Preparation method and application of a whole-cell biosensor for detecting protease activity

Abstract

The present application relates to a kind of whole-cell biosensors of E. coli surface display, for realizing the high sensitivity detection of multiple proteases.The sensor is displayed on the surface of E. coli by synthetic biology method and includes anchor module, protease-specific cleavage peptide and fluorescent reporter module of recombinant fusion protein.When target protease exists, its specific cleavage peptide causes fluorescent reporter module to dissociate from cell surface, and fluorescence signal is weakened, and the degree of signal attenuation is positively correlated with protease concentration and activity.The detection sensitivity of the present application is high, and the detection limit of matrix metalloproteinase-2, thrombin and other proteases reaches the level of femtogram per milliliter, which is more than three orders of magnitude higher than traditional methods;It is simple to operate, only needs to be added sample-incubation-detection three steps, and the cost of single detection is less than 0.1 dollar;It can directly detect protease activity in complex biological matrix without expensive antibody, and has good clinical application and large-scale screening potential.

Description

Technical Field

[0001] This invention belongs to the field of biosensors, specifically relating to a method for preparing and applying a whole-cell biosensor for detecting protease activity. Background Technology

[0002] Protease activity detection plays a crucial role in disease diagnosis, drug development, and basic life science research. Abnormal expression of specific proteases is closely related to the progression of various diseases, such as matrix metalloproteinases and tumor metastasis, and thrombin and thrombosis. Therefore, accurate detection of these proteases is of great significance for early diagnosis and efficacy evaluation.

[0003] Traditional protease detection methods are mainly divided into two categories: activity detection based on substrate cleavage and total amount detection based on antibody recognition. The former includes the use of chromogenic / fluorescent substrates, FRET probes, and enzyme profiling, which can directly reflect enzyme activity but are easily affected by interference and difficult to quantify; the latter, such as ELISA and Western blot, can accurately quantify but cannot distinguish the functional activity state of the enzyme, and are cumbersome and time-consuming to operate.

[0004] Despite the emergence of new detection technologies, significant limitations remain. Existing methods generally suffer from insufficient sensitivity, difficulty in achieving stable detection in complex biological samples, complex procedures, high reagent costs, and the inability to reflect the true functional activity of proteases in real time. These shortcomings limit their application in large-scale clinical screening and dynamic studies.

[0005] Therefore, developing a novel technology platform that combines high sensitivity, high specificity, ease of operation, low cost, and direct detection of protease functional activity has become an urgent technological need in this field and a key breakthrough for promoting the development of precision medicine. Summary of the Invention

[0006] This invention provides a method for preparing and applying a whole-cell biosensor for detecting protease activity, enabling ultrasensitive detection of protease activity. The sensor displays a fusion protein containing a protease-specific cleavage site and a fluorescent reporter protein on the bacterial surface. When the target protease is present, specific cleavage causes the fluorescent reporter module to dissociate from the cell surface, resulting in a decrease in the fluorescence signal. The degree of attenuation is proportional to the protease concentration and activity. This technology achieves femtogram-per-milliliter level detection sensitivity, requires only three steps, is cost-effective, and can directly and accurately detect the functional activity of proteases in complex biological matrices, providing an innovative tool for disease diagnosis, drug screening, and basic research.

[0007] On the one hand, the present invention provides a whole-cell biosensor for detecting protease activity, employing the following technical solution: A whole-cell biosensor for detecting protease activity, comprising: Engineered E. coli cells display recombinant fusion proteins on their surface; The recombinant fusion protein contains, from N-terminus to C-terminus, the following: An anchoring module is used to locate the fusion protein to the bacterial cell outer membrane; The protease-specific peptide cleavage module contains the specific recognition and cleavage sequences of the target protease; The signal reporting module is for fluorescent proteins.

[0008] Preferably, the anchoring module is formed by fusing the N-terminal sequence of Escherichia coli lipoprotein Lpp with the transmembrane domain sequence of the outer membrane protein OmpA.

[0009] Preferably, the N-terminal sequence of Lpp consists of the first 1-15 amino acids, more preferably the first 9 amino acids; the transmembrane domain sequence of OmpA consists of the 46th-159th amino acids.

[0010] Preferably, the signal reporting module is a superfolded green fluorescent protein sfGFP.

[0011] Preferably, the target protease is matrix metalloproteinase-2, and the amino acid sequence of the protease-specific cleavage peptide module includes SEQ ID NO: 1, wherein the cleavage site of matrix metalloproteinase-2 is located between V and G.

[0012] Preferably, the target protease is thrombin, and the amino acid sequence of the protease-specific cleavage peptide module includes SEQ ID NO: 2, wherein the cleavage site of thrombin is located between R and G.

[0013] Preferably, the target protease is a prostate-specific antigen, and the amino acid sequence of the protease-specific cleavage peptide module includes SEQ ID NO: 3, wherein the cleavage site of the prostate-specific antigen is located after Q.

[0014] On the other hand, the present invention also provides a method for preparing a whole-cell biosensor for detecting protease activity, employing the following technical solution: A method for preparing a whole-cell biosensor for detecting protease activity includes the following steps: S1. Construct a recombinant expression plasmid containing a nucleotide sequence encoding a recombinant fusion protein; S2. The recombinant expression plasmid was transformed into competent E. coli cells, and positive clones were obtained through culture and screening. S3. Cultivate positive clones, induce the expression of recombinant fusion proteins and display them on the surface of bacterial cells to obtain whole-cell biosensors.

[0015] This invention also provides a method for detecting protease activity, employing the following technical solution: A method for detecting protease activity, using the aforementioned whole-cell biosensor, includes the following steps: The whole-cell biosensor and the sample to be tested were mixed and incubated in a suitable buffer solution; After incubation, bacterial cells were isolated and the fluorescence signal intensity of the bacterial cells was detected. The activity of the target protease in the sample was quantified based on the degree of decrease in fluorescence signal intensity.

[0016] Preferably, the fluorescence signal intensity is detected at the single-cell level by flow cytometry; quantification is based on a pre-established standard curve that describes the correspondence between changes in fluorescence signal intensity and the concentration or activity of the target protease.

[0017] In summary, the beneficial effects of the present invention are as follows: The whole-cell biosensor of this invention achieves a significant breakthrough in detection performance. Its core advantage lies in its ultra-high detection sensitivity at the femtogram per milliliter level, which is more than three orders of magnitude higher than traditional ELISA methods. It can accurately capture changes in protease activity at extremely low abundance levels, meeting the needs of early disease diagnosis. Simultaneously, this technology directly detects the catalytic cleavage function of proteases, strictly distinguishing between their active and inactive forms, providing more biologically relevant functional information. Furthermore, the sensor exhibits excellent anti-interference ability and stability in complex biological matrices (such as serum and cell supernatant), achieving accurate quantification in near-realistic sample environments.

[0018] From a practical and application perspective, this invention greatly simplifies the detection process, requiring only three steps: "sample addition - incubation - detection," avoiding the cumbersome multi-step operations of traditional methods. Based on the unlimited reproduction of engineered bacteria, the cost per detection is extremely low (less than $0.1), less than one-tenth the cost of commercial kits, laying a solid foundation for large-scale screening and widespread application in resource-constrained environments. The platform also boasts high modularity and versatility, flexibly adapting to different protease targets by changing the cleavage peptide sequence, demonstrating broad potential for multiplex detection and customized development. Attached Figure Description

[0019] Figure 1 A schematic diagram of a protease sensing platform based on bacterial surface display; Figure 2 Image of pZC52 plasmid; Figure 3 Image A is a Western blot image of the cellular components of the strain carrying the pZC52 plasmid; Figure 3Image B is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis image of the cell components of the strain carrying the pZC52 plasmid; Figure 3 Image C shows Escherichia coli observed using a laser confocal microscope; Figure 3 Image D is a photograph of Escherichia coli observed under a fluorescence microscope; Figure 4 To verify the feasibility of detecting MMP-2 using a whole-cell biosensor; Figure 5 Figure A shows the flow cytometry analysis of the biosensor's response to MMP-2. Figure 5 In the figure, B represents the linear correlation between the average fluorescence intensity and the logarithm of the MMP-2 concentration; Figure 5 C represents the selectivity of the biosensor to potential interfering substances; Figure 5 D represents the inhibitory effect of BB-2516 on the 10 ng / mL MMP-2-induced biosensor response; Figure 6 Comparison of MMP-2 detection performance in 1% human serum (Figure A). Figure 6 Analysis of the recovery rate of spiked serum in 1% human serum (subject to B). Figure 6 C represents the sensor fluorescence response in different cell supernatants; Figure 6 D is a heatmap of MMP-2 concentration in cell supernatant; Figure 6 E in the figure represents the concentration of MMP-2 in the supernatant as verified by ELISA. Figure 7 A in the diagram is a schematic diagram of a thrombin sensing mechanism based on surface-displaying substrates; Figure 7 In Figure B, the linear relationship between average fluorescence intensity and the logarithm of thrombin concentration is represented. Figure 7 C represents the specificity evaluation of the thrombin sensor for other proteases; Figure 7 D is a schematic diagram of the design of a biosensor for detecting prostate-specific antigen; Figure 7 E represents the correlation between the fluorescence signal and the logarithm of the prostate-specific antigen concentration; Figure 7 F represents the specificity assessment of the prostate-specific antigen sensor in the presence of multiple interfering proteases; Figure 8 In the middle, A represents the fluorescence distribution spectrum under the thrombin concentration gradient; Figure 8 Image B shows the fluorescence distribution spectrum under the concentration gradient of prostate-specific antigen (PSA). Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the embodiments.

[0021] Example Example 1 The specific steps for sensor molecule construction and engineered bacteria preparation are as follows: S1. Construction of recombinant expression plasmid pZC52 First, the recombinant expression plasmid pZC52 was constructed using standard molecular biology techniques (seamless cloning and restriction endonuclease ligation). This plasmid contains the following core components: (1) Surface display anchoring module: This module is formed by the direct fusion of the first nine amino acids from the N-terminus of the E. coli lipoprotein Lpp with amino acids 46–159 of the outer membrane protein OmpA. The Lpp part is responsible for covalently anchoring the fusion protein to the peptidoglycan layer, while the OmpA part forms a β-barrel structure that allows the subsequent module to pass through and be fixed to the outer membrane, exposing it to the cell surface. (2) Protease cleavage spatial sensing module: This module contains the CueR protein, a linker, and a cleavable polypeptide sequence encoding a highly specific MMP-2. The polypeptide sequence is designed as GPLG↓VRG, where the arrow (↓) indicates the cleavage site of MMP-2 (located in the amide bond between valine V and glycine G). The remaining sequence is used to reduce steric hindrance to ensure that the enzyme's catalytic activity is effectively utilized.

[0022] (3) Signal reporting module: Encodes superfolded green fluorescent protein (sfGFP), which has excellent folding efficiency and fluorescence intensity, serving as a detection signal source. For example... Figure 1 The diagram shown is a schematic of a protease sensing platform based on bacterial surface display.

[0023] S2. Preparation and Surface Display Verification of Engineered Strains The correctly constructed plasmid pZC52 was transformed into competent *E. coli* DH5α cells. The transformed bacterial culture was plated on LB agar plates containing 10 μg / mL chloramphenicol and incubated upside down at 37°C for 12–16 hours. Single positive clones were picked and inoculated into LB liquid medium containing the same antibiotic for amplification. After sequencing verification, glycerol was added to a final concentration of 50%, and the culture was frozen at -80°C as a seed strain. Figure 2 The image shown is a map of the pZC52 plasmid.

[0024] After streaking activation of the seed strain, single colonies were picked and inoculated into LB medium containing 10 μg / mL chloramphenicol. The cultures were incubated at 37°C and 250 rpm for 12–16 hours to promote proper folding and display of the fusion protein. Subsequently, 1 mL of the bacterial culture was collected by centrifugation at 12,000 ×g for 1 minute, washed twice with PBS buffer (pH 7.4), and resuspended in PBS. Strong green fluorescence was observed in the bacterial cells using fluorescence microscopy, confocal microscopy, Western blotting, and polyacrylamide gel electrophoresis to verify successful display of sfGFP on the cell surface.

[0025] Figure 3 The successful display of the fusion protein on the surface of *E. coli* was systematically verified using Western blot, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and microscopic imaging techniques. In the figure, M represents the protein molecular weight standard; T represents the whole cell lysis buffer; C represents the soluble component; IM represents the inner membrane component; and OM represents the outer membrane component. Figure 3 -A and Figure 3 Cellular component analysis of -B showed that the outer membrane component exhibited a bright band at approximately 62 kDa (as indicated by the red arrow), a position consistent with the theoretical molecular weight of the fusion protein, indicating that the protein had been successfully localized and stably anchored to the bacterial outer membrane. Figure 3 -C laser confocal microscope and Figure 3 The fluorescence microscopy images of the -D strain further provided a direct comparison of the fluorescence signals of the engineered bacteria and the control bacteria: the engineered bacteria showed clear outlines under bright field and exhibited obvious green fluorescence in fluorescent field, while the control bacteria did not show such a signal. This result directly confirms that the superfolded green fluorescent protein has been efficiently and stably displayed on the surface of the engineered bacteria, laying the foundation for subsequent sensing functions.

[0026] Example 2 The specific steps for MMP-2 activity detection are as follows: After overnight culture, the bacterial culture was centrifuged (12,000 × g, 1 min) to collect the cells. The cells were then washed twice thoroughly with 1×TCNB buffer (50 mM Tris-HCl, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35, pH 7.5) to remove residual culture medium. Subsequently, the cells were resuspended in the same TCNB buffer, and the OD of the suspension was adjusted. 600 The value was set to 2.3, and it was used as a standardized working bacterial suspension for later use.

[0027] In the reaction system, 6 μL of the above-mentioned working bacterial suspension and 3 μL of solution containing different concentrations of MMP-2 standards (or test samples) were added to 291 μL of 1×TCNB buffer, making the total volume 300 μL. The mixed reaction system was incubated at 37°C for 2.5 hours to complete the enzymatic digestion reaction. After the reaction, the reaction tube was centrifuged (12,000 × g, 1 min) to collect the bacterial cells. The supernatant was discarded, and the cells were washed once with 1×PBS buffer and resuspended to remove free sfGFP from the solution. Flow cytometry was then used immediately for detection: sfGFP was excited using a 488 nm laser, and the fluorescence signal was collected through a 530 / 30 nm wavelength filter. 30,000 bacteria were collected for each sample to ensure statistical reliability.

[0028] Figure 4 Through comparative experiments, the feasibility of detecting matrix metalloproteinase-2 (MMP-2) using a whole-cell biosensor was visually verified. The figure shows that the fluorescence intensity of the engineered strain carrying plasmid pZC52 (a fusion protein displaying a specific MMP-2 cleavage site on its surface) was significantly reduced after MMP-2 treatment compared to the untreated control group. In contrast, the fluorescence intensity of the control strain carrying plasmid pZC47 (lacking this cleavage site) did not change significantly under the same MMP-2 treatment. This result directly demonstrates that the constructed sensor can specifically respond to the enzymatic activity of MMP-2, and the weakening of its fluorescence signal is due to the specific cleavage of the surface-displayed substrate by MMP-2, thus establishing the effectiveness of this sensing mechanism.

[0029] Example 3 A systematic and in-depth evaluation of the core performance of the MMP-2 whole-cell biosensor was conducted, including verification of sensitivity, linear range, specificity, and response mechanism. The specific steps are as follows: S1. Establishment of standard curve and determination of sensitivity and linear range A series of standard solutions with varying concentrations were prepared using high-purity recombinant human MMP-2 standards in 1×TCNB detection buffer. The concentration range should cover at least five orders of magnitude (0.01, 0.1, 1, 10, 100, 1000, 10000, 10000 pg / mL) from below the expected detection limit to near signal saturation. Following the standardized detection procedure described in Example 2, each concentration of MMP-2 standard was reacted with the working bacterial suspension of the sensor. At least three replicates were performed for each concentration point.

[0030] The mean fluorescence intensity (MFI) of each sample was detected and recorded using flow cytometry. A scatter plot was drawn with the logarithm of the MMP-2 concentration on the x-axis and the corresponding MFI value on the y-axis. Linear regression was performed within the linear interval where the signal changes significantly to obtain the standard curve equation.

[0031] Detection limit calculation: Usually, the concentration value obtained by subtracting the signal value corresponding to 3 times the standard deviation from the average MFI value of the blank control (0 pg / mL) and substituting it into the standard curve is used as the method detection limit.

[0032] S2, Sensor Specificity Verification Proteases that may be structurally and functionally related to the target protease (MMP-2) were selected as potential interfering agents. These included other types of proteases (such as thrombin, trypsin, and cathepsin B) and non-protease substances that may be present in high abundance in the sample (such as bovine serum albumin BSA). The target protease (MMP-2) was also set up as a positive control. All interfering proteases and the MMP-2 positive control were diluted to the same high concentration (e.g., 100 ng / mL), which should be much higher than their potential cross-reactivity threshold. Detection was performed according to standard procedures.

[0033] Calculate the signal change rate of each experimental group relative to the blank control. Use t-test statistical analysis to determine whether the signal changes of each interfering experimental group are significantly different from those of the blank control group.

[0034] S3, Response Mechanism Verification Three reaction groups were set up: positive group: MMP-2 (10 ng / mL) + sensor; experimental group: MMP-2 at the same concentration and its specific small molecule inhibitor (BB-2516) were pre-incubated at 37°C for 20 minutes before the sensor was added; blank group: detection buffer + sensor only. All groups were incubated, washed and detected according to standard procedures.

[0035] Compare the MFI values ​​of the three groups. Theoretically, the MFI of the inhibition group should be significantly higher than that of the positive group (i.e., the signal is weakened and suppressed), and close to the level of the blank group.

[0036] Calculate the inhibition rate: Inhibition rate (%) =

[0037] in, F 0 indicates the blank control group MFI (no protease, no inhibitor); F 1 indicates the experimental group MFI (containing both protease and inhibitor); F 2 represents the positive control group MFI (containing protease but no inhibitor).

[0038] Figure 5The system demonstrates the quantitative detection performance and specificity validation of the whole-cell biosensor for matrix metalloproteinase-2 (MMP-2): Figure 5 The flow cytometry fluorescence spectrum of -A visually demonstrates that with the increase of MMP-2 concentration (0.1), the concentration of MMP-2 decreases. As the concentration of the bacterium increased (to 100,000 pg / mL), the fluorescence peak of the bacterial population showed a regular leftward shift, indicating that the fluorescence signal gradually weakened. Figure 5 -B further confirms that there is an excellent linear correlation between the signal attenuation level and the logarithm of the MMP-2 concentration, which enables high-precision quantification. Figure 5 The selectivity experiment of -C showed that the sensor was almost unresponsive to high concentrations (100 ng / mL) of various potential interfering proteases (BSA, thrombin, etc.), and only produced a significant signal change for MMP-2, proving that its recognition is highly specific. Figure 5 -D effectively blocked MMP-2-induced signal attenuation via the inhibitor BB-2516, with a half-maximal inhibitory concentration (IC50) of 1,500%. 50 The value was 9.92 nM, confirming that the sensor's response was entirely dependent on the catalytic activity of MMP-2.

[0039] Example 4 The specific steps for verifying the accuracy of whole-cell biosensors in complex matrices are as follows: S1. Sample Preprocessing and Spiking Design Six serum samples from healthy individuals were diluted to working concentrations with detection buffer, and the background concentration (C_initial) of the target protease was determined using an ELISA control method. This step was used to confirm the initial values ​​of the samples. Different concentrations of protease standard solutions were added to each of the six samples to prepare a series of spiked samples, ensuring the spiked concentrations covered multiple orders of magnitude of the expected detection range. A sample containing only solvent (without standard) was also included as a negative control (i.e., containing only the background value). After all spiked samples were prepared, they were incubated on ice for 15–30 minutes to allow the standard and matrix to mix thoroughly.

[0040] S2, Sensor Detection Process For each sample to be tested (including samples spiked at various concentrations and negative controls): Reaction system construction: In a flow cytometer, add the following sequentially: 291 μL detection buffer 3 μL of test sample (spiked serum or control) 6 μL Standardized Engineered Bacterial Working Solution The total volume is 300 μL. Mix gently by vortexing.

[0041] Place the reaction tubes in a 37 °C incubator and incubate in the dark for 2.5 hours. After incubation, centrifuge the reaction tubes at 12,000 × g for 1 minute and carefully discard the supernatant. Add 1 mL of PBS buffer, gently resuspend the bacterial cells, and centrifuge again, discarding the supernatant. This step aims to remove free sfGFP from the reaction solution. Resuspend the bacterial pellet in 500 μL of PBS and immediately perform flow cytometry analysis. Using a flow cytometer, acquire fluorescence signals (FITC channel) for at least 30,000 bacterial particles per sample. Record the mean fluorescence intensity (MFI).

[0042] S3. Parallel Experiment and Control Setup All spiked levels and controls must be performed in at least three technical replicates (n≥3) to assess precision. Commercially available ELISA kits should be used, strictly following their instructions, and the same set of spiked samples should be tested in parallel for methodological comparison.

[0043] Based on the standard curve established using pure standards in buffer, the MFI value measured for each sample was converted into the corresponding protease concentration.

[0044] Calculate the recovery rate: Recovery rate (%) =

[0045] in, C D Indicates the measured concentration; C I Indicates the initial concentration; C A Indicates the concentration added.

[0046] Precision calculation: Calculate the relative standard deviation (RSD) of the concentration in three repeated tests at each spiking level.

[0047] Table 1 shows the results of spiked recovery detection of matrix metalloproteinase-2 (MMP-2) using the whole-cell biosensor described in this invention in human serum diluted to 1% by spike. Experiments were conducted at six different spike concentrations (0.4 to 40 ng / mL), and the sensor's detection recoveries ranged from 91.5% to 107.5%, with relative standard deviations (RSDs) ranging from 4.72% to 10.8%, all within acceptable analytical performance ranges. Notably, at a low spike concentration of 0.4 ng / mL, commercial ELISA kits could not detect it (ND), while this sensor still provided accurate results. This directly demonstrates that the sensor of this invention possesses superior sensitivity and reliability compared to traditional methods in complex matrices, especially in the low concentration range.

[0048] Table 1. Recovery of MMP-2 in diluted human serum (1%)

[0049] Figure 6 The system validated the practical application performance of the whole-cell biosensor in detecting matrix metalloproteinase-2 (MMP-2) in complex biological matrices: Figure 6 -A comparison shows that the sensor and the commercial ELISA kit have highly consistent results in detecting MMP-2 in 1% human serum; Figure 6 -B further confirmed through spiked recovery experiments that the sensor achieved a recovery rate of 91.5%–107.5% in the serum matrix, with relative standard deviations (RSD) generally below 10%, demonstrating excellent accuracy and repeatability; Figure 6 -C uses a three-dimensional bar chart to visually present the differences in the fluorescence response of the sensor to the supernatant of seven different cell lines. Figure 6 -D then converted it into a heatmap distribution of MMP-2 concentration, which clearly revealed that the MMP-2 level in the supernatant of cancer cell lines (such as HeLa and A549) was significantly higher than that in normal cells; Figure 6 -E was validated using the traditional ELISA method on the same samples, and the resulting concentration heatmap was compared with... Figure 6 The -D results showed a high degree of agreement. Overall, this sensor not only enables accurate quantification in complex serum samples, but can also be directly used for reliable detection and differential analysis of MMP-2 activity in real biological samples such as cell supernatants.

[0050] Example 5 S1, Modular Sensor Construction Using standard molecular biology techniques, peptide sequences encoding different PCP modules (Table 2) were cloned into the anchoring and reporter modules of a universal plasmid backbone to construct plasmids pZC57-Thrombin and pZC58-PSA. For the thrombin sensor (pZC57-Thrombin): a DNA sequence encoding a thrombin-specific cleavage peptide was inserted into the universal plasmid backbone. This peptide sequence was designed as CGLVPR↓GS (where the arrow ↓ indicates the thrombin cleavage site, located between arginine R and glycine G), placing it between the anchoring module and the sfGFP reporter module. For the PSA sensor (pZC58-PSA): using the same method, a DNA sequence encoding a prostate-specific antigen (PSA)-specific cleavage peptide was inserted. This peptide sequence was designed as HSSKLQ↓KK (where the arrow ↓ indicates the PSA cleavage site, located after glutamine Q). The correctly constructed plasmids were transformed into *E. coli* DH5α competent cells and screened on LB agar plates containing chloramphenicol. Single clones were selected, and after sequencing verification, glycerol seed strains were prepared and preserved.

[0051] Table 2. Cutting sequences of the three enzymes

[0052] S2, New Sensor Performance Verification Optimize cell washing and resuspending steps using the corresponding detection buffer, co-incubate with different concentrations of target proteases (thrombin or PSA), and detect a series of target proteases at varying concentrations, following the same procedure as the MMP-2 detection protocol. Measure MFI using flow cytometry and plot the MFI versus protease concentration curve. Calculate the detection limit, linear range, and sensitivity of the new sensor. Validate the specificity of the newly constructed thrombin or PSA sensor for their respective target proteases according to the protocol in Part I.

[0053] S3, Data Analysis and Proof of Generality The analytical performance of the new sensor (for thrombin and PSA) was validated, specifically including the determination of recovery, limit of detection, linear range, and selectivity in diluted serum.

[0054] Table 3 presents the spiked recovery performance data of the sensor for thrombin in diluted human serum. At five spiking levels ranging from 0.5 to 1000 pg / mL, the sensor recoveries ranged from 93.92% to 108.0%, with RSDs ranging from 4.9% to 10.8%. These results demonstrate that the sensor maintains high accuracy and precision even in trace (pg / mL) detection. In contrast, commercial ELISA kits failed to detect thrombin at the two lowest concentration points (0.5 and 2 pg / mL), further highlighting the significant advantages of this invention in detecting ultra-low abundance targets.

[0055] Table 3 Recovery rate of thrombin in diluted human serum

[0056] Table 4 summarizes the spiked recovery results of the sensor for prostate-specific antigen (PSA) in diluted human serum. At three spiking levels ranging from 0.1 to 5.0 ng / mL, the sensor recoveries ranged from 92.0% to 110.0%, with RSDs ranging from 5.38% to 11.31%, highly consistent with the results of the standard electrochemiluminescence (ECL) method, which has recoveries between 96.6% and 110.0%. These data indicate that the detection accuracy of this sensor for PSA is comparable to that of the mature ECL method, further validating that this platform can achieve accurate and stable quantitative analysis in complex biological matrices for the detection of different target proteases.

[0057] Table 4. Recovery rate of PSA in diluted human serum

[0058] Figure 7The schematic diagrams and data graphs comprehensively demonstrate the specific sensing mechanism and performance of the platform of this invention when it is extended to the detection of thrombin and prostate-specific antigen (PSA). Figure 7 -A and 7-D illustrate the design principles of surface-display sensors for thrombin and PSA, respectively, in schematic form. The core of these sensors is to integrate their respective specific cleavage peptide sequences into the fusion protein. Figure 7 -B and 7-E were demonstrated through linear fitting curves that the degree of fluorescence signal attenuation of the sensor has a good linear relationship with the logarithm of the corresponding protease concentration, verifying its quantitative detection capability. Figure 7 -C and 7-F demonstrate that the two sensors still produce significant responses only to their respective target proteases in the presence of multiple high concentrations of interfering proteases, fully demonstrating the high specificity of the sensor design.

[0059] Figure 8 The fluorescence distribution pattern obtained by flow cytometry visually demonstrated the dynamic response of the whole-cell biosensor to thrombin and prostate-specific antigen (PSA): Figure 8 -A shows that as the thrombin concentration increases from 0.01 to 10,000 pg / mL, the fluorescence peak of the bacterial community shows a regular and obvious leftward shift, indicating that the fluorescence signal gradually and uniformly weakens. Figure 8 -B shows a similar leftward shift of the fluorescence peak induced by PSA in the concentration range of 0.02 to 10,000 pg / mL. This dose-dependent change in fluorescence distribution directly confirms that the target protease can effectively recognize and cleave its corresponding surface-displaying substrate peptide, causing the reporter protein sfGFP to dissociate from the cell surface, thereby achieving quantitative attenuation of the fluorescence signal at the single-cell level, further verifying the universality and reliability of this sensing mechanism for different proteases.

[0060] Table 5 lists the specific nucleotide sequences of each core genetic module involved in constructing the whole-cell biosensor described in this invention. This sequence list covers the complete DNA sequences from promoters, cell surface anchoring proteins, sensing modules containing spatial regulatory domains and substrates specifically cleaved by different proteases, to the fluorescent reporter protein sfGFP. It also provides the coding sequences of thrombin and prostate-specific antigen-specific cleavage peptides for modular replacement, providing a precise molecular blueprint for realizing the programmable design of the sensor and flexible expansion for different target proteases.

[0061] Table 5. Component sequence list of this patent design

[0062] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A whole-cell biosensor for detecting protease activity, characterized in that, include: Engineered E. coli cells, wherein the surface of the E. coli cells displays recombinant fusion proteins; The recombinant fusion protein comprises, from N-terminus to C-terminus, the following: An anchoring module is used to position the fusion protein onto the bacterial cell outer membrane; The protease-specific peptide cleavage module contains the specific recognition and cleavage sequences of the target protease; The signal reporting module is for fluorescent proteins.

2. The whole-cell biosensor for detecting protease activity according to claim 1, characterized in that, The anchoring module is formed by fusing the N-terminal sequence of Escherichia coli lipoprotein Lpp with the transmembrane domain sequence of the outer membrane protein OmpA.

3. A whole-cell biosensor for detecting protease activity according to claim 2, characterized in that, The N-terminal sequence of the Lpp consists of its first 1-15 amino acids, preferably the first 9 amino acids; the transmembrane domain sequence of the OmpA consists of its 46th-159th amino acids.

4. A whole-cell biosensor for detecting protease activity according to claim 1, characterized in that, The signal reporting module is a superfolded green fluorescent protein sfGFP.

5. A whole-cell biosensor for detecting protease activity according to claim 1, characterized in that, The target protease is matrix metalloproteinase-2, and the amino acid sequence of the protease-specific cleavage peptide module includes SEQ ID NO: 1, wherein the cleavage site of the matrix metalloproteinase-2 is located between V and G.

6. A whole-cell biosensor for detecting protease activity according to claim 1, characterized in that, The target protease is thrombin, and the amino acid sequence of the protease-specific cleavage peptide module includes SEQ ID NO:2, wherein the cleavage site of the thrombin is located between R and G.

7. A whole-cell biosensor for detecting protease activity according to claim 1, characterized in that, The target protease is a prostate-specific antigen, and the amino acid sequence of the protease-specific cleavage peptide module includes SEQ ID NO: 3, wherein the cleavage site of the prostate-specific antigen is located after Q.

8. A method for preparing a whole-cell biosensor as described in any one of claims 1-7, characterized in that, Includes the following steps: S1. Construct a recombinant expression plasmid containing a nucleotide sequence encoding the recombinant fusion protein; S2. The recombinant expression plasmid is transformed into competent E. coli cells, and positive clones are obtained through culture and screening. S3. Cultivate the positive clone, induce the expression of the recombinant fusion protein and display it on the surface of bacterial cells to obtain the whole-cell biosensor.

9. A method for detecting protease activity, characterized in that, The whole-cell biosensor as described in any one of claims 1-7 comprises the following steps: The whole-cell biosensor and the sample to be tested were mixed and incubated in a suitable buffer solution; After incubation, bacterial cells were isolated, and the fluorescence signal intensity of the bacterial cells was detected. The activity of the target protease in the sample was quantified based on the degree of decrease in fluorescence signal intensity.

10. The method according to claim 9, characterized in that, The fluorescence signal intensity was detected at the single-cell level by flow cytometry; the quantification was based on a pre-established standard curve, which described the correspondence between changes in fluorescence signal intensity and the concentration or activity of the target protease.

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