comprising the steps of:
(1) Mixing the recognition probe dispersion and the capture probe dispersion to form a first complex;
adding a sample into the first compound, incubating for a period of time, and taking a supernatant;
Adding trypsinogen and a signal probe into the supernatant, incubating for a period of time, measuring the fluorescence intensity after incubation, and if FXIa inhibitor exists in the sample, sequentially amplifying the first-order signal and the second-order signal by the measured fluorescence signal, wherein the recovery degree of the fluorescence signal is related to the content of FXIa inhibition active ingredients;
(2) Sequentially replacing the sample in the step (1) with a sample containing FXIa inhibitor with gradient concentration, and repeating the step (1) for a plurality of times to obtain a standard curve of the concentration of the FXIa inhibitor corresponding to the recovery degree of the fluorescent signal;
(3) And (3) determining the recovery degree of the fluorescent signal of the sample to be tested by using the method in the step (1), and comparing the recovery degree with the standard curve obtained in the step (2) to obtain the concentration of the FXIa inhibitor in the sample to be tested.
As a still further improvement of the above-mentioned scheme, in the step (1), the mass ratio of the recognition probe in the recognition probe dispersion to the capture probe in the capture probe dispersion is 1.about.2:2.about.1.
As a further improvement of the above-described scheme, the conditions for measuring the fluorescence intensity are: the fluorescence intensity at 600nm was measured, and the excitation wavelength was 365nm.
In another aspect, the present invention provides a method for screening for inhibition activity of coagulation factor FXIa in a complex system, the method comprising the steps of:
(1) Mixing the recognition probe dispersion and the capture probe dispersion to form a first complex;
Adding a sample to be screened into the first compound, incubating for a period of time, and taking supernatant;
Adding trypsinogen and a signal probe into the supernatant, incubating for a period of time, and measuring the fluorescence intensity after incubation is finished, wherein the measured fluorescence intensity is marked as F;
(2) Replacing the first compound in the step (1) with pure water with the same volume, repeating the step (1), and marking the measured fluorescence intensity as F0;
(3) Replacing the sample to be screened in the step (1) with a negative control group, repeating the step (1), and marking the measured fluorescence intensity as Fm;
(4) Replacing the first compound in the step (1) with pure water with the same volume, replacing the sample to be screened with a negative control group, repeating the step (1), and marking the measured fluorescence intensity as F1;
(5) The calculation formula of the index n for evaluating the inhibition capacity of the sample to be screened on FXIa is as follows:
the research aims to establish a quantitative analysis and activity screening of FXIa protein inhibitor through a double-enzyme cascade signal amplification system. In the invention, enterokinase and an aptamer AptE capable of recognizing FXIa active site are jointly modified on ZnSe@ZnS QD to obtain a AptE-QDs-EK recognition probe. FXIa is modified on Magnetic Nanoparticles (MNPs) through amide bonds to obtain the MNPs-FXIa capture probe. After the MNPs-FXIa and AptE-QDs-EK are incubated together, the MNPs-FXIa and AptE-QDs-EK are specifically combined through interaction of FXIa and AptE, and the MNPs-FXIa-QDs-AptE-EK complex is obtained. In the presence of an inhibitor of FXIA activity, the inhibitor competes with AptE40 for binding to the FXIA active site, causing AptE-QDs-EK to be released from MNPs-FXIA and into the supernatant, and when equilibrium is reached, the concentration of AptE-QDs-EK in the supernatant is positively correlated with the concentration of FXIA inhibitor in the assay system. After magnetic separation, the supernatant was incubated with trypsinogen, the enterokinase on AptE-QDs-EK of which catalyzes the N-terminal hexapeptide hydrolysis of the trypsinogen and amplification to produce large amounts of trypsin, a primary signal cascade. Trypsin hydrolyzes hexapolyarginine peptide (RRRRRR, R6) and dissociates CdTe@ZnS QDs from the R6-RQDs complex, and fluorescence of supernatant increases sharply, which is a secondary signal cascade amplification. Through a double-enzyme cascade fluorescence signal amplification system, the active ingredients of the FXIa inhibitor in a complex system or a trace amount can be amplified into extremely strong fluorescence signals, so that high-sensitivity activity screening and quantitative detection are realized. The feasibility of the method is examined by taking the previously reported FXIa inhibitor aptamer FELIAP as a positive model drug, and the result shows that the method of the invention realizes ultrasensitive detection on FELIAP, and has good linear correlation between fluorescence response signals and FELIAP concentration (R 2 =0.9925) in the concentration range of 1-375 nM. Meanwhile, the established novel method is applied to screening of the F XI a inhibitors in the crude protein extract of the ground beetles, 2 components with higher active ingredients are successfully screened, and verification is carried out by adopting a conventional ELISA method, so that the results are consistent. The development of the method provides a key technical means for the high-sensitivity discovery, high-throughput quantitative analysis and high-connotation activity screening of the active ingredients of the FXIa inhibitor in a complex system, and has a certain application value in the drug research of novel anticoagulant peptides and the screening research of the active ingredients of the FXIa inhibitor in a traditional Chinese medicine complex system.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: the invention develops a detection analysis method based on enterokinase-trypsin double enzyme cascade fluorescence signal amplification, which is used for connecting the output fluorescence signal with the concentration of a compound capable of combining with FXIa active site in a natural product through enterokinase activity in supernatant fluid, so that the detection signal can be greatly enhanced. On the other hand, the magnetic beads are used for replacing the ELISA plate to be used as a solid-phase carrier, so that a larger effective specific surface area can be provided, and other endogenous interfering substances in the sample are removed through magnetic separation, so that higher capture efficiency is achieved. Finally, the invention evaluates the feasibility of ultrasensitive analysis in a complex system and deeply discusses a signal amplification mechanism. The constructed method is successfully applied to high-flux, simple, convenient and rapid ultrasensitive detection analysis of the coagulation factor FXIa inhibitor, has good linearity in the range of 1-375 nM, and the detection results of high, medium and low concentration quality control samples show that the method has good precision and accuracy. Meanwhile, when the method is applied to screening of traditional Chinese medicine ground beetle extracts with FXIa inhibiting activity, two ground beetle extracts possibly containing FXIa inhibiting agents are successfully screened out. The method provided by the invention has great application value and prospect in screening and finding active ingredients in traditional Chinese medicines and complex systems.
Drawings
FIG. 1 is a schematic diagram of FXIa inhibitor detection; (A) formation of MNPs-FXIa-QDs-AptE-EK complex; (B) FXIa inhibitor-mediated release of QDs-AptE-EK; (C) principle of enterokinase-trypsin double enzyme cascade amplification; (D) legend.
FIG. 2 is a graph of the results of nanocharacterization and enzyme activity monitoring, TEM image (A) of ZnSe@ZnS QDs and particle size distribution (B); (C) Ultraviolet absorption spectra of ZnSe@ZnS QDs and AptE-QDs-EK NPs; (D) enterokinase activity verification of QDs-AptE-EK.
FIG. 3 is a graph showing the result of aggregation quenching of CdTe@ZnS QDs induced by hexapolyarginine peptide, showing a transmission electron microscope image (A) and a particle size distribution (B) of CdTe@ZnS QDs; a change in RQDs Zeta potential (C) and a change in hydrated particle size (D) caused by hexapolyarginine peptide; the fluorescence intensity changes (E) and fluorescence spectra (F) at 600nm wavelength after CdTe@ZnS QDs (150. Mu.g/mL) were mixed with different concentrations (0, 10, 20, 30, 40, 50, 60 and 70. Mu.g/mL) of hexapolyarginine peptides, the excitation wavelength was 365nm (interpolated images are photographs of the mixture at 365nm wavelength under UV light).
FIG. 4 is a graph showing the results of the double enzyme mediated fluorescence recovery of the R6-RQDs complex, wherein (A) trypsin has a fluorescence recovery effect on the R6-RQDs complex; (B) Fluorescence recovery of enterokinase-activated trypsinogen on the R6-RQDs complex; (C) Quick response time image of R6-RQDs complex to enterokinase.
FIG. 5 is a diagram showing the results of the principle of amplification of the double enzyme cascade signal, (A) SDS-PAGE shows the activation of trypsinogen by enterokinase (lane 1: enterokinase; lane 2: trypsinogen; lane 3: enterokinase + trypsinogen; lane 4: trypsin); a transmission electron microscope image (B) and a particle size distribution (C) of SiO 2 NPs; (D) Schematic diagram of trypsin degradation of dansyl chloride labeled hexapolyarginine peptide; (E) Trypsin catalyzes the degradation of dansyl chloride labeled hexapolyarginine peptide (interpolation chart SiO 2 -R6-DNS NPs and photographs of supernatants under 365nm uv light after different times of incubation with trypsin); (F) The fluorescence intensity of the supernatant at 535nm (excitation wavelength 310 nm) changed after incubation of SiO 2 -R6-DNS NPs with trypsin for different times; (G) Effect of trypsin activity inhibition experiment heat inactivation on recovery of R6-RQDs complex fluorescence by trypsin; (H) Effect of PMSF concentration on trypsin recovery of R6-RQDs complex fluorescence.
FIG. 6 is a graph showing the results of detection of 5FXIa inhibitory aptamer FELIAP, (A) verification of enterokinase activity of the MNPs-FXIa-AptE40-QDs-EK complex; (B) Screening active ingredients of FXIa inhibitor of crude protein extract of Eupolyphaga Seu Steleophaga; the effect of crude woodlouse protein extracts on APTT (C) and PT (D) (data expressed as mean±sd, n=3. Statistical analysis was tested using one-way ANOVA, ns: P >0.05, P <0.01, P <0.001, P < 0.0001).
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
The embodiment of the invention provides a fluorescent probe for detecting the activity of a coagulation factor FXIa inhibitor in a complex system, which comprises an identification probe, a capture probe and a signal probe; the recognition probe consists of fluorescent quantum dots, enterokinase and an aptamer which are respectively modified on the fluorescent quantum dots; the capture probe consists of a nano magnetic sphere and FXIa covalently modified on the nano magnetic sphere; the binding affinity of the aptamer to the FXIa active site is lower than the binding affinity of the FXIa inhibitor to the FXIa active site. The recognition probe is AptE-QDs-EK. The capture probe is MNPs-FXIa. The signal probe is CdTe@ZnS.
As shown in FIG. 1, the invention aims at FXIa inhibitor active ingredients and establishes a fluorescence screening method by a double enzyme cascade signal amplification system. Firstly, aptE-QDs-EK recognition probes which are jointly modified by enterokinase and an aptamer AptE with low binding affinity to FXIa active site are prepared, and FXIa is covalently modified on nano Magnetic Spheres (MNPs) through an amide bond to form capture probes MNPs-FXIa. Because AptE40 is specifically combined with FXIa active site, the capture probe MNPs-FXIa is coupled with AptE-QDs-EK recognition probes to obtain MNPs-FXIa-AptE-QDs-EK complex. When a compound having a higher binding affinity to the FXIa active site than the aptamer AptE is present in the system, the compound competitively binds to the FXIa active site, at which time AptE-QDs-EK is detached from the MNPs-FXIa surface. After magnetic separation, the supernatant was aspirated and trypsinogen and a weakly fluorescent luminescent substrate R6-RQDs were added. At this time, aptE-QDs-EK enterokinase can activate trypsinogen to be converted into trypsin, so that primary signal amplification is realized. After the trypsin degrades R6 into small peptide and free amino acid, RQDs fluorescence is quickly recovered, and secondary signal amplification is realized. Finally, the extent of recovery of the fluorescent signal is correlated with the content of FXIa inhibiting active ingredient. The invention realizes ultrasensitive detection of FXIa inhibitor FELIAP by a double-enzyme signal amplification strategy, and has good linear relation (R 2 = 0.9916) within the range of 1-375 nM. In addition, the established method is successfully applied to active ingredient screening of FXIa inhibitors of 10 crude extracts of ground beetle proteins, and two extracts with potential FXIa inhibition activities are successfully screened. The establishment of the method provides a key technical means for the high-sensitivity discovery, high-throughput quantitative analysis and high-connotation activity screening of the active ingredients of the FXIa inhibitor in a complex system, and has a certain application value in the drug research of novel anticoagulant peptides and the screening research of the active ingredients of the FXIa inhibitor in a traditional Chinese medicine complex system.
The sources of the materials related to the embodiment of the invention are as follows:
ammonia (25-28 wt%), 3-aminopropyl triethoxysilane (APTES), coomassie Brilliant blue R250 (electrophoresis grade, 90%), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), ammonium persulfate (APS, electrophoresis grade, 98%), sodium dodecyl sulfate (SDS, electrophoresis grade, 98.5%), tetramethyl ethylenediamine (TEMED, 99%), n-hexanol (99%), triton X-100 (B.C) were purchased from Albumin Biochemical technologies Co., ltd.
Tetraethylorthosilicate (TEOS, a.r), cyclohexane (A.R) were purchased from national pharmaceutical chemicals limited.
HEPES buffer (1M, pH 7.5, cell culture grade), recombinant human enterokinase, 30% Acr-Bis (29:1) gum stock, tris-Gly electrophoresis powder were purchased from Shanghai Biyun biotechnology Co.
Carboxylated magnetic nanoparticles (MNPs-COOH, 500-600 nm) were purchased from Shanghai Meilin Biochemical technologies Co.
Trypsinogen (from bovine pancreas) was purchased from Sigma-Aldrich company.
Trypsin, 6X Protein Loading Buffer, available from Jiangsu Kaiki Biotechnology Co., ltd.
Factor XIa is available from ROSSIX. The hexapolyarginine peptide (R6), N-terminal dansyl chloride modified hexapolyarginine peptide (DNS-R6), val-Asp-Asp-Asp-Asp-Lys peptide (VDDDDK) were all synthesized by Gill Biochemical (Shanghai) and purified by HPLC.
BCA protein content detection kit (Kaiki Bio Inc.), factor XIa Calibrator (Primei Bio Inc., ),AptE40(5'-H2N-(CH2)6-TGT CAC TCT GAT CAA AAA TTT TGT AGT CAT CTT GTT ATG C-3')、FELIAP(5'-AAC CTA TCG GAC TAT TGT TAG TGA TTT TTA TAG TGT-3', Bio Inc.), EDC (Allatin), carboxylated magnetic microspheres (Nanfeng nanometer).
The experimental water was taken from MERCK MILLIPORE Direct Q3 ultra-pure water integrated system, and the resistivity was 18.2 M.OMEGA.cm 2/M. The experimental nitrogen was taken from Sciway BIO NG + nitrogen generator.
All oligonucleotides were synthesized by the company Shanghai, inc. of Biotechnology and purified by HPLC.
Example 1
(1) Preparation and characterization of recognition probes AptE-QDs-EK
To a three-necked flask sealed with N 2 were added 0.014g of sodium borohydride (NaBH 4, 0.4 mmol) and 0.0078g of selenium powder (Se, 0.1 mmol), 500. Mu.L of redistilled water was injected, and the mixture was magnetically stirred under N 2 for about 40min, and after the black selenium powder was completely reacted, the ice bath was carried out for 10min, and the obtained supernatant was NaHSe solution. 0.1517g L-glutathione (GSH, 0.4 mmol) was added to 100mL of 0.0877g Zn (Ac) 2·2H2 O (4 mM) aqueous solution, the pH was adjusted to 10.5 with NaOH solution (1M), N 2 was passed through and heated and stirred on a magnetic stirrer for 30min, then the prepared NaHSe (1 mmol) solution was added, stirring was continued for 30min, and then the mixture was refluxed at 100℃for 90min to give a transparent colorless solution, namely the prepared QDs solution (1 mM). Concentrating the prepared quantum dot solution in a rotary evaporator to about 1/3 of the original volume, adding absolute ethyl alcohol (1.5 times of the volume), stirring uniformly, centrifuging for 30min at 13000r/min, collecting the white precipitate at the bottom, and vacuum drying at 60deg.C for use.
To ultrapure water was added 10mg ZnSe@ZnS QDs, and the dispersion was sonicated to give a ZnSe@ZnS QDs dispersion, 100. Mu.L ZnSe@ZnS QDs dispersion (10 mg/mL), 2nmol AptE40, 5. Mu.g recombinant human Enterokinase (EK) and 10mg EDC, and 25mM HEPES buffer (pH 7.5) was added to a total volume of 1mL. After incubation for 2h at room temperature, 16500g was centrifuged for 5min to collect the pellet. The pellet was washed 3 times with 25mM Tris-HCl buffer (pH 8.0) and redispersed in 200. Mu.L 25mM Tris-HCl buffer (pH 8.0) to give a ZnSe@ZnS QDs capture probe (AptE-QDs-EK) co-functionalized with AptE for enterokinase and stored at 4℃for further use.
(2) Construction of Signal Probe hexapolyarginine peptide-CdTe@ZnS QDs Complex (R6-RQDs)
CdCl 2·2.5H2 O (0.8 mmol), reduced glutathione (GSH, 1.6 mmol) and 100mL of ultra pure water were added and adjusted to ph10.0 with 1M NaOH solution to give a cadmium precursor solution. ZnSO 4·7H2 O (1.26 mmol), GSH (1.26 mmol) and 100mL of ultrapure water were added, and the pH of the mixture was adjusted to 10.0 with a 1M NaOH solution to obtain a zinc precursor solution. Tellurium powder (0.3 mmol), naBH 4 (1.8 mmol) and 3mL of ultrapure water were added, immediately capped with a rubber stopper inserted with a syringe needle, and stirred at room temperature for 65min. In the process, the hydrogen generated in the bottle is discharged through the needle head on the rubber plug, and the original air in the bottle is discharged; the solution color changed from dark purple to light pink indicating that the reaction had ended, yielding a NaHTe solution.
2ML of the freshly prepared NaHTe solution was rapidly poured into the cadmium precursor solution prepared above with vigorous stirring and refluxed for 25min. 10mL of zinc precursor solution was injected while the reaction solution was boiling, and the mixture was refluxed for 15min. Then 10mL of zinc precursor solution was again injected and reflux continued for 15min. Stopping heating, and cooling the reaction liquid to room temperature in an ice water bath to obtain the red fluorescent CdTe@ZnS QDs dispersion liquid. The CdTe@ZnS QDs solution was concentrated to 1/3 of the original volume by rotary evaporation, 3 volumes of isopropanol was added, and the precipitate was collected by centrifugation at 2500g for 10 min. And (3) vacuum drying the precipitate at 40 ℃ to obtain a brick red solid, namely red fluorescent CdTe@ZnS QDs (RQDs for short). The above RQDs powder was dissolved in 25mM Tris-HCl buffer (pH 8.0) and diluted to 150. Mu.g/mL, and the R6-RQDs complex was prepared immediately before use.
The preparation method of the R6-RQDs compound comprises the following steps:
And mixing 50 mu L of hexapolyarginine peptide solution with different concentrations (0, 10, 20, 30, 40, 50, 60 and 70 mu g/mL) with 150 mu g/mL CdTe@ZnS QDs solution with equal volume, and then uniformly vortex mixing to obtain the hexapolyarginine peptide-CdTe@ZnS QDs compound.
Wherein, the final concentration of the hexapolyarginine peptide-CdTe@ZnS QDs compound adopted in the subsequent embodiment of the invention is 150 mug/ml, and the compound is prepared by the following method:
The hexa-poly-arginine peptide solution 40 mug/mL is mixed with 150 mug/mL CdTe@ZnS QDs solution with equal volume, and then vortex mixing is carried out, and the hexa-poly-arginine peptide-CdTe@ZnS QDs compound is formed through electrostatic attraction of positive and negative charges.
(3) Preparation of Capture probes MNPs-FXIa
First, MNPs-COOH were washed 3 times with coupling buffer (25 mM HEPES, pH7.5, containing 0.1% Tween-20), and the coupling buffer was redispersed to a final concentration of 5mg/mL. 1mL of MNPs-COOH dispersion, 0.1mL of FXIa solution (5 mg/mL), 50mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were added to a 2mL centrifuge tube. The mixture was reacted at room temperature for 4h, after magnetic separation, MNPs-FXIa was washed 3 times with coupling buffer, redispersed in 25mM Tris-HCl buffer (pH 8.0, containing 0.1% Tween-20) to a final concentration of 5mg/mL and stored at 4℃for further use. Dispersing MNPs-FXIa in 1mLHEPES buffer solution, taking 500 mu L, magnetically separating, discarding supernatant, adding 100 mu L of purified water as a sample to be detected, measuring FXIa concentration by using a BCA protein content detection kit, and calculating to obtain protein load rate on the MNPs.
(4) Nanometer characterization and enzyme activity monitoring
FIG. 2A shows a transmission electron microscope image of ZnSe@ZnS QDs, which are seen to be spheroid with an average particle size of 3.0.+ -. 0.5nm (FIG. 2B). The diffraction ring diameters were measured with ImageJ software and were 0.255, 0.412, 0.479nm from inside to outside, respectively, and the squares of the radii were 0.0163, 0.0431, 0.0583, respectively (ratio: 1:2.6:3.6), which is a typical face-centered cubic crystal form with crystal plane indices (111), (220), and (311), respectively, from inside to outside. The result shows that ZnSe@ZnS QDs are successfully prepared, and the smaller nanometer particle size has larger specific surface area, so that the functional modification of the nanometer surface is facilitated, the steric hindrance is reduced, and the bonding efficiency is improved.
FIG. 2C shows the ultraviolet absorption spectra of ZnSe@ZnS QDs and AptE-QDs-EK NPs, from which it is seen that ZnSe@ZnS QDs and AptE-QDs-EK have a distinct exciton absorption peak at 360nm (FIGS. 2C-a and 2C-b). In addition, aptE-QDs-EK NPs exhibited a new absorption peak around 260nm (fig. 2C-C), since aptamer AptE is a segment of ssDNA, with a characteristic absorption at 260nm, the appearance of this absorption peak means AptE40 has been successfully modified at the surface of znse@zns QDs without affecting the optical properties of QDs. Since enterokinase has ultraviolet absorption at 280nm, but is closer to 260n wavelength, the characteristic peak of enterokinase absorption at 280nm is not obvious for AptE-QDs-EK.
To verify the modification of enterokinase on the surface of ZnSe@ZnS QDs, the enterokinase activity of the recognition probe AptE-QDs-EK was measured. As shown in FIG. 2D, aptE-QDs-EK was incubated with trypsinogen, R6-RQDs complex for a period of time, and AptE-QDs-EK was found to activate trypsinogen rapidly, restoring RQDs fluorescence, whereas the negative control group (25 mM Tris-HCl buffer) did not result in activation of trypsinogen after the same incubation period, and RQDs fluorescence did not return. The above results indicate that enterokinase has been successfully modified on the surface of ZnSe@ZnS QDs and that enterokinase of AptE-QDs-EK recognition probes still has enzymatic activity. The above results strongly demonstrate the successful preparation of AptE-QDs-EK recognition probes.
Example 2 enterokinase-trypsin double enzyme cascade Signal amplification System feasibility analysis
And mixing 50 mu L of hexapolyarginine peptide solutions (0, 10, 20, 30, 40, 50, 60 and 70 mu g/mL) with the same volume of CdTe@ZnS QDs solution, and then uniformly vortex mixing to obtain a hexapolyarginine peptide-CdTe@ZnS QDs compound (R6-RQDs), and examining quenching conditions of the hexapolyarginine peptide on the CdTe@ZnS QDs by measuring fluorescence intensity at 600 nm.
50. Mu.L of trypsin solutions (0, 5, 10, 15 and 20. Mu.g/mL) were incubated with 50. Mu. L R6, 6-RQDs complex for 10min at 25℃and the recovery of trypsin to R6-RQDs was investigated by measuring the fluorescence intensity at 600 nm.
Mu.L of enterokinase solutions (0, 50, 100, 150, 200, 300, 400 and 500. Mu.g/mL) were incubated with 50. Mu.L of trypsin stock solution (0.1 mg/mL), at 25℃for 2h, 50. Mu. L R6-RQDs complex was added, and incubated at 25℃for 5min, and the recovery of the fluorescence of enterokinase-activated trypsin to R6-RQDs was examined by measuring the fluorescence intensity at 600 nm.
The fluorescence intensity is measured by a SpectraMax M 2e multifunctional enzyme-labeled instrument, and the excitation wavelength is 365nm.
Hexapolyarginine peptide induced CdTe@ZnS QDs aggregation quenching
FIG. 3A shows a high resolution transmission electron microscope image of CdTe@ZnS QDs, as can be seen by the figure RQDs assuming a spheroid shape with an average particle size of 3.6.+ -. 0.5nm (FIG. 3B). The diffraction ring diameters were measured with ImageJ software and were 0.254, 0.414, 0.487nm from the inside to the outside, and the squares of the radii were 0.0161, 0.0428, 0.0592, respectively (ratio: 1:2.7: 3.7), which is a typical face-centered cubic crystal form with crystal indices (111), (220), and (311), respectively, from the inside to the outside. The results show that CdTe@ZnS QDs have been prepared successfully,
The cdte@zns QDs surface is stabilized by glutathione (pKa 1=2.21) and 3-mercaptopropionic acid (pKa 1=4.34), the carboxyl groups of both of which are usually present in partially ionized form under neutral or weakly alkaline conditions, which gives cdte@zns QDs surface a rich negative charge, the presence of which allows repulsive forces between the quantum dots to exist without aggregation, so that the negative charge of the quantum dot surface is critical for maintaining the stability of the quantum dots in solution. Arginine (Arg, R) is a typical basic amino acid, the side chain guanidine groups of which are positively charged by protonation under weak acidic, neutral or even slightly basic conditions, and as such, hexapolyarginine peptides are in fact linear oligopeptides with a large number of positive charges. When hexapolyarginine peptides coexist with CdTe@ZnS QDs in solution, hexapolyarginine peptides bind to the surface of quantum dots by electrostatic force, and the binding results in a great reduction of the negative charge on the surface of the quantum dots. As shown in FIG. 3C, when CdTe@ZnS QDs (1 mg/mL) were mixed with hexapolyarginine peptide (0.3 mg/mL), the Zeta potential of RQDs was greatly reduced from-34.6.+ -. 0.3mV to-21.3.+ -. 0.8mV, which resulted in a dramatic decrease in the stability of the quantum dots in aqueous solution, which tended to aggregate into larger particles due to weakening of electrostatic repulsion between the quantum dots, as shown in FIG. 3D, the hydration diameter of the quantum dots was greatly increased from 9.97.+ -. 2.01nm to 2.88.+ -. 1.67. Mu.m, which suggests that the quantum dots have aggregated from a monodisperse state into larger particles. More importantly, the aggregation of RQDs also resulted in a significant change in fluorescence properties, as shown in FIG. 3E, after 150 μg/mL RQDs was mixed with different concentrations of hexapolyarginine peptide (0, 10, 20, 30, 40, 50, 60 and 70 μg/mL), the fluorescence intensity of CdTe@ZnS QDs decreased dramatically while the emission wavelength of CdTe@ZnS QDs also shifted progressively red from 600nm to 605nm as the concentration of hexapolyarginine peptide increased (FIG. 3F). When the hexa-arginine concentration was too high, it was even possible to observe directly with the naked eye that RQDs dispersion turned from clear to turbid. The above results strongly demonstrate that hexapolyarginine peptides can induce aggregation quenching of quantum dots by neutralizing RQDs surface negative charges.
Double enzyme mediated fluorescence recovery of R6-RQDs complexes
Trypsin is a serine protease with endopeptidase activity capable of hydrolysing peptide bonds on the carboxy side of the lysine and arginine residues of polypeptides. As shown in FIG. 4A, after incubation of various concentrations of trypsin (0, 5, 10, 15, 20. Mu.g/mL) mixed with the R6-RQDs complex, the fluorescence of the quantum dots at 600nm was significantly recovered. This is probably due to the fact that trypsin degrades hexapolyarginine peptides into shorter small peptides or arginines, the mixture of which has a lower ratio of guanidine groups to carboxyl groups than hexapolyarginine peptides, and their number of positive charges is less than that of hexapolyarginine peptides, thus reducing the ability to induce RQDs aggregation quenching, resulting in the recovery of fluorescence of the latter. Enterokinase (Enterokinase, EK) is a mammalian secreted serine protease capable of catalyzing the hydrolysis of the carboxy-terminal peptide bond of the N-terminal hexapeptide Val-Asp-Lys (vdddk) of trypsinogen (Trypsinogen, TPS), resulting in the activation of the trypsinogen as trypsin. Thus, a small amount of enterokinase activates trypsinogen to produce a large amount of trypsin by enzymatic reaction, while trypsin can restore the fluorescence of the R6-RQDs complex and has been verified by the previous experiments. Thus, the R6-RQDs complex is much more sensitive to enterokinase than trypsin in the presence of trypsinogen, which is the basic principle of the enterokinase-trypsin double enzyme cascade amplification system. As shown in FIG. 4B, the addition of different concentrations of enterokinase (0, 50, 100, 150, 200, 300, 400 and 500. Mu.g/mL) in the presence of trypsinogen resulted in a significant recovery of the fluorescence intensity of the R6-RQDs complex, and this recovery of fluorescence was significantly enterokinase concentration dependent.
More importantly, the response of the R6-RQDs complex to enterokinase was very rapid, as shown in FIG. 4C, adding 1nM enterokinase to the mixture of trypsinogen and R6-RQDs complex, and after a period of about 200s of silencing, the red fluorescence of RQDs increased rapidly and reached plateau at 320 s. Such a fast response is due to the efficient signal amplification of the enterokinase-trypsin double enzyme system. Meanwhile, the lowest response concentration of the R6-RQDs complex to trypsin is 5 mug/mL, and the lowest response concentration to enterokinase is as low as 50pM (about 1.34 ng/mL), so that the sensitivity is greatly improved. The above results clearly show that the R6-RQDs complex is able to respond to enterokinase in a very short time (within 5 min) and is much higher than the sensitivity to trypsin, indicating the effectiveness of the enterokinase-trypsin double enzyme signal amplification system.
Example 3 verification of the principle of amplification of the double enzyme Cascade fluorescence Signal
(1) Gel electrophoresis to verify activation of trypsinogen by enterokinase
Activation of trypsinogen by recombinant human enterokinase was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Enterokinase (50. Mu.g/mL), trypsinogen (200. Mu.g/mL), trypsin (100 mg/mL) were prepared in 25mM Tris-HCl buffer (pH 8.0) to the desired concentration for use. To prepare enterokinase-activated trypsinogen, 50. Mu.L of enterokinase solution (10 nM) was mixed with 50. Mu.L of trypsinogen solution (400. Mu.g/mL) and incubated at room temperature for 30 min. 50. Mu.L of the above sample and 10. Mu.L of 6X Protein Loading Buffer, boiling water bath for 5min, ice bath for 5min, and 9000g of the mixture were centrifuged for 1min, and 10. Mu.L of the supernatant was sampled. Before the bromophenol blue indicator enters the separation gel, the current is set to be constant for 30mA, after the bromophenol blue enters the separation gel, the current is constant for 45mA, and the electrophoresis is stopped when the indicator is 1cm away from the lower edge of the gel. The gel pieces were peeled off, and after washing the surface with ultrapure water, the gel pieces were placed in 0.25% coomassie brilliant blue R250 staining solution and stained at 25 ℃ for 2 hours. The stained gum pieces were decolorized in a decolorizing solution (methanol: water: glacial acetic acid=30:60:10, v/v/v) at 25℃for 1h. The slab of gum photo was processed by Image J2.0 software.
(2) Trypsin degradation of dansyl chloride labeled hexapolyarginine peptide
First, the aminated silica nanoparticles were prepared by the reverse phase microemulsion method. 75mL of cyclohexane, 17.7mL of TritonX-100, 18mL of n-hexanol, 0.48mL of ultrapure water, and 2.4mL of aqueous ammonia (25 to 28 wt%) were added. After stirring vigorously for 30min, 1mL TEOS and 1mL APTES were added and the mixture was stirred at room temperature in the dark for 72h. And (3) centrifuging 2500g for 5min, collecting precipitate, washing with absolute ethyl alcohol for 3 times, and drying to obtain the aminated silica nano particles (SiO 2 NPs).
20Mg of the above SiO 2 NPs,20mg of N-terminal dansyl chloride labeled hexapolyarginine peptide (DNS-R6), 200mg of EDC and 2mL of 25mM HEPES buffer (pH 7.5) were added. Stirring the mixed solution at room temperature in the dark for 1h, centrifuging 16500g for 5min, collecting precipitate, washing with 25mM HEPES buffer solution for 3 times, and dispersing in 25mM Tris-HCl buffer solution (pH 8.0) to obtain fluorescent peptide modified silica nanoparticle (SiO 2 -R6-DNS NPs), and preserving at 4 ℃ for later use.
100. Mu.L of SiO 2 -R6-DNS NPs dispersion (2 mg/mL) and 100. Mu.L of trypsin solution (1 mg/mL) were added, after incubation for different times (0, 30, 60, 90, 120 min) at 37℃in the absence of light, 16500g was centrifuged for 5min, 150. Mu.L of the supernatant was taken out in 96-well plates, fluorescence intensities at 535nm and fluorescence emission spectra at 420 to 650nm were measured, and excitation wavelength was set at 310nm.
(3) Trypsin activity inhibition assay
To further verify the degradation of the hexapolyarginine peptide by trypsin, trypsin activity inhibition experiments were designed. The inhibition of trypsin activity by heating and addition of inhibitors, respectively, was compared to untreated trypsin to demonstrate that the recovery of trypsin fluorescence from the R6-RQDs complex was caused by enzymatic activity of trypsin. In the trypsin heat-inactivation experiment, a trypsin solution (20. Mu.g/mL) was boiled in water for 15min and cooled to room temperature to obtain a heat-inactivated trypsin solution. 50. Mu.L of heat-inactivated trypsin solution (20. Mu.g/mL) or an equal concentration of non-heat-inactivated trypsin solution and 50. Mu. L R6-RQDs complex were added to a 96-well plate, and after incubation of the mixture at 25℃for 10min, the fluorescence intensity at 600nm was measured, and the excitation wavelength was 365nm.
In the trypsin inhibitor experiments, 200. Mu.L of trypsin solution (25. Mu.g/mL) and various volumes (0, 10, 20, 40, 50. Mu.L) of 10mM trypsin inhibitor phenylmethanesulfonyl fluoride (PMSF) solution were added, and 25mM Tris-HCl buffer (pH 8.0) was added to a total volume of 250. Mu.L. Incubation was carried out at 25℃for 5min to give a PMSF-treated trypsin solution. 50. Mu.L of the PMSF-treated trypsin solution and 50. Mu. L R6-RQDs complex were added to a 96-well plate, and the mixture was incubated at 25℃for 10min to determine 600nm fluorescence intensity, and excitation wavelength was 365nm.
Verification of double-enzyme cascade signal amplification principle
To verify that the course of operation of the enterokinase-trypsin double enzyme cascade amplification system was consistent with expectations, activation of trypsinogen by enterokinase was verified by SDS-PAGE electrophoresis. As shown in FIG. 5A, the band around 20-25 kDa was significantly lighter for enterokinase-activated trypsinogen (lane 3) compared to trypsinogen (lane 2), while a new band around 10kDa was present (red arrow), which was consistent with trypsin (lane 4), indicating that trypsin was indeed produced by enterokinase activation of trypsinogen, which was consistent with the description of the detection principle.
In order to verify the degradation of hexa-arginine peptide by trypsin, a fluorescent peptide degradation experiment was designed. The aminated silica nanoparticles (SiO 2 NPs) used in the experiment are in a sphere-like shape (figure 5B), and the APTES modified silicon spheres are mutually adhered due to the modification effect of APTES on the surfaces of the silicon spheres, so that the pore diameter structure of the silicon spheres is fuzzy, which shows that amino groups are successfully modified on the silicon spheres, the average particle size is about 38.99 +/-4.67 nm (figure 5C), and the modification of the polypeptide is utilized. The principle is shown in fig. 5D, the N-terminal dansyl chloride labeled hexapolyarginine peptide (R6-DNS) is modified on the surface of SiO 2 NPs through an amide bond, and after the peptide segment connecting the dansyl chloride and the SiO 2 NPs is degraded by trypsin, the dansyl chloride is separated from the surface of the silica nanoparticle, and appears in the supernatant after centrifugal separation. The degradation degree of trypsin on hexapolyarginine peptide can be evaluated by measuring fluorescence emitted by dansyl chloride in the supernatant. As shown in fig. 5E, R6-DNS modified silica nanoparticles (SiO 2 -R6-DNS NPs) were incubated with trypsin (1 mg/mL) at 37 ℃, the fluorescence intensity of the supernatant after centrifugation increased significantly at 535nm with prolonged incubation time, and the fluorescence emission wavelength of the supernatant also red shifted from 510nm to 535nm with prolonged incubation time (fig. 5F), which may be related to increased degradation of the dansyl chloride labeled peptide fragment length with prolonged incubation time, and shortened peptide fragment length. FIG. 5G also clearly shows that the fluorescence of the supernatant was brighter with prolonged incubation time, whereas the fluorescence after incubation with SiO 2 -R6-DNS NPs with Tris-HCl buffer instead of trypsin was hardly enhanced, which precludes the possibility of spontaneous shedding of the dansyl chloride label under weakly alkaline conditions leading to false positive results.
To further verify that the fluorescence recovery of trypsin on the R6-RQDs complex was related to its enzyme activity, the trypsin activity was inhibited by heat inactivation and the addition of the serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), compared to trypsin with normal activity. FIG. 5H shows that trypsin addition resulted in a sharp recovery of the R6-RQDs complex fluorescence, consistent with previous experimental results, whereas trypsin inactivated by boiling water bath resulted in little recovery of fluorescence, with fluorescence intensity very close to that of the negative control (Tris-HCl). Furthermore, the ability of trypsin after treatment with PMSF to restore fluorescence to the R6-RQDs complex was significantly reduced (FIG. 5H), and the extent of reduction increased with increasing PMSF concentration. The above results strongly demonstrate that the fluorescence recovery of trypsin on the R6-RQDs complex is closely related to its enzyme activity, indicating that trypsin causes the fluorescence recovery of the R6-RQDs complex by catalyzing the hydrolysis of hexapolyarginine peptide.
EXAMPLE 4 detection of FXIa inhibitor aptamer FELIAP
Mu.L of MNPs-FXIa (5 mg/mL), 10 mu L AptE-QDs-EK (5 mg/mL) and 5 mu L0.1M Tris-HCl buffer (containing 1M NaCl, pH 7.4) were added to the EP tube and incubated for 30min at room temperature. After magnetic separation, the supernatant was discarded, washed 4 times with 200. Mu.L of 25mM Tris-HCl buffer (pH 8.0), and redispersed in 25mM Tris-HCl buffer (pH 8.0) to prepare MNPs-FXIa-AptE-QDs-EK complex, which was stored at 4℃for use.
For FELIAP assays, 10. Mu.L of MNPs-FXIa-AptE40-QDs-EK complex and 50. Mu.L of FELIAP solution of different concentrations were first added to a 1.5mL centrifuge tube and incubated at room temperature for 20min. After magnetic separation, 50. Mu.L of the supernatant was taken, 50. Mu.L of an original trypsin solution (0.1 mg/mL) and 50. Mu.L of a freshly prepared R6-RQDs complex were added, and after incubation at room temperature for 10min, the fluorescence intensity at 600nm was measured and the excitation wavelength was set at 365nm.
Detection of FXIa inhibitory aptamer FELIAP
In order to verify the feasibility of the method of the invention for evaluating the activity of FXIa inhibitors, the invention was examined using as a verification model an aptamer FELIAP that has been specifically shown to have FXIa inhibiting activity. According to previous reports AptE and FELIAP are two nucleic acid aptamers capable of specifically binding to the active site of FXIa, of which FELIAP has a very strong affinity for FXIa (kd=1.8 nM). In the method, in the process of forming the MNPs-FXIa-AptE-QDs-EK complex, aptE is specifically combined with FXIa active sites, and the combination of the two serves as a bridge to connect an MNPs-FXIa capture probe with an enterokinase modified AptE-QDs-EK fluorescent signal probe. The formation of MNPs-FXIa-AptE-QDs-EK nanocomposite and enterokinase activity were examined by the present invention via fluorescence signal. As shown in FIG. 6A, incubation of MNPs-FXIa-QDs-AptE-EK complex with trypsinogen and R6-RQDs complex resulted in rapid activation of trypsinogen, and degradation of R6 by generated trypsin resulted in rapid recovery of RQDs fluorescence by releasing aggregation quenching of RQDs. The negative control group (25 mM Tris-HCl buffer, pH 8.0) did not result in trypsinogen activation after incubation, and the fluorescence intensity at 600nm was still low. The results indicated that MNPs-FXIa-AptE-QDs-EK complex formation was not affected and enterokinase activity was not affected.
And analyzing and detecting FELIAP model medicaments by adopting a constructed method. The results showed that, in the range of 1-375 nM, all concentrations of FELIAP solutions resulted in release of AptE-QDs-EK from MNPs-FXIa into the supernatant, and AptE-QDs-EK in the supernatant resulted in recovery of RQDs fluorescence by the enterokinase-trypsin cascade signal amplification system. Has good linearity in the drug concentration range of 1-375 nM (Fluorescence Intensity =0.2727 [ FELIAP ] +411.3, R 2 =9916). In addition, quality control samples with three concentration levels of high (300 nM), medium (180 nM) and low (15 nM) in the linear range were verified for precision and accuracy. The results are shown in Table 1, with accuracy in all concentration levels in the range of 77.2% to 101.2% and RSD less than 25.0%. The results show that the double-enzyme cascade signal amplification system developed by the research method has higher sensitivity and reliable quantitative performance in FXIa inhibitor detection.
Table 1 precision and accuracy experimental results (n=5)
EXAMPLE 5 Activity screening of Eupolyphaga Seu Steleophaga extract FXIa inhibitor
The active screening object of this embodiment is 10 kinds of ground beetle extracts, which are prepared by the applicant and are all water-soluble freeze-dried powder.
Mu.L of the MNPs-FXIa-AptE-QDs-EK complex prepared in example 4 and 50. Mu.L of an aqueous ground beetle extract (1. Mu.g/mL) or a control sample were added to the EP tube, wherein FELIAP aqueous solution (1. Mu.M) was used as a positive control group and ultrapure water was used as a negative control group. The mixture was incubated at room temperature for 20min, 50. Mu.L of the supernatant was taken, 50. Mu.L of the trypsinogen (0.1 mg/mL) and 50. Mu. L R6-RQDs complex were added, and incubated at room temperature for 10min, and the fluorescence intensity at 600nm was measured, with excitation wavelength of 365nm.
In order to eliminate the influence caused by the direct interaction of the components to be tested with trypsinogen or the R6-RQDs complex, a correction group is arranged for each component to be tested, MNPs-FXIa-AptE-QDs-EK in the correction group is replaced by ultrapure water with the same volume, and the rest detection processes are identical to the experimental group. Delta F/Fn is used as an index for evaluating the inhibition capability of the ground beetle extract on FXIa, wherein delta F is the difference value between F and F0, F and F0 are the fluorescence intensities of an experimental group and a corresponding correction group at 600nm, and Fn is the fluorescence intensity of a negative control group after correction.
And selecting a traditional APTT and PT kit for anticoagulant activity screening experiment verification. The experimental animals are provided by animal experiment centers of Chinese university of medical science, and male Sprague Dawley rats weighing 180-200 g are given sufficient food and water daily at a continuous temperature (24+ -1deg.C), relative humidity (50+ -10%) and for 12 hours of day and night period, and are fasted for 24 hours before the experiment, without water forbidding. Animal experiments were conducted in accordance with guidelines of the national institutes of health, guidelines for care and use of laboratory animals (revised in NIH publication No. 8023, 1978), and were approved by the animal ethics committee of the university of Chinese medical science (acceptance number: 2023-05-012). Taking blood from abdominal aorta after anesthesia of rat, taking 3.8% sodium citrate as anticoagulant, centrifuging for 10min at 900g, and collecting supernatant to obtain Platelet Poor Plasma (PPP). 10. Mu.L of sample solution (10 mg/mL), 40. Mu.L of platelet-poor plasma and 50. Mu.L of APTT reagent were added, mixed well, incubated at 37℃for 3min, 50. Mu.L of calcium chloride solution was added, and APTT time was measured using an SC40 semi-automatic coagulation analyzer. 10. Mu.L of the sample solution (10 mg/mL) and 40. Mu.L of platelet-poor plasma were added, mixed well, incubated at 37℃for 3min, 100. Mu.L of PT reagent was added, and the PT time was measured.
Screening of Eupolyphaga Seu Steleophaga extract with FXIa inhibiting activity
Screening FXIa inhibitors on 10 water-soluble ground beetle crude protein extracts by using a developed method. As shown in fig. 6B, the detection signal Δf/Fn of the positive control compound FELIAP was significantly increased (p < 0.005) compared to the negative control group (ultrapure water), proving that the detection process was effective, which is consistent with the previous experimental results. Of the 10 ground beetle extracts, both extracts No. 1 and No. 2 may lead to a different degree of increase in the detection signal, especially the detection signal of extract No. 1 is significantly increased compared to ultra pure water (p < 0.05), which means that it may contain a more active or higher content of FXIa inhibitor. The results of conventional anticoagulation activity studies also show that extract No. 1 can significantly prolong the APTT time (fig. 6C), with significant differences compared with the blank and other components, and that all components have no trend to significantly prolong the PT time (fig. 6D), which shows that extract No. 1 can prolong the APTT time by affecting the relevant factors in the intrinsic coagulation pathway, consistent with the results of the methods established in the present invention. The development of the method provides a key technology and an analysis method for evaluating the activity for screening FXIa inhibitors in ground beetle extracts and researching medicaments.
Claims (9)
1. A fluorescent probe for detecting the activity of a coagulation factor FXIa inhibitor in a complex system, characterized in that the fluorescent probe comprises a recognition probe, a capture probe and a signaling probe; the recognition probe consists of fluorescent quantum dots, enterokinase and an aptamer which are respectively modified on the same fluorescent quantum dots; the capture probe consists of a nano magnetic sphere and FXIa covalently modified on the nano magnetic sphere; the binding affinity of the aptamer to the FXIa active site is lower than the binding affinity of the FXIa inhibitor to the FXIa active site; the signal probe is a hexapolyarginine peptide-CdTe@ZnS QDs compound; when the fluorescent probe is used, trypsinogen is added into a complex system, enterokinase in the recognition probe catalyzes the hydrolysis of N-terminal hexapeptide of the trypsinogen, and the trypsin is generated by amplification.
2. The fluorescent probe for detecting the activity of a coagulation factor FXIa inhibitor in a complex system according to claim 1, wherein the recognition probe is AptE-QDs-EK, wherein the enterokinase is recombinant human enterokinase, the fluorescent quantum dot is znse@zns quantum dot, and the aptamer is AptE.
3. The fluorescent probe for detecting the activity of a coagulation factor FXIa inhibitor in a complex system according to claim 2, wherein the AptE-QDs-EK is prepared by the following method:
ZnSe@ZnS dispersion, aptE, recombinant human enterokinase, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride in a buffer solution, and incubating for a period of time;
after the incubation, the pellet was collected as AptE-QDs-EK.
4. The fluorescent probe for detecting the activity of a coagulation factor FXIa inhibitor in a complex system according to claim 1, wherein the capture probe is MNPs-FXIa, and the preparation method of MNPs-FXIa is as follows:
MNPs-COOH dispersion, FXIa solution and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride are mixed and then reacted for a period of time at room temperature, and the MNPs-FXIa is obtained after magnetic separation.
5. The fluorescent probe for detecting the activity of a coagulation factor FXIa inhibitor in a complex system according to claim 1, wherein the preparation method of the hexapolyarginin-cdte@zns QDs complex is as follows:
Fully mixing the hexapolyarginine peptide solution with the CdTe@ZnS QDs solution, and collecting the product, namely the hexapolyarginine peptide-CdTe@ZnS QDs compound; wherein the mass ratio of the hexapolyarginine peptide in the hexapolyarginine peptide solution to CdTe@ZnS in the CdTe@ZnS QDs solution is 1-7:15.
6. A method for detecting the activity of a coagulation factor FXIa inhibitor in a complex system, comprising the steps of:
(1) Mixing the recognition probe dispersion and the capture probe dispersion to form a first complex;
adding a sample into the first compound, incubating for a period of time, and taking a supernatant;
Adding trypsinogen and a signal probe into the supernatant, incubating for a period of time, measuring the fluorescence intensity after incubation, and if FXIa inhibitor exists in the sample, sequentially amplifying the first-order signal and the second-order signal by the measured fluorescence signal, wherein the recovery degree of the fluorescence signal is related to the content of FXIa inhibition active ingredients;
The recognition probe consists of fluorescent quantum dots, enterokinase and an aptamer which are respectively modified on the same fluorescent quantum dots; the capture probe consists of a nano magnetic sphere and FXIa covalently modified on the nano magnetic sphere; the binding affinity of the aptamer to the FXIa active site is lower than the binding affinity of the FXIa inhibitor to the FXIa active site; the signal probe is a hexapolyarginine peptide-CdTe@ZnS QDs compound;
(2) Sequentially replacing the sample in the step (1) with a sample containing FXIa inhibitor with gradient concentration, and repeating the step (1) for a plurality of times to obtain a standard curve of the concentration of the FXIa inhibitor corresponding to the recovery degree of the fluorescent signal;
(3) And (3) determining the recovery degree of the fluorescent signal of the sample to be tested by using the method in the step (1), and comparing the recovery degree with the standard curve obtained in the step (2) to obtain the concentration of the FXIa inhibitor in the sample to be tested.
7. The method according to claim 6, wherein in the step (1), the mass ratio of the recognition probe in the recognition probe dispersion to the capture probe in the capture probe dispersion is 1:1.
8. The method for detecting the activity of a coagulation factor FXIa inhibitor in a complex system according to claim 6, wherein the conditions for measuring the fluorescence intensity are as follows: the fluorescence intensity of 600 nm was measured and the excitation wavelength was 365 nm.
9. A method for screening for inhibition activity of coagulation factor FXIa in a complex system, comprising the steps of:
(1) Mixing the recognition probe dispersion and the capture probe dispersion to form a first complex;
Adding a sample to be screened into the first compound, incubating for a period of time, and taking supernatant;
Adding trypsinogen and a signal probe into the supernatant, incubating for a period of time, and measuring the fluorescence intensity after incubation is finished, wherein the measured fluorescence intensity is marked as F;
The recognition probe consists of fluorescent quantum dots, enterokinase and an aptamer which are respectively modified on the same fluorescent quantum dots; the capture probe consists of a nano magnetic sphere and FXIa covalently modified on the nano magnetic sphere; the binding affinity of the aptamer to the FXIa active site is lower than the binding affinity of the FXIa inhibitor to the FXIa active site; the signal probe is a hexapolyarginine peptide-CdTe@ZnS QDs compound;
(2) Replacing the first compound in the step (1) with pure water with the same volume, repeating the step (1), and marking the measured fluorescence intensity as F0;
(3) Replacing the sample to be screened in the step (1) with a negative control group, repeating the step (1), and marking the measured fluorescence intensity as Fm;
(4) Replacing the first compound in the step (1) with pure water with the same volume, replacing the sample to be screened with a negative control group, repeating the step (1), and marking the measured fluorescence intensity as F1;
(5) The calculation formula of the index n for evaluating the inhibition capacity of the sample to be screened on FXIa is as follows: 。
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