Triple-reaction peptide probe and application thereof

Abstract

The invention discloses a triple-reaction peptide probe and application thereof, and the triple-reaction peptide probe is a T-shaped structure composed of three peptide branches for identifying three target proteases, each of the three peptide branches comprises a restriction enzyme cutting site fragment used for being specifically recognized by target protease and a peptide fragment used for signal distinguishing; the triple-reaction peptide probe is applied to simultaneously detecting and identifying three target proteases; by designing the triple-reaction peptide probe, multiple proteases and activities thereof can be evaluated and accurately distinguished simultaneously by using one nanopore, the probe can be specifically recognized by three proteases simultaneously and release distinguishable output peptide fragments, different current signals are generated in a single aerolysin nanopore, and the sensitivity of the aerolysin is improved. The method has the advantages of high sensitivity, high selectivity and the like, and has great potential in the aspect of non-invasive disease diagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and particularly to a triple-reaction peptide probe and its application. Background Art

[0002] Accurate disease diagnosis usually requires the simultaneous analysis of multiple biomarkers in a single sample to improve diagnostic accuracy and guide personalized treatment. However, the simultaneous detection of three or more proteases in a sample, especially using single-substrate probes, remains a major challenge. Changes in the expression of various biomolecules are often associated with the occurrence and progression of diseases. Accurate disease diagnosis requires the simultaneous analysis of multiple biomarkers in a sample. This can improve the diagnostic accuracy and provide a direction for personalized treatment. Proteases are essential at all stages of tumorigenesis and development, such as angiogenesis, invasion, and metastasis. In particular, several types of proteases have been used as biomarkers for cancer diagnosis and prognosis. For example, caspase-8 (CASP8) is located at the top of the caspase cascade and is considered a caspase initiator. It can activate downstream caspases, cleave key cellular substrates, and ultimately induce apoptosis. Cathepsin B (Cat.B) is overexpressed in a variety of tumor cells and plays different roles at different stages of malignant tumors, such as tumor invasion, metastasis, apoptosis, etc. Matrix metalloproteinase-2 (MMP-2) is an extracellular matrix degrading enzyme that can degrade basement membrane components and is related to tumor angiogenesis and metastasis. Recent research progress has focused on the development of innovative sensing methods for monitoring protease activity, such as fluorescence spectroscopy and immunoassay.

[0003] However, when it comes to multi-target sensing, these technologies are severely affected by overlapping emission spectra or laborious sample preparation. Therefore, a reliable and convenient method for simultaneously monitoring the activities of multiple proteases is highly desirable. Summary of the Invention

[0004] To solve the above problems, the present invention provides a triple-reaction peptide probe and its application.

[0005] A triple-reaction peptide probe, wherein the triple-reaction peptide probe is a T-shaped structure composed of three peptide branches for identifying three target proteases, and each of the three peptide branches includes a cleavage site fragment for specific recognition by the target protease and a peptide fragment for signal discrimination.

[0006] Description: The above peptide probes have a T-shaped structure with three independent arms, and each arm contains a target-specific recognition site. After adding the target protease, they cleave the probes at their respective cleavage sites, releasing distinguishable peptide fragments composed of different amino acids. These peptide fragments generate different current signals within a single aerolysin nanopore. By utilizing these current signals, the corresponding target protease can be identified. This method has advantages such as high sensitivity and high selectivity, and has great potential in non-invasive disease diagnosis.

[0007] In some embodiments, the three target proteases are: CASP8, Cat.B, MMP-2.

[0008] Description: Simultaneously identifying the above three target proteases can improve diagnostic accuracy, guide personalized treatment, predict disease prognosis, and facilitate drug development. These proteases are respectively involved in important physiological and pathological processes such as apoptosis, autophagy, inflammatory response, and extracellular matrix remodeling. Therefore, simultaneously detecting their activity levels can provide more comprehensive disease information and help formulate more effective treatment strategies.

[0009] Furthermore, the amino acid sequences of the three peptide branches are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0010] Description: In the above peptide branches, by reducing the occurrence of arginine, the synthesis complexity can be avoided; by reducing the occurrence of tryptophan, the T-shaped probe after enzymatic cleavage can be prevented from passing through the aerolysin nanopore. By adding histidine at the central position, since histidine is positively charged, the electrostatic force will prevent macromolecules or molecules with opposite charges from passing through within the aerolysin nanopore channel. Through the enzyme recognition site, it is used for the recognition of CASP8, Cat.B, and MMP-2. In addition, in order for the short peptide, the output product after enzymatic cleavage, to pass through the aerolysin nanopore and generate characteristic current, glutamic acid is added to each short peptide, increasing the interaction with the inside of the pore.

[0011] In other embodiments, the three target proteases are: CASP3, CASP6, PSA.

[0012] Description: Simultaneously identifying the above three target proteases can more comprehensively evaluate cell apoptosis and prostate health status, improve the diagnostic accuracy of specific diseases such as prostate cancer, guide the formulation of personalized treatment plans, predict disease development and patient prognosis, and provide important biological markers for the development of new treatment methods and drugs.

[0013] Furthermore, the amino acid sequences of the three peptide branches are respectively shown as SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0014] The present invention also provides an application of a triple-reaction peptide probe in simultaneously detecting and recognizing three target proteases.

[0015] The present invention also provides an application of a triple-reaction peptide probe in simultaneously detecting the activities of three target proteases.

[0016] Further, the detection method includes:

[0017] First, add 10 μM of the triple-reaction peptide probe and 10 μM of the liquid to be detected into 80 μL of Tris-HCl buffer solution with a concentration of 0.05 M - 0.1 M, and incubate at 37 °C for 2 h to obtain a reaction solution;

[0018] Then, take 20 μL of the reaction solution and add it to the cis end of the nanopore detection cell, apply a transmembrane voltage of +50 mV to the nanopore detection cell, and use an aerolysin nanopore to record the change of ionic current to obtain the amplitudes and durations of multiple blocking events;

[0019] Furthermore, identify the target protease in the liquid to be detected through the amplitudes and durations of multiple blocking events.

[0020] Further, the detection method includes:

[0021] First, add 10 μM of the triple-reaction peptide probe and 10 μM of the liquid to be detected into 80 μL of Tris-HCl buffer solution with a concentration of 0.05 M - 0.1 M, and incubate at 37 °C for 2 h to obtain a reaction solution;

[0022] Then, take 20 μL of the reaction solution and add it to the cis end of the nanopore detection cell, apply a transmembrane voltage of +50 mV to the nanopore detection cell, and use an aerolysin nanopore to record the change of ionic current to obtain the amplitudes and durations of multiple blocking events;

[0023] Furthermore, identify three target proteases through the amplitudes and durations of multiple blocking events and obtain the activity of each target protease.

[0024] Note: By using the above method to record the change of ionic current with an aerolysin nanopore, the target protease in the liquid to be detected can be recognized and its activity can be detected with high sensitivity, quickly, and directly quantified. It has the advantages of low sample consumption, real-time monitoring, environmental friendliness, and wide application.

[0025] The beneficial effects of the present invention are:

[0026] The triple-reaction peptide probe of the present invention has a T-shaped structure. After adding the target protease, the probe is cleaved at its respective cleavage sites, releasing distinguishable peptide fragments composed of different amino acids. The peptide fragments generate different current signals within a single aerolysin nanopore. Using these current signals, the corresponding target protease can be identified. This method has the advantages of high sensitivity and high selectivity and has great potential in non-invasive disease diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a data graph of using a nanopore to identify individual peptide fragments in Example 1 of the present invention;

[0028] Figure 2 It is a current trace and scatter plot of CASP8, Cat.B, and MMP-2 in Example 1 of the present invention;

[0029] Figure 3 It is an enzymatic reaction data graph of CASP8, Cat.B, and MMP-2 in Example 1 of the present invention;

[0030] Figure 4 It is a data graph of current trace and frequency comparison for simultaneously detecting multiple proteases in Example 1 of the present invention;

[0031] Figure 5 It is a data graph of the quantitative determination results of CASP8, Cat.B, and MMP-2 in Example 1 of the present invention;

[0032] Figure 6 It is a data graph of the recovery test results of three proteases in human serum in Example 1 of the present invention;

[0033] Figure 7 It is a data graph of the machine learning-assisted recognition results of the enzyme digestion products in Example 1 of the present invention;

[0034] Figure 8 It is a schematic diagram of the nanopore detection principle in the examples of the present invention;

[0035] Figure 9 It is a data graph that three peptide fragments generated different blocking signals in the nanopore test in Example 1 of the present invention;

[0036] Figure 10 It is a data graph that no signal was generated by the triple-reaction peptide probe in Example 1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0037] To further elaborate on the methods adopted and the effects achieved by the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with experiments.

[0038] Nanopore analysis is an effective single-molecule platform that can obtain detailed information about individual molecules without labeling. The passage of charged molecules through a nanopore causes temporary fluctuations in the ionic current. These modifications can be recorded and analyzed to identify the analyte and determine its concentration. This simple yet sophisticated method has been effectively applied to various research fields, including DNA sequencing, peptides, single-molecule chemistry, metal ions, and various biomolecule detection methods. The single-molecule performance of the nanopore platform makes it an ideal tool for observing enzyme activity. So far, only a few reports have described the use of nanopores to monitor the activity of one or two enzymes. However, the simultaneous detection of three or more proteases in a sample using a single substrate probe remains a major challenge.

[0039] Embodiments of the present invention provide a triple-reaction peptide probe that can simultaneously monitor the activities of three proteases through a single aerolysin nanopore. The peptide probe has a T-shaped structure with three independent branches, each branch containing a target-specific recognition site (i.e., the cleavage site in the following description). After adding the target protease, as Figure 8 shown, the target protease cleaves the probe at the cleavage site, releasing distinguishable peptide fragments composed of different amino acids. The unique current signatures generated by each output fragment allow for the simultaneous quantification of proteases at picomolar or femtomolar concentrations without labeling or amplification. This strategy enables the accurate and multiplex quantification of multiple enzymes, which is crucial for non-invasive disease diagnosis.

[0040] Combining the above, the present invention provides the following Embodiment 1 and Embodiment 2 for detailed description;

[0041] Embodiment 1: A triple-reaction peptide probe, which is a T-shaped structure composed of three peptide branches for recognizing three target proteases. Each of the three peptide branches includes a cleavage site fragment specifically recognized by the target protease and a peptide fragment for signal discrimination;

[0042] Specifically, each of the three peptide branches is sequentially arranged in the order of a linker fragment, a cleavage site fragment, a peptide fragment, and a terminal amino acid; the peptide fragment includes glutamate connected to the terminal amino acid; the terminal amino acid is aspartic acid or cysteine; the three peptide branches are connected to the midpoint lysine of the T-shaped structure through histidine in the linker fragment;

[0043] Exemplarily, the three target proteases are: CASP8 (caspase 8), Cat.B (cathepsin B), MMP-2 (matrix metalloproteinase 2); the amino acid sequences of the three peptide branches are shown as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 respectively; the T-shaped structural formula of the triple-reaction peptide probe is as follows:

[0044]

[0045] In the above T-shaped structure, N is asparagine, C is cysteine, E is glutamic acid, Y is tyrosine, H is histidine, F is phenylalanine, K is lysine, L is leucine, G is glycine, W is tryptophan, P is proline, V is valine, R is arginine, T is threonine, and D is aspartic acid;

[0046] It can be understood that in the design of the above T-type probe, in order to avoid the background signal interference generated by the residual trypsin digestion of the P0 probe during the activation of the aerolysin protein when passing through the nanopore, the occurrence of arginine (R) was reduced during the probe design. At the same time, to avoid complex synthesis, the occurrence of tryptophan (W) was also reduced. To prevent this T-type probe from passing through the aerolysin nanopore, histidine (H) was added at the central position. Since histidine is positively charged, the electrostatic force will prevent macromolecules or molecules with opposite charges from passing through inside the aerolysin nanopore. Because the inside of the aerolysin nanopore is overall positively charged and the R282 at the cis end is also positively charged, when the T-type probe enters the aerolysin nanopore, mutual repulsion will occur, thus preventing translocation within the pore. On each arm of the T-type probe, recognition sites for enzymes are respectively included for the recognition of CASP8, Cat.B, and MMP-2. In order for the short peptide of the output product after enzymatic cleavage to pass through the aerolysin nanopore and generate characteristic current, glutamic acid (E) was added to each short peptide to increase the interaction with the inside of the pore.

[0047] Combining the above content, the triple reaction peptide probe obtained in Example 1 was used to simultaneously detect and identify three target proteases and their activities;

[0048] The detection method includes: First, add 10 μM of the triple reaction peptide probe and 10 μM of the liquid to be detected into 80 μL of Tris-HCl buffer solution with a concentration of 0.05 M to 0.1 M, and incubate at 37 °C for 2 h to obtain a reaction solution;

[0049] Then, take 20 μL of the reaction solution and add it to the cis end of the nanopore detection cell, apply a transmembrane voltage of +50 mV to the nanopore detection cell, and use the aerolysin nanopore to record the change of ionic current to obtain the amplitudes and durations of multiple blocking events;

[0050] Furthermore, identify the three target proteases through the amplitudes and durations of multiple blocking events and obtain the activity of each target protease.

[0051] Experimental example: The triple reaction peptide probe of Example 1 above was tested to judge its detection effect;

[0052] 1. Determine whether the triple-reaction peptide probe can achieve detection;

[0053] S1. First, add 10 μM of the triple-reaction peptide probe and 10 μM of the target protease to 80 μL of Tris-HCl buffer with a concentration of 0.05 M to 0.1 M, and incubate at 37 °C for 30 minutes; among the target proteases, the concentration of CASP8 is 10 pM to 100 nM, the concentration of Cat.B is 1 pM to 150 nM, and the concentration of MMP-2 is 1 fM to 1 μM;

[0054] S2. Then add 1 μL of 5 mM EDTA to terminate the activity of the target protease and incubate on ice for 5 min to obtain a reaction solution. Take 20 μL of the reaction solution and add it to the cis end of the nanopore detection cell. Apply a transmembrane voltage of +50 mV to the nanopore detection cell, and use aerolysin nanopore to record the change of ionic current to obtain the amplitudes and durations of multiple blocking events;

[0055] S3. Distinguish the three peptide fragments cleaved and released by CASP8, Cat.B, and MMP-2 through the amplitudes and durations of the blocking events and obtain the activity of the target protease.

[0056] The experimental results show that (as shown in combination with Figure 1 ), in the presence of caspase 8 (CASP8), cathepsin B (Cat.B), and matrix metalloproteinase-2 (MMP-2), each protease cleaves at the corresponding recognition site, releasing a connected cleavage fragment and peptide fragment (such as N-EYGLETD-C, N-EYYHEFK-C, N-VRGYEYYHE-C), and a T-shaped connecting fragment (i.e., the remaining part after removing the above three fragments in the T-shaped structural formula of the above triple-reaction peptide probe); the shorter peptide segments (such as N-EYGLETD-C, N-EYYHEFK-C, N-VRGYEYYHE-C) can smoothly pass through the aerolysin nanopore to generate current signals, thus serving as reporters of the target protease. Due to size limitations, the remaining larger fragments (the remaining part after removing the above three fragments in the T-shaped structural formula of the triple-reaction peptide probe) may only generate collision signals caused by branched structures. Different fragments are produced under the action of two or more proteases. However, only three short output peptides (N-EYGLETD-C, N-EYYHEFK-C, N-VRGYEYYHE-C) can generate effective and unique signals, and the frequencies of these signals can be recorded. Therefore, the experiment proves that the triple-reaction peptide probe in Example 1 can identify and distinguish three target proteases;

[0057] As Figure 1As shown, single peptides (ED, EK, and VE) were identified using nanopores. (A, D, G) are representative current traces of peptides ED, EK, and VE. (B, E, H) are signal events generated by ED, EK, and VE. (C, F, I) are current blockade histograms of ED, EK, and VE events with current blockade values. (J) is the Gaussian fitting analysis of the current blockade histogram. (K) is the comparison of the I / I0 histograms generated by the three peptides. (L) is a scatter plot showing the relationship between the dwell time and I / I0 of the three peptides. All data were collected at +50 mV.

[0058] First, by detecting the ability of the aerolysin nanopore to recognize all peptides, including one of the above triple-reaction peptide probes and three short peptide fragments: 7-mer ED (N-EYGLETD-C), 7-mer EK (N-EYYHEFK-C), and 9-mer VE (N-VRGYEYYHE-C); it was found that the addition of the triple-reaction peptide probe (P0) produced only a small amount of background signal, with low blockage and short duration. These signals were hypothesized to be due to the collision of the analyte with the pore, which is due to its size limitation. As expected, the three short peptides produced different blocking events in the nanopore test (as Figure 9 shown), while the triple-reaction peptide probe did not produce a through-pore signal (i.e., no through-pore), and would not interfere with the subsequent through-signal, indicating that the triple-reaction peptide probe (P0) would not affect the through-pore of the enzyme cleavage fragment and the peptide fragment (as Figure 10 shown);

[0059] Specifically, in the presence of 7-mer ED, many short blocking events lasting from 0.22 to 1.00 ms occurred, and low current blockade occurred ( Figure 1 A-C). The blockade ratio is defined as I / I0 (where I and I0 represent the current values of the blocked and open pores, respectively), and its range of variation is from 0.27 to 0.49. The ratio of the exponential function fit is 0.40 ± 0.01. When 7-mer EK was introduced, I / I0 increased sharply from 0.38 to 0.63. The data were statistically modeled using an exponential function, and the result was 0.47 ± 0.01 ( Figure 1 D-F). The incorporation of VE increased the blockade degree to 0.59 ± 0.01 ( Figure 1 G-I). Comparative analysis showed that the separation between the peaks of the three polypeptides was good ( Figure 1(J-L), indicating different populations with a preferential I / I0 ratio, which helps in the differentiation of ED, VE, and EK. A small theoretical residual branched polypeptide (R-PP) was designed, which should be produced after digestion by three proteases. Its signal is completely different from other analytes and can thus be ignored; it was found that the remaining T-shaped fragment in the triple reaction peptide probe (P0) after enzymatic cleavage also does not pass through the pores, i.e., it has no effect on the pore passage of the enzymatic cleavage fragments and peptide fragments;

[0060] In summary, it can be shown that neither the triple reaction peptide probe nor the remaining T-shaped fragment of the triple reaction peptide probe after enzymatic cleavage passes through the pores, while the enzymatic cleavage fragments and peptide fragments exhibit pore passage signals. Therefore, the triple reaction peptide probe in this experiment can achieve detection.

[0061] II. Determine whether this experiment can be used to detect the target protease;

[0062] Based on the above results, the utility of peptide probe P0 in the simultaneous determination of three proteases involved in tumor initiation and malignant progression, namely CASP8, Cat.B, and MMP-2, was evaluated.

[0063] First, it was tested by the above-described method for simultaneously detecting and identifying three target proteases and their activities. In the experiment, the test solution was replaced with 10 μM of the target protease. Among the target proteases, Caspase8 (CASP8) (concentration range from 10 pM to 100 nM) and Cat.B (from 1 pM to 150 nM), MMP-2 (from 1 fM to 1 μM);

[0064] Some collision events with short-term partial blocking characteristics were observed in the experiment, which may be caused by the collision between the analyte and the pores. Absorbing more signals is a target-specific current characteristic with a relatively high current amplitude and a longer duration.

[0065] Combined Figure 2 As shown, the analysis of the scatter plot and current blockade histogram shows that the properties such as the characteristic current amplitude and duration generated by the reaction products of CASP8, Cat.B, and MMP-2 do not show significant differences compared to those generated by ED, VE, and EK respectively; Figure 2 It can be found that when the three proteases are present simultaneously, the generated current signals are located in well-separated regions in the scatter plot ( Figure 2B), new clusters with distinct boundaries and higher current blockade can be distinguished, and it is speculated that the formation of this cluster is caused by the interaction between the nanopores and the residual small branched polypeptides (R-PP) generated after digestion by the three proteases. These findings suggest that the current signals of ED, VE, and EK can be used as markers for identifying CASP8, Cat.B, and MMP-2. The duration of the CASP8 signal is the shortest, and the current blockade time is between 0.30 and 0.43; the Cat.B signal is distinguished in the range of 0.42 - 0.50, while the MMP-2 signal exhibits the largest current blockade in the range of 0.52 - 0.65.

[0066] Therefore, whether this experiment can be used for the detection of target proteases; preferably, the following analysis can be used to further evaluate the detection effect:

[0067] Figure 3 In the study of single enzyme analysis. (A, C, E) are for the study of enzyme reaction kinetics. Time response curves of event frequency when [P0] = 20 μM and CASP8 = 50 nM. B = 150 nM, MMP-2 = 1 μM. (B, D, F) are Lineweaver - Burk plots used to determine Km and Vmax. The inset provides an enlarged view of the x - intercept and y - intercept regions. (B) For CASP8, Km = 6.99 μM, Vmax = 44.52 μM min -1 . (D) For Cat.B, Km = 6.37 μM, Vmax = 43.84 μM min -1 ; (F) For MMP-2, Km = 7.22 μM, Vmax = 50.51 μM min -1 .

[0068] Subsequently, the enzyme activities of individual proteases were studied. Aliquots were periodically extracted from the reaction mixture every 20 minutes for up to 180 minutes to analyze the frequency of the corresponding feature (fsig) over time ( Figure 3 ). A time curve of substrate digestion was constructed.

[0069] CASP8 cleaves the substrate almost linearly within the first 60 minutes and reaches saturation at 120 minutes ( Figure 3 A). The Michaelis constant (Km) was determined to be 6.99 μM and the maximum initial reaction rate (Vmax) was 44.52 μM - min using the Lineweaver - Burk plot of fsig or different substrate concentrations catalyzed by CASP8 ( Figure 3 B). Similarly, for Cat.B, the Km value was 6.37 μM and the Vmax was 43.84 μM - min ( Figure 3D). The Km of protease MMP-2 is 7.22 μM and the Vmax is 50.51 μM-min( Figure 3 F).

[0070] Figure 4 Simultaneously detect multiple proteases. (A) Current trace in the presence of 10-8 M concentration of CASP8 (yellow). B (purple) and MMP-2 (light green). (B) Comparison of the current characteristics (f) frequencies of the target proteins generated by single measurements (black bars) and simultaneous measurements (gray bars). (C, D, E) Signal frequencies of different concentrations of CASP8 (C) or Cat.B (D) or MMP-2 (E) and two other proteases at constant concentrations. (F, G, H) Characteristic frequencies of two proteases at different concentrations and one protease at a constant concentration. All data were collected at +50 mV. Each experiment was performed three times.

[0071] P0 is mainly used to monitor enzyme-catalyzed reactions( Figure 4 ). The current trace records show the expected characteristic events of the output short peptides ED, EK, and VE. In addition, when the concentration of one protease increases within a certain range while the concentrations of the other two proteases remain unchanged, the characteristic frequencies of the three proteases are measured( Figure 4 C, D, F). The results show that the characteristic frequency of one protease (CASP8, Cat.B, or MMP-2) changes proportionally with the concentration, while the characteristic frequencies of the other two proteases remain unchanged at fixed concentrations( Figure 4 C, D, and F). The same is true when one target is at a constant concentration and the other two concentrations change( Figure 4 E, G, and H). These results indicate that the measurement of at least one protease is not affected by the presence of other protease probe complexes. Comparing the target frequencies in the mixed enzyme and individual protease solutions, similarities were found and the signal frequencies remained basically unchanged( Figure 4 B);

[0072] The above content shows that during the identification of multiple proteases, the identification method of this experiment is not interfered with.

[0073] III. Judge the effect of this experiment on the detection of target proteases;

[0074] (1). Sensitivity detection;

[0075] Under optimized conditions, the detection performance was evaluated by monitoring the change in the event frequency of a single protease at different concentrations. To reduce the influence of time-dependent data, each analyte was continuously recorded for 10 minutes using a single nanopore. As Figure 5 shown, as the concentration of the target increases, the characteristic frequencies of the three proteases gradually increase. Figure 5Quantitative determination of CASP8 Cat.B and MMP-2. (A - C) Current traces in the presence of CASP8, Cat.B, and MMP-2. (D - F) Correlation of event frequency with CASP8, Cat.B, and MMP-2 protein concentrations. There was a significant correlation between the event frequency (y) and target concentration (x) of CASP8, Cat.B, and MMP-2, which was expressed as follows: CASP8: y = 113.256 + 7.519x (R 2 = 0.995); Cat.B: y = 82.159 + 5.001x (R 2 = 0.986); MMP-2: y = 85.973 + 6.695x (R 2 = 0.990). The lowest detection concentrations of CASP8, Cat.B, and MMP-2 were 50 fM, 1 fM, and 1 pM, respectively. Notably, CASP8 and Cat.B were the minimum for the simultaneous detection of these two proteases. The detection range included six orders of magnitude for CASP8 (5×10 -8 to 5×10 -4 M) and MMP-2 (1×10 -6 to 1×10 -2 M), and eight orders of magnitude for Cat.B (1×10 -7 to 1×10 -1 M). This special sensitivity was achieved without the need for complex labeling or signal amplification, mainly due to the specific cleavage sites in the carefully designed peptide probes and the excellent sensing ability of nanopore technology; that is to say, the detection in this experiment could respond instantaneously with the concentration of proteases and had good detection performance.

[0076] (2) Selective detection;

[0077] CASP8, Cat.B, and MMP-2 were evaluated by detecting other proteases, including main protease (Mpro), papain-like protease (PLpro), acetylcholinesterase (AChE), and horseradish peroxidase (HRP). Different enzyme cleavage signals could only be observed in the presence of the three target proteases. The digestion of interfering substances matched that of the control group, indicating that probe P0 had high selectivity for the detection of the three targets.

[0078] (3) Application in real samples

[0079] To evaluate the practical applicability of the proposed strategy, three target proteases were simultaneously added to human serum and analyzed as described above. The current traces of the original serum samples showed low levels of noisy spikes. Using the standard addition method, CASP8, Cat.B, and MMP-2 were added to human serum samples, and the samples were centrifuged at 12,000 r for 15 min to obtain the liquid supernatant. 10 μL of peptide substrate was added to 90 μL of human serum sample, and the three enzymes were identified under similar conditions to those for enzyme activity detection. The supernatant was combined with 10 μM peptide probe and incubated with different concentrations of CASP8 (5×10 -8 , 5×10 -9 and 5×10 -10 M), Cat.B (1×10 -9 , 1×10 -10 and 1×10 -11 M) and MMP-2 (1×10 -6 , 1×10 -7 and 1×10 -8 M) at 37 °C for 120 min, and then the mixture was used for the aerolysin nanopore assay;

[0080] As Figure 6 shown, after adding these three target proteins, a large number of easily distinguishable specific features ( Figure 6 ) were generated. The recovery rate was evaluated by calculating the ratio of the actual digestion rate to the theoretical value obtained from the digestion curve. The detected protease content was closely related to the spiked concentration. The spiked recovery rate of CASP8 was 87.0% - 96.0%. For Cat.B, it was 77.7% - 84.0%, and for MMP-2, it was 82.0% - 94.3% ( Figure 6 ). This nanopore sensing strategy is applicable to the simultaneous monitoring of the activities of multiple proteases in complex samples because the recovery rate is within the acceptable range for actual sample analysis.

[0081] Figure 6 Shown in

[0082] is the recovery test of three proteases in human serum. (A) Background current traces of serum samples. (B - D) Different concentrations of CASP8, Cat.B, and MMP-2 were added to human serum and detected by the nanopore method. Data were obtained at +50 mV.

[0083] ​It is understandable that machine learning relies on the preliminary understanding and inspection of learning objectives through data analysis. This process includes selecting an appropriate mathematical model, formulating hyperparameters, and training the model using sample data (enzyme concentration data in the above experiments) with defined strategies and a suitable learning algorithm for data analysis and prediction; in this dataset, each event is labeled with a unique peptide probe with a specific identifier; MATLAB automatically extracts the mean and standard deviation of each event to construct a feature matrix; considering comprehensively, a linear support vector machine (SVM) model is selected to process the experimental data;

[0084] The linear support vector machine (SVM) model is selected because it performs excellently on the test set. The evaluation of this linear support vector machine model shows satisfactory validation accuracy, indicating high-quality input data. Figure 7 A shows the confusion matrix results of testing this linear SVM model, reporting an accuracy range of 97.7%-99.0% for the peptide probe perception results. Figure 7 B shows the decision boundary graph generated using the linear SVM model. As Figure 7 shown in C, we use the trained model to accurately predict unknown data and distinguish the products of simultaneous digestion by three enzymes. Finally, Figure 7 D shows the histogram of the corresponding event counts. As a tool for detecting peptide probes, the linear support vector machine model has remarkable effectiveness and precision, and has broad application potential in biochemical research and analysis.

[0085] Figure 7 Among them, (A) is the polypeptide classification confusion matrix generated by the SVM model. 400 events in each peptide class are regarded as a test set. The true positive rate (TPR) and false negative rate (FNR) are shown on the right. (B) is the decision boundary generated by the SVM model. Each colored area represents the region for predicting the corresponding peptide event. The mean value of the test data is covered on the decision boundary, accompanied by a scatter plot to illustrate the standard deviation (SD) for demonstration. (C) is the scatter plot of the mean and standard deviation of CASP8, Cat.B, and MMP-2 in the presence of enzymatic digestion products. ED, EK, and VE events are represented by yellow, blue-violet, and lake blue dots respectively. (D) is the histogram of the corresponding event counts.

[0086] In summary, the triple - reaction peptide probe of the embodiment of the present invention can simultaneously evaluate and accurately distinguish multiple proteases through a single nanopore. The T - shaped structure composed of three free arms is the key component of the probe, endowing the probe with a multi - component response function. Due to the introduction of specific target recognition sites and specific amino acid compositions on different arms, the probe can be specifically digested by three proteases simultaneously and release distinguishable output peptide fragments, generating significantly different current signals within a single aerolysin nanopore. The detection sensitivities of CASP8 and Cat.B reach unprecedented highs without any labeling or amplification. The potential of the peptide probe can be further explored to simultaneously measure various types of analytes, with advantages such as good detection performance, high sensitivity, and good selectivity, showing great potential for non - invasive disease diagnosis.

[0087] Example 2: Based on the mechanism of Example 1, another probe was set for different target proteases. The three target proteases are: CASP3 (caspase 3), CASP6 (caspase 6), and PSA (prostate - specific antigen); the amino acid sequences of the three peptide branches are shown as SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 respectively; the T - shaped structure formula of the triple - reaction peptide probe is as follows:

[0088]

[0089] That is, when the target protease is CASP3, the cleavage site fragment of one branch among the three peptide branches is DEVD; when the target protease is CASP6, the cleavage site fragment of one branch among the three peptide branches is VQVD; when the target protease is PSA, the cleavage site fragment of one branch among the three peptide branches is HSSKLQ.

Claims

1. A triple-responsive peptide probe, characterized in that, The triple-responsive peptide probe is a T-shaped structure composed of three peptide branches for recognizing three target proteases. Each of the three peptide branches includes a cleavage site fragment specifically recognized by the target protease and a peptide fragment for signal discrimination.

2. The triple-responsive peptide probe according to claim 1, wherein The three target proteases are: CASP8, Cat.B, MMP-2.

3. The triple-reaction peptide probe according to claim 2, wherein The amino acid sequences of the three peptide branches are shown as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

4. The triple-responsive peptide probe according to claim 1, wherein The three target proteases are: CASP3, CASP6, PSA.

5. The triple-responsive peptide probe according to claim 4, wherein The amino acid sequences of the three peptide branches are shown as SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

6. Application of a triple-responsive peptide probe in simultaneously detecting and recognizing three target proteases.

7. Application of a triple-responsive peptide probe in simultaneously detecting the activities of three target proteases.

8. The application according to claim 6, characterized in that, The detection method includes: First, add 10 μM of the triple-responsive peptide probe and 10 μM of the test solution to 80 μL of Tris-HCl buffer with a concentration of 0.05 M - 0.1 M, and incubate at 37 °C for 2 h to obtain a reaction solution. Then, take 20 μL of the reaction solution and add it to the cis end of the nanopore detection cell. Apply a transmembrane voltage of +50 mV to the nanopore detection cell, and use an aerolysin nanopore to record the change in ionic current to obtain the amplitudes and durations of multiple blocking events; then identify the three target proteases through the amplitudes and durations of multiple blocking events.

9. The application according to claim 7, wherein The detection method includes: First, add 10 μM of the triple-responsive peptide probe and 10 μM of the test solution to 80 μL of Tris-HCl buffer with a concentration of 0.05 M - 0.1 M, and incubate at 37 °C for 2 h to obtain a reaction solution. Then, take 20 μL of the reaction solution and add it to the cis end of the nanopore detection cell. Apply a transmembrane voltage of +50 mV to the nanopore detection cell, and use an aerolysin nanopore to record the change in ionic current to obtain the amplitudes and durations of multiple blocking events. Then identify the three target proteases through the amplitudes and durations of multiple blocking events and obtain the activity of each target protease.

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