Electrochemical urine biopsy system for cathepsin B analysis and detection method thereof

Abstract

The invention discloses an electrochemical urine biopsy system for cathepsin B analysis and a detection method of the electrochemical urine biopsy system. The electrochemical urine biopsy system comprises gold nanoparticles AuNPs, a peptide-peptide nucleic acid PNA series probe and an electrochemical sensor. The peptide-peptide nucleic acid tandem probe is combined with gold nanoparticles through a mercaptan modified sequence to form a spherical nanostructure with the diameter of 15 nm, a polypeptide substrate sequence serves as a specific reaction substrate of cathepsin B, and PNA provides stability and a signal shielding function. The electrochemical sensor is based on a cutting enzyme-assisted DNAwalker mechanism, signal amplification is started through methylene blue MB release, and high-sensitivity detection of the released PNA probe is achieved. The system provided by the invention has the characteristics of high specificity, high sensitivity and rapid response, can realize quantitative analysis of cathepsin B through noninvasive urine biopsy, and provides a new technical means for early diagnosis and treatment monitoring of tumors.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an electrochemical urine biopsy system and detection method for cathepsin B analysis. Background Technology

[0002] Cathepsin B (CB) is a cysteine ​​protease that plays a crucial role in various physiological and pathological processes. It participates in protein degradation and recycling within cells and exhibits significant overexpression in tumorigenesis, development, and metastasis. Abnormal CB activity is closely associated with multiple diseases, particularly in the tumor microenvironment, where elevated levels are considered potential biomarkers of tumor invasion and metastasis. Therefore, the detection and quantitative analysis of cathepsin B are of great significance for early disease diagnosis, treatment monitoring, and prognostic assessment.

[0003] Traditional methods for detecting cathepsin B mainly include enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and fluorescent probe detection. However, these methods have some limitations. For example, ELISA requires complex antibody labeling and washing steps, resulting in long detection times and high costs; while mass spectrometry offers high sensitivity and specificity, the equipment is expensive and the operation is complex, making rapid detection difficult; and although fluorescent probe detection has the advantages of speed and sensitivity, it is easily interfered with by biological background fluorescence when used in vivo, leading to unstable detection signals.

[0004] In recent years, with the rapid development of nanotechnology and biosensor technology, nanomaterial-based biosensors have provided new approaches for the detection of biomarkers. Gold nanoparticles (AuNPs) are widely used in the field of biosensing due to their unique optical properties, good biocompatibility, and ease of surface modification. However, most existing detection methods based on gold nanoparticles focus on fluorescence or colorimetric detection, and the specific detection and quantitative analysis of cathepsin B still present challenges. Furthermore, these methods often require complex sample pretreatment and instrumentation, making it difficult to achieve rapid, non-invasive in vivo detection.

[0005] Urine biopsy, as a non-invasive testing method, has gradually gained attention due to its advantages such as ease of operation, readily available samples, and non-invasiveness. However, most existing urine testing methods rely on traditional biomarker detection technologies, such as enzyme-linked immunosorbent assay (ELISA) and fluorescent probe detection. The application of these methods in urine is limited by biological background interference and detection sensitivity.

[0006] Therefore, it is necessary to develop an electrochemical urine biopsy system and detection method with high sensitivity and specificity for cathepsin B (CB) analysis. Summary of the Invention

[0007] The purpose of this invention is to provide an electrochemical urine biopsy system and detection method for cathepsin B (CB) analysis.

[0008] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, this application provides an electrochemical urine biopsy system for cathepsin B (CB) analysis.

[0009] Secondly, this application provides an electrochemical urine biopsy method for cathepsin B (CB) analysis.

[0010] Thirdly, this application provides a peptide-peptide nucleic acid (PNA) tandem probe for cathepsin B (CB) analysis.

[0011] Fourthly, this application provides an electrochemical sensor for cathepsin B (CB) analysis.

[0012] The first aspect of this application provides an electrochemical urine biopsy system for cathepsin B (CB) analysis, the electrochemical urine biopsy system for cathepsin B analysis includes gold nanoparticles (AuNPs), peptide-peptide nucleic acid (PNA) tandem probes, and an electrochemical sensor.

[0013] A peptide-peptide-nucleic acid (PNA) tandem probe binds to gold nanoparticles via its terminal thiol groups, forming a nanoprobe. The peptide-peptide-nucleic acid (PNA) tandem probe comprises a polypeptide substrate sequence for a cathepsin B (CB)-specific reaction and a peptide-nucleic acid (PNA) sequence covalently linked to the polypeptide substrate sequence. The amino acid sequence of the polypeptide substrate is shown in SEQ ID NO.1, and the nucleotide sequence of the peptide-nucleic acid (PNA) is shown in SEQ ID NO.2. The amino acid sequence of the peptide-peptide-nucleic acid (PNA) tandem probe is shown in SEQ ID NO.3. An electrochemical sensor is used to detect the PNA probe released after cleavage by cathepsin B. The electrochemical sensor is based on a nick-assisted DNAwalker signal amplification mechanism. Methylene blue (MB) release initiation signal amplification is achieved by initiating the release of methylene blue (MB) when the released PNA probe binds to a specific sequence on the electrochemical sensor, thereby generating a detectable electrochemical signal.

[0014] Gold nanoparticles (AuNPs) serve as a carrier, providing a stable nanostructure platform. They are bound to peptide-peptide nucleic acid (PNA) tandem probes via thiol-modified sequences to form spherical nanostructures.

[0015] The peptide-peptide nucleic acid (PNA) tandem probe binds to gold nanoparticles (AuNPs) via a thiol-modified sequence. The peptide substrate sequence serves as a specific reaction substrate for cathepsin B (CB), with one end modified with a thiol group (-SH) for binding to the gold nanoparticles; the other end binds to the peptide nucleic acid (PNA) via a covalent bond.

[0016] Peptide-peptide nucleic acid (PNA) tandem probes provide stability and signal shielding until the peptide substrate sequence is cleaved by cathepsin B to release the PNA.

[0017] Furthermore, the peptide-peptide nucleic acid (PNA) tandem probe is bound to gold nanoparticles through a thiol-modified sequence to form a spherical nanostructure with a diameter of 15 nm.

[0018] Furthermore, the gold nanoparticles have a particle size of 15 nm, and the particle size increases to 30 nm after modification with peptide-peptide nucleic acid (PNA) tandem probes; the loading ratio of peptide-peptide nucleic acid (PNA) tandem probes is 1:50.

[0019] Furthermore, the electrochemical sensor includes: a swing electrode for immobilizing the DNAwalker; a locking sequence for locking the DNAwalker; a methylene blue (MB) labeled substrate sequence for signal amplification; and an electrochemical detection unit for detecting the electrochemical signal triggered by the released PNA probe. The nucleotide sequence of the locking sequence is shown in SEQ ID NO.4; the nucleotide sequence of the DNAwalker is shown in SEQ ID NO.5; and the nucleotide sequence of the substrate sequence is shown in SEQ ID NO.6.

[0020] Furthermore, the peptide substrate sequence of the peptide-peptide nucleic acid (PNA) tandem probe has been optimized, and the nucleotide sequence of the PNA is shown in SEQ ID NO.2, to improve the specificity of the response to cathepsin B; the detection limit of the electrochemical sensor is 17.5 pg / ml.

[0021] The second aspect of this application provides an electrochemical urine biopsy method for cathepsin B (CB) analysis, comprising the following steps: providing an electrochemical urine biopsy system; binding a peptide-peptide nucleic acid (PNA) tandem probe to gold nanoparticles to form a nanoprobe for detecting cathepsin B; injecting the nanoprobe into a living organism, causing it to release the PNA probe under the action of cathepsin B; collecting a urine sample from the organism; and using the electrochemical sensor to detect the PNA probe in the urine sample to analyze the activity of cathepsin B.

[0022] Furthermore, the electrochemical sensor amplifies the signal through a nicking enzyme-assisted DNAwalker mechanism; the urine sample is diluted 100 times after collection.

[0023] Furthermore, the detection limit of the electrochemical sensor is 17.5 pg / ml; the PNA probe in the urine sample reaches its peak value 2 hours after injection of the nanoprobe.

[0024] A third aspect of this application provides a peptide-peptide nucleic acid (PNA) tandem probe for cathepsin B (CB) analysis. The probe comprises gold nanoparticles (AuNPs), a polypeptide substrate sequence, and a peptide nucleic acid (PNA). The polypeptide substrate sequence forms a strong bond with gold atoms on the surface of the gold nanoparticles through a chemical bond formed by its terminal thiol group (-SH). The peptide nucleic acid binds to the other end of the polypeptide substrate sequence through a covalent bond, forming an integrated probe structure.

[0025] The fourth aspect of this application provides an electrochemical sensor for cathepsin B (CB) analysis, comprising: a wobbling electrode for immobilizing a DNA walker; a locking sequence for locking the DNA walker; a substrate sequence labeled with methylene blue (MB) for signal amplification; and an electrochemical detection unit for specifically detecting a PNA probe released by a peptide-peptide nucleic acid (PNA) tandem probe.

[0026] Beneficial effects: This invention provides a cathepsin B detection system with high specificity, high sensitivity, rapid response, and strong anti-interference ability. It achieves quantitative analysis of cathepsin B through non-invasive urine biopsy, providing a new technical means for early diagnosis, treatment monitoring, and prognostic assessment of tumors.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] (1) The peptide-PNA tandem probe designed in this invention is optimized for the specific reaction of cathepsin B, and can specifically recognize and cleave the peptide substrate, thereby releasing the PNA probe. This specific design ensures that the detection signal comes only from the activity of cathepsin B, avoiding interference from other proteases or biomolecules, and improving the accuracy of detection.

[0029] (2) Through the signal amplification mechanism of the electrochemical sensor, this invention can achieve highly sensitive detection of low concentrations of PNA probes. The detection limit of the electrochemical sensor is as low as 17.5 pg / ml, which is far lower than the detection limit of traditional detection methods, enabling the detection of early low levels of cathepsin B activity, providing strong support for the early diagnosis of diseases.

[0030] (3) Experiments show that GPPs (gold nanoparticles-peptide-PNA tandem probes) can rapidly generate fluorescence signals within 15 minutes under the action of different concentrations of cathepsin B, and the signal intensity is linearly related to the concentration of cathepsin B. This rapid response capability makes the detection process more efficient and suitable for rapid diagnosis and real-time monitoring.

[0031] (4) The detection system of the present invention exhibits good stability in a variety of complex biological environments. In the presence of DNase, there is almost no leakage of fluorescence signal in the reaction system; even in serum and low pH media, the fluorescence signal output remains stable. This anti-interference capability ensures the reliability of the detection results, enabling accurate detection of cathepsin B activity even in complex biological samples.

[0032] (5) This invention uses urine samples for detection, avoiding the invasive procedures required by traditional detection methods, such as tissue biopsy or blood sampling. Urine sample acquisition is non-invasive, simple, and easily repeatable, making it suitable for large-scale screening and long-term monitoring.

[0033] (6) This invention utilizes a nicking enzyme-assisted DNAwalker mechanism to amplify the signal, initiating signal amplification through the release of methylene blue (MB), further improving the sensitivity and quantification capability of the detection. This signal amplification mechanism enables the detection system to accurately quantify low concentrations of PNA probes, providing reliable technical support for the quantitative analysis of cathepsin B.

[0034] (7) This invention not only verified the responsiveness of GPPs to cathepsin B through fluorescence signals, but also verified the PNA probe in urine in multiple dimensions through electrochemical signals and mass spectrometry analysis. This multi-dimensional verification method ensures the accuracy and reliability of the detection results, providing a solid foundation for clinical application. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 This diagram illustrates the reaction of the gold nanoparticle-peptide-PNA (GPP) of the present invention with cathepsin B at the tumor site, and the detection process of the in vitro electrochemical biosensor used for urine biopsy.

[0037] Figure 2 This is a diagram showing the synthesis and characterization of the GPP of the present invention. Figure 2A is a TEM image of the GPP of this invention. Scale bar: 100nm. Figure 2 B represents a comparative analysis of the zeta potentials of AuNP and GPP in this invention. Figure 2 C is the particle size distribution diagram of AuNP and GPP according to DLS test of the present invention. Figure 2 D represents the UV-Vis absorption spectra of AuNP and GPPs of this invention. Figure 2 E is a diagram showing the optimization and selection of peptide substrate sequences in response to CB according to the present invention. Figure 2 F is a standard curve showing the loading of peptide-PNA tandem probes on AuNP constructed according to the present invention.

[0038] Figure 3 This is a diagram showing the in vitro performance of the GPP of the present invention. Figure 3 A is a graph showing the fluorescence release time kinetics of GPPs at different CB concentrations according to the present invention. Figure 3 B is a graph showing the time-dependent changes in fluorescence intensity of CB and DNaseI when incubated with GPPs according to the present invention. Figure 3 C is a graph showing the stability test results of the GPPs of this invention in 10% FBS, PBS and pH 5.5. Figure 3 D is the fluorescence intensity diagram of GPP incubated with cell lysates treated with the CB inhibitor according to this invention. The concentration of the CB inhibitor used in this experiment was 100 μM.

[0039] Figure 4 This is an in vivo and in vitro detection diagram of the GPP of the present invention. Figure 4 A is a graph showing the impedance changes during the stepwise construction of the electrochemical biosensor of the present invention (a: bare electrode; b: oscillation + locking; c: oscillation + locking + MB-substrate; d: after adding PNA probe). Figure 4 B shows the current changes after incubating mouse urine (collected after GPP injection) and tandem probe standards with the constructed sensor of this invention. Figure 4 C is a graph showing the time-dependent changes in fluorescence signals in urine samples collected from tumor-bearing mice according to this invention. GPPs were co-incubated with different concentrations of CB, and collection was triggered by the release of tandem probes. Figure 4 D represents the electrochemical signal of this invention and Figure 4 E is the standard curve diagram of this invention. Figure 4 F is a graph showing the current signals generated by urine under different time gradients according to the present invention. Figure 4 G is a mass spectrometry diagram of urine collected two hours after GPP injection according to this invention. Figure 4 H is the mass spectrometry analysis diagram of the standard sample of the tandem probe of this invention.

[0040] Figure 5This is a schematic diagram of the cytotoxicity experiment of the present invention. (The horizontal axis represents the amount of probe added with gold nanoparticles as carriers, and the vertical axis represents the cell viability). Detailed Implementation

[0041] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the scope of protection of the present invention.

[0042] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0044] In this application, "~ one less" means one or more, and "more than" means two or more. "The following ~ one less item (item)" or similar expressions refer to any combination of these items, including any combination of single items (items) or multiple items (items). For example, "~ one less item (item) in a, b, or c", or "~ one less item (item) in a, b, and c", can all mean: a, b, c, a~b (i.e., a and b), a~c, b~c, or a~b~c, where a, b, and c can be single or multiple.

[0045] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0046] The first aspect of this application provides an electrochemical urine biopsy system for cathepsin B (CB) analysis, which includes gold nanoparticles (AuNPs), peptide-peptide nucleic acid (PNA) tandem probes, and an electrochemical sensor.

[0047] A peptide-peptide nucleic acid (PNA) tandem probe binds to the gold nanoparticles via its terminal thiol group, forming a nanoprobe. The peptide-peptide nucleic acid (PNA) tandem probe comprises a polypeptide substrate sequence for a cathepsin B (CB) specific reaction and a peptide nucleic acid (PNA) sequence covalently linked to the polypeptide substrate sequence. The amino acid sequence of the polypeptide substrate is shown in SEQ ID NO. 1, and the nucleotide sequence of the peptide nucleic acid (PNA) is shown in SEQ ID NO. 2; TTTATATGTTGGA. The amino acid sequence of the peptide-peptide nucleic acid (PNA) tandem probe is shown in SEQ ID NO. 3; Cys-Gly-Arg-Val-Cit.

[0048] An electrochemical sensor is used to detect PNA probes released after being cleaved by cathepsin B. The electrochemical sensor is based on a nicking enzyme-assisted DNAwalker signal amplification mechanism.

[0049] Gold nanoparticles (AuNPs) serve as a carrier, providing a stable nanostructure platform. They are bound to peptide-peptide nucleic acid (PNA) tandem probes via thiol-modified sequences to form spherical nanostructures.

[0050] The peptide-peptide nucleic acid (PNA) tandem probe binds to gold nanoparticles (AuNPs) via a thiol-modified sequence. The peptide substrate sequence serves as a specific reaction substrate for cathepsin B (CB), with one end modified with a thiol group (-SH) for binding to the gold nanoparticles; the other end binds to the peptide nucleic acid (PNA) via a covalent bond.

[0051] Peptide-peptide nucleic acid (PNA) tandem probes provide stability and signal shielding until the peptide substrate sequence is cleaved by cathepsin B to release the PNA.

[0052] An electrochemical sensor is used to detect released peptide-peptide nucleic acid (PNA) probes. The electrochemical sensor is based on a nicking enzyme-assisted DNAwalker mechanism, which is initiated by the release of methylene blue (MB) to amplify the signal. When the released PNA probe binds to a specific sequence on the electrochemical sensor, the release of methylene blue (MB) is initiated, thereby generating a detectable electrochemical signal.

[0053] In some embodiments, the peptide-peptide nucleic acid (PNA) tandem probe binds to the gold nanoparticles via a thiol-modified sequence to form a spherical nanostructure with a diameter of 15 nm.

[0054] In some embodiments, the gold nanoparticles have a particle size of 15 nm, and the particle size is increased to 30 nm after modification with the peptide-peptide nucleic acid (PNA) tandem probe; the loading ratio of the peptide-peptide nucleic acid (PNA) tandem probe is 1:50.

[0055] In some embodiments, the electrochemical sensor includes: a wobbling electrode for immobilizing the DNAwalker; a locking sequence for locking the DNAwalker; a methylene blue (MB) labeled substrate sequence for signal amplification; the substrate sequence is labeled with methylene blue (MB) for signal amplification; and an electrochemical detection unit for detecting an electrochemical signal triggered by a released PNA probe. The nucleotide sequence of the locking sequence is shown in SEQ ID NO. 4; the nucleotide sequence of the DNAwalker is shown in SEQ ID NO. 5; and the nucleotide sequence of the substrate sequence is shown in SEQ ID NO. 6.

[0056] In some embodiments, the peptide substrate sequence of the peptide-peptide nucleic acid (PNA) tandem probe is optimized, and the nucleotide sequence of the PNA is shown in SEQ ID NO.2, to improve the specificity of the response to cathepsin B; the detection limit of the electrochemical sensor is 17.5 pg / ml.

[0057] The second aspect of this application provides an electrochemical urine biopsy method for cathepsin B (CB) analysis, comprising the following steps: providing an electrochemical urine biopsy system; binding a peptide-peptide nucleic acid (PNA) tandem probe to gold nanoparticles to form a nanoprobe for detecting cathepsin B; injecting the nanoprobe into a living organism, causing it to release the PNA probe under the action of cathepsin B; collecting a urine sample from the organism; and using an electrochemical sensor to detect the PNA probe in the urine sample to analyze the activity of cathepsin B.

[0058] In some embodiments, the electrochemical sensor amplifies the signal via a nicking enzyme-assisted DNAwalker mechanism; the urine sample is diluted 100-fold after collection.

[0059] In some embodiments, the detection limit of the electrochemical sensor is 17.5 pg / ml; the PNA probe in the urine sample reaches its peak value 2 hours after injection of the nanoprobe.

[0060] The third aspect of this application provides a peptide-peptide nucleic acid (PNA) tandem probe for cathepsin B (CB) analysis. The probe includes gold nanoparticles (AuNPs), a polypeptide substrate sequence, and a peptide nucleic acid (PNA). The polypeptide substrate sequence forms a strong bond with gold atoms on the surface of the gold nanoparticles through a chemical bond formed by its terminal thiol group (-SH). The peptide nucleic acid binds to the other end of the polypeptide substrate sequence through a covalent bond, forming an integrated probe structure.

[0061] The fourth aspect of this application provides an electrochemical sensor for cathepsin B (CB) analysis, comprising: a wobbling electrode for immobilizing a DNA walker; a locking sequence for locking the DNA walker; a substrate sequence labeled with methylene blue (MB) for signal amplification; and an electrochemical detection unit for specifically detecting a PNA probe released by a peptide-peptide nucleic acid (PNA) tandem probe.

[0062] Example 1

[0063] The present invention provides an electrochemical urine biopsy system for cathepsin B (CB) analysis, comprising gold nanoparticles (AuNPs), peptide-peptide nucleic acid (PNA) tandem probes, and an electrochemical sensor.

[0064] Gold nanoparticles (AuNPs) serve as a carrier, providing a stable nanostructure platform. They are bound to peptide-peptide nucleic acid (PNA) tandem probes via thiol-modified sequences to form spherical nanostructures.

[0065] The peptide-peptide-nucleic acid (PNA) tandem probe binds to gold nanoparticles (AuNPs) via a thiol-modified sequence. The polypeptide substrate sequence, acting as a specific substrate for cathepsin B (CB), has a thiol group (-SH) modified at one end for binding to the gold nanoparticles; the other end is covalently bound to the peptide-peptide-nucleic acid (PNA). The peptide-peptide-nucleic acid (PNA) tandem probe provides stability and signal shielding until the polypeptide substrate sequence is cleaved by cathepsin B, releasing the PNA. The nucleotide sequence of the peptide-peptide-nucleic acid (PNA) is shown in SEQ ID NO. 2: TTTATATGTTGGA. The amino acid sequence of the peptide-peptide-nucleic acid (PNA) tandem probe is shown in SEQ ID NO. 3: Cys-Gly-Arg-Val-Cit.

[0066] The electrochemical sensor is used to detect released peptide-peptide nucleic acid (PNA) probes. Based on the nicking enzyme-assisted DNAwalker mechanism, the electrochemical sensor amplifies the initiation signal through the release of methylene blue (MB). When the released PNA probe binds to a specific sequence on the electrochemical sensor, it initiates the release of methylene blue (MB), thereby generating a detectable electrochemical signal.

[0067] The peptide-peptide nucleic acid (PNA) tandem probe binds to the gold nanoparticles via a thiol-modified sequence to form a spherical nanostructure with a diameter of 15 nm.

[0068] The gold nanoparticles have a particle size of 15 nm, and the particle size increases to 30 nm after modification with peptide-peptide nucleic acid (PNA) tandem probes; the loading ratio of peptide-peptide nucleic acid (PNA) tandem probes is 1:50.

[0069] The electrochemical sensor includes: a swing electrode for immobilizing a DNAwalker; a locking sequence for locking the DNAwalker; a methylene blue (MB)-labeled substrate sequence for signal amplification; and an electrochemical detection unit for detecting the released PNA probe. The nucleotide sequence of the locking sequence is shown in SEQ ID NO.4; the nucleotide sequence of the DNAwalker is shown in SEQ ID NO.5; and the nucleotide sequence of the substrate sequence is shown in SEQ ID NO.6.

[0070] The peptide substrate sequence of the peptide-peptide nucleic acid (PNA) tandem probe was optimized to improve the specificity of the response to cathepsin B; the detection limit of the electrochemical sensor is 17.5 pg / ml.

[0071] The sequences involved in this invention are shown in Table 1:

[0072] Table 1

[0073]

[0074] Thiol modification sequences: The 5′-thiol modification of SEQ ID NO:5 is used to immobilize the Swing chain on the surface of the gold electrode (or gold nanoparticles). The 5′-thiol modification of SEQ ID NO:6 is used to immobilize the MB-labeled substrate chain on the electrode surface.

[0075] Example 2

[0076] An electrochemical urine biopsy method for cathepsin B (CB) analysis according to the present invention includes the following steps: providing an electrochemical urine biopsy system; binding the peptide-peptide nucleic acid (PNA) tandem probe to gold nanoparticles to form a nanoprobe for detecting cathepsin B; injecting the nanoprobe into a living organism, causing it to release the PNA probe under the action of cathepsin B; collecting a urine sample from the organism; and using the electrochemical sensor to detect the PNA probe in the urine sample to analyze the activity of cathepsin B.

[0077] The electrochemical sensor amplifies the signal through a nicking enzyme-assisted DNAwalker mechanism; the urine sample is diluted 100 times after collection.

[0078] The detection limit of the electrochemical sensor is 17.5 pg / ml; the PNA probe in the urine sample reached its peak value 2 hours after the nanoprobe was injected.

[0079] Example 3

[0080] The present invention discloses a peptide-peptide nucleic acid (PNA) tandem probe for cathepsin B (CB) analysis. The probe comprises gold nanoparticles (AuNPs), a polypeptide substrate sequence, and a peptide nucleic acid (PNA). The polypeptide substrate sequence is chemically bonded to gold atoms on the surface of the gold nanoparticles through its terminal thiol group (-SH), achieving a strong binding. The peptide nucleic acid is covalently bonded to the other end of the polypeptide substrate sequence, forming an integrated probe structure.

[0081] Example 4

[0082] An electrochemical sensor for cathepsin B (CB) analysis according to the present invention comprises: a wobbling electrode for immobilizing a DNA walker; a locking sequence for locking the DNA walker; a substrate sequence labeled with methylene blue (MB) for signal amplification; and an electrochemical detection unit for specifically detecting a PNA probe released by a peptide-peptide nucleic acid (PNA) tandem probe.

[0083] Example 5

[0084] Experimental Section

[0085] 1.1 Materials and Instruments

[0086] Tris(2-carboxyethyl)phosphine (TCEP), 6-mercapto-1-hexanol (MCH), and hexaammonium chloride ruthenium(III) were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Sodium citrate and CB inhibitors were purchased from Shanghai Aladdin Biotechnology Co., Ltd. (Shanghai, China). CHAPS lysis buffer was purchased from Zeye Biotechnology Co., Ltd. (Shanghai, China). DNase I was obtained from Sigma-Aldrich, Inc. (St. Louis, Missouri, USA). DNA markers (25–500 bp) were purchased from Sangon Biotech Co., Ltd. Phosphate-buffered saline (PBS), culture medium (DMEM), fetal bovine serum (FBS), and trypsin-EDTA solution were obtained from Seville Biotechnology Co., Ltd. Hoechst 33342 was purchased from Beyotime Biotechnology Co., Ltd. All other chemicals were used as is without further purification. All oligonucleotides used in this study were synthesized and purified at Sangon Biotech Co., Ltd. (Shanghai, China). Peptide nucleic acid sequences were synthesized and purified by Peptide Biotechnology Co., Ltd.

[0087] Transmission electron microscopy (TEM) images were obtained using a JEOL JEM-1400 microscope. Fluorescence measurements and MTS determinations were recorded on a microplate reader (TECAN SPARK, Switzerland). Morphological and hydrodynamic dimensions were determined using a Nano ZS90 particle size analyzer (Malvern Instruments, Malvern, UK). Gold nanoparticle (AuNPs) and DNA absorption were monitored using a UV-Vis spectrophotometer (UV-2600i, Shimadzu, Japan). Electrochemical measurements were performed on a CHI 760E workstation (CHInstruments, China). Fluorescence imaging of mice was performed using a Tanon ABL X5 high-resolution in vivo imaging system (Shanghai, China). MALDI-TOF probe mass spectrometry quantification in urine was performed on an Ultraflexreme (Brook, USA).

[0088] 1.2 Synthesis of Artificially Synthesized Urine Markers (GPPs)

[0089] AuNPs (15 nm) were synthesized by citrate reduction. The gold nanoparticles (15 nm) were prepared using the classic citrate reduction method (Turkevich et al., 1951). Briefly, 3.5 mL of trisodium citrate (1%, w / v) was added to 100 mL of boiling HAuCl4 solution (0.01%, w / v) under vigorous stirring. The AuNPs were then purified by centrifugation twice at 12000 rpm for 30 min each time. The resulting AuNP solution was stored at room temperature (RT) protected from light for further use. The synthesized AuNPs were then characterized by transmission electron microscopy (TEM), UV-Vis spectroscopy, dynamic light scattering (DLS), and zeta potential analysis.

[0090] 200 μL of 5 nM MAuNP solution was incubated with 5 μL of a 10 μM peptide / peptide-nucleic acid PNA tandem probe at room temperature for 2 h. Subsequently, the reaction mixture was centrifuged at 13000 rpm for 30 min to separate the supernatant, and the probe conjugate precipitate was resuspended in 50 μL of ultrapure water to remove unbound peptide-PNA conjugates. The resulting GPPs were stored in the dark at 4 °C. The synthesized GPPs were characterized using TEM, UV-Vis spectroscopy, DLS, and zeta potential analysis.

[0091] Experimental Example 1

[0092] 1.3 Feasibility verification of cathepsin B (CB) response to GPPs

[0093] First, 1×10 6Cells were harvested from MDA-MB-231 culture flasks and washed three times with pre-chilled PBS. After centrifugation at 1000 rpm for 5 minutes, 50 μL of CHAPS lysis buffer was added. The cell suspension was incubated on ice for 30 minutes, followed by centrifugation at 12000 g for 20 minutes at 4°C to remove cell debris. Fifteen minutes before use, the supernatant containing CB was activated by adding activation buffer (25 mM MES, 5 mM DTT, pH 5.0). CB-triggered fluorescence signal detection was performed in 60 μL reaction buffer (25 mM MES, pH 5.0) containing GPPs solution and 30 μL of cell lysate, followed by incubation at 37°C for 2 hours. Fluorescence signals were measured using a microplate reader with an excitation wavelength set to 615 nm (Cy5 channel) and an emission filter set to 650–750 nm.

[0094] Experimental Example 2

[0095] GPPs performance verification

[0096] A mixture of 1 nM GPPs and different concentrations of active CB (0.2 to 1000 ng / mL) was incubated in 100 μL buffer for 3 hours. The solution was then centrifuged at 15000 rpm for 25 minutes, and the supernatant was collected for fluorescence signal determination. The stability of the GPPs was assessed using a multimode plate reader. Calibration curves were generated by plotting the fluorescence signal versus the corresponding CB concentration at specific time intervals. The limit of detection (LOD) was determined using the 3σ method. To activate CB, CB was diluted to 500 ng / mL in activation buffer, incubated at room temperature for 15 minutes, and then incubated at 37 °C for 30 minutes with PBS (pH 5.5) and fetal bovine serum (FBS, 10% v / v), respectively.

[0097] Urine sample analysis

[0098] Urine samples were collected from mice at different time intervals after GPP injection, and fluorescence signals were measured using a multifunctional microplate reader. After confirming the successful assembly of the electrochemical sensor by impedance testing, the urine samples (diluted 100-fold) were electrochemically tested using square wave voltammetry, and the corresponding electrochemical signals were recorded.

[0099] GPP cytotoxicity analysis

[0100] like Figure 5 As shown, Figure 5This is a schematic diagram of the cytotoxicity assay of the present invention. (The horizontal axis represents the amount of probe added using gold nanoparticles as carriers, and the vertical axis represents cell viability). To evaluate the cytotoxicity of GPP, MTA-MB-231 cells were used as a model cell line for the MTT assay. Cells were seeded at a density of 10,000 cells per well in 96-well plates and cultured for 12 hours. After removing the original culture medium, the cells were treated with different concentrations of GPP for 24 hours. Subsequently, 100 μL of MTT solution (0.5 mg·mL⁻¹, final concentration) was added to each well, and the plates were then incubated at 37°C in 5% CO₂ / 95% air for another 4 hours. After incubation, the culture medium was carefully aspirated, and 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve the methyl sulfoxide crystals. The absorbance of the dissolved methyl sulfoxide was measured at a wavelength of 490 nm.

[0101] result

[0102] 2.1 Working Mechanism

[0103] A systematic peptide-PNA tandem probe was developed, utilizing gold nanoparticles (AuNPs) as a carrier, the peptide as the CB reaction substrate, and PNA providing enhanced stability and signal shielding. Notably, both the peptide and PNA are composed of a peptide bond backbone, and the peptide-PNA tandem probe is synthesized in an integrated manner, eliminating cumbersome linking processes and successfully combining complex functions into a simple structure. As shown in the schematic diagram, CB enzyme, an overexpressed protease in tumors, was selected as the model target. At the tumor site, the CB enzyme efficiently cleaves the specific reactive peptide substrate, thereby releasing the PNA reporter probe, which is excreted into the urine and quantified using an electrochemical sensor. The sensor employs a nick-enzyme-assisted DNAwalker mechanism, where PNA competitively displaces the locked DNA walker, amplifying the initiation signal via methylene blue (MB) release. Therefore, the designed system integrates enzyme reaction activation, urine biopsy, and electrochemical detection, providing a simplified method for real-time and accurate quantification of endogenous CB.

[0104] Figure 1 This diagram illustrates the reaction of the gold nanoparticle-peptide-PNA (GPP) of the present invention with cathepsin B at the tumor site, and the detection process of the in vitro electrochemical biosensor used for urine biopsy.

[0105] Successful synthesis and characterization of GPPs

[0106] The CB-responsive peptide-PNA tandem probe binds to a synthesized AuNP via a thiol-modified sequence. The AuNP serves as a carrier, with a diameter of 15 nm, forming a spherical GPP nanostructure. TEM micrographs show the uniformity of the GPP. Figure 2A). Compared to bare gold nanoparticles, PNA significantly improves the potential of GPP due to its electroneutrality, and its particle size also increases to approximately 30 nm due to probe modification. Figure 2 B and C). Meanwhile, the UV-vis absorption peak of GPP showed a significant red shift (B and C). Figure 2 D). All these findings confirm the successful synthesis of GPP.

[0107] Subsequently, the feasibility of GPP reacting with CB in vitro was verified. To improve the specificity of the CB enzyme, five substrate sequences were designed based on the CB enzyme's activity mode as an endopeptidase, including sequences a and b targeting the P1 site, and sequences c, d, and e targeting the P2 site. The ratio of these two sequences was used to characterize the specificity of the designed sequences for the CB enzyme. Figure 2 E). The results showed that sequence a had high specificity for CB enzyme. To verify the probe loading density on AuNP, standard curves were obtained by testing different concentrations of probes, with a loading ratio calculated to be 1:50 (E). Figure 2 F).

[0108] Figure 2 This is a diagram showing the synthesis and characterization of the GPP of the present invention. Figure 2 This is a diagram showing the synthesis and characterization of the GPP of the present invention. Figure 2 A is a TEM image of the GPP of this invention. Scale bar: 100nm. Figure 2 B represents a comparative analysis of the zeta potentials of AuNP and GPP in this invention. Figure 2 C is the particle size distribution diagram of AuNP and GPP according to DLS test of the present invention. Figure 2 D represents the UV-Vis absorption spectra of AuNP and GPPs of this invention. Figure 2 E is a diagram showing the optimization and selection of peptide substrate sequences in response to CB according to the present invention. Figure 2 F is a standard curve showing the loading of peptide-PNA tandem probes on AuNP constructed according to the present invention.

[0109] Experimental Example 3

[0110] In vitro performance verification of GPPs

[0111] The in vitro reactivity of GPPs to CB was investigated, including their kinetic characteristics and sensitivity at different CB concentrations. Kinetic experiments showed that fluorescence signals were acquired within 15 minutes at different CB concentrations, demonstrating the rapid CB response capability of GPPs. Figure 3A). A standard curve was then established using the signal intensity over 15 minutes. Considering the GPPs would be used for in vivo analysis, the reaction system's resistance to interference in challenging media such as DNase, low pH, and serum was verified. Results showed that, in the presence of DNase, there was almost no fluorescence signal leakage in the reaction system within 60 minutes. Figure 3 B). Furthermore, it maintains stability of the fluorescence signal output even in serum and low pH media. Figure 3 C). Furthermore, the fluorescence signal is significantly reduced in the presence of CB inhibitors, such as... Figure 3 As shown in D, the fluorescence signal originates from a specific reaction of CB.

[0112] Figure 3 This is a diagram showing the in vitro performance of the GPP of the present invention. Figure 3 A is a graph showing the fluorescence release time kinetics of GPPs at different CB concentrations according to the present invention. Figure 3 B is a graph showing the time-dependent changes in fluorescence intensity of CB and DNaseI when incubated with GPPs according to the present invention.

[0113] Test Example 4

[0114] Urine electrochemical detection

[0115] To improve analytical sensitivity, an electrochemical sensor was designed for in vitro detection in urine. This sensor utilizes a DNA walker system that generates a strong signal gain through a nicking enzyme. First, electrochemical impedance spectroscopy (EIS) validated the stepwise sensor assembly (…). Figure 4 A). The feasibility of the sensor operation was verified by using the constructed sensor to detect tandem probe standards and urine collected after GPP injection. Figure 4 B). Urine fluorescence detection signals collected at different time intervals indicated that the GPP tandem probe successfully responded to CB and released the PNA probe in the urine. Figure 4 C). Simultaneously, electrochemical tests were performed on the PNA probes released from the reaction of GPPs with different concentrations of CB, a standard curve was established, and the corresponding equation between CB concentration and current signal was obtained ( Figure 4 D and E). The limit of detection (LOD) was calculated to be 17.5 pg / ml. Subsequently, electrochemical detection measurements were performed on urine samples collected at different time intervals. The results showed that the PNA probe in urine reached its peak at 2 hours, which was consistent with the trend of urine fluorescence detection results, further demonstrating the reliability of the established electrochemical detection system. Figure 4F). Finally, urine was collected from mice injected with GPP and analyzed by mass spectrometry. Three forms of PNA probes were found in the urine compared to the tandem probe standard, due to varying degrees of degradation of residual amino acids on the PNA. These findings strongly confirm that GPPs successfully release PNA probes into the urine after responding to CB in vivo. Figure 4 The above results indicate that PNA probes in the form of these amino acid residues do not affect the accurate recognition of PNA sequences by the electrochemical sensor, thus providing reliable technical support for urine biopsy-mediated in vitro tumor detection based on the specificity of this electrochemical sensor.

[0116] Figure 4 This is an in vivo and in vitro detection diagram of the GPP of the present invention. Figure 4 A is a graph showing the impedance changes during the stepwise construction of the electrochemical biosensor of the present invention (a: bare electrode; b: oscillation + locking; c: oscillation + locking + MB-substrate; d: after adding PNA probe). Figure 4 B shows the current changes after incubating mouse urine (collected after GPP injection) and tandem probe standards with the constructed sensor of this invention. Figure 4 C is a graph showing the time-dependent changes in fluorescence signals in urine samples collected from tumor-bearing mice according to this invention. GPPs were co-incubated with different concentrations of CB, and collection was triggered by the release of tandem probes. Figure 4 D represents the electrochemical signal of this invention and Figure 4 E is the standard curve diagram of this invention. Figure 4 F is a graph showing the current signals generated by urine under different time gradients according to the present invention. Figure 4 G is a mass spectrometry diagram of urine collected two hours after GPP injection according to this invention. Figure 4 H is the mass spectrometry analysis diagram of the standard sample of the tandem probe of this invention.

[0117] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. An electrochemical urine biopsy system for cathepsin B analysis, characterized in that: The electrochemical urine biopsy system for cathepsin B analysis includes gold nanoparticles (AuNPs), peptide-peptide nucleic acid (PNA) tandem probes, and an electrochemical sensor. The peptide-peptide nucleic acid (PNA) tandem probe binds to the gold nanoparticles via its terminal thiol groups to form a nanoprobe. The peptide-peptide nucleic acid (PNA) tandem probe comprises a polypeptide substrate sequence for a cathepsin B-specific reaction and a peptide nucleic acid (PNA) sequence covalently linked to the polypeptide substrate sequence. The amino acid sequence of the polypeptide substrate sequence is shown in SEQ ID NO. 1, and the nucleotide sequence of the peptide nucleic acid (PNA) is shown in SEQ ID NO.

2. As shown in SEQ ID NO. 2, the amino acid sequence of the peptide-peptide nucleic acid (PNA) tandem probe is shown in SEQ ID NO.

3. The electrochemical sensor is used to detect the PNA probe released after being cleaved by cathepsin B, and the electrochemical sensor is based on a nicking enzyme-assisted DNAwalker signal amplification mechanism.

2. The electrochemical urine biopsy system for cathepsin B analysis according to claim 1, characterized in that: The peptide-peptide nucleic acid (PNA) tandem probe binds to the gold nanoparticles via a thiol-modified sequence to form a spherical nanostructure with a diameter of 15 nm.

3. The electrochemical urine biopsy system for cathepsin B analysis according to claim 1, characterized in that: The gold nanoparticles have a particle size of 15 nm, and the particle size is increased to 30 nm after modification with the peptide-peptide nucleic acid (PNA) tandem probe; the loading ratio of the peptide-peptide nucleic acid (PNA) tandem probe is 1:

50.

4. The electrochemical urine biopsy system for cathepsin B analysis according to claim 1, characterized in that: The electrochemical sensor includes: a wobbling electrode for immobilizing a DNAwalker; a locking sequence for locking the DNAwalker; a methylene blue MB-labeled substrate sequence for signal amplification; the nucleotide sequence of the locking sequence is shown in SEQ ID NO.4; the nucleotide sequence of the DNAwalker is shown in SEQ ID NO.5; the nucleotide sequence of the substrate sequence is shown in SEQ ID NO.6; it is labeled with methylene blue MB for signal amplification; and an electrochemical detection unit for detecting an electrochemical signal triggered by a released PNA probe.

5. The electrochemical urine biopsy system for cathepsin B analysis according to claim 1, characterized in that: The detection limit of the electrochemical sensor is 17.5 pg / ml.

6. A method for detecting cathepsin B using the electrochemical urine biopsy system as described in any one of claims 1-5, characterized in that... The process includes the following steps: providing the electrochemical urine biopsy system as described in claim 1; combining the peptide-peptide nucleic acid (PNA) tandem probe with gold nanoparticles to form a nanoprobe for detecting cathepsin B; and injecting the nanoprobe into a living organism to release the PNA probe under the action of cathepsin B. Collect urine samples from organisms; The electrochemical sensor was used to detect the PNA probe in a urine sample to analyze the activity of cathepsin B.

7. The detection method according to claim 6, characterized in that: The electrochemical sensor amplifies the signal through a nicking enzyme-assisted DNAwalker mechanism; the urine sample is diluted 100 times after collection.

8. The detection method according to claim 6, characterized in that: The detection limit of the electrochemical sensor is 17.5 pg / ml; the PNA probe in the urine sample reaches its peak value 2 hours after the nanoprobe is injected.

9. A peptide-peptide nucleic acid (PNA) tandem probe for cathepsin B analysis, characterized in that: The probe comprises gold nanoparticles (AuNPs), a polypeptide substrate sequence, and a peptide nucleic acid (PNA). The polypeptide substrate sequence forms a strong bond with gold atoms on the surface of the gold nanoparticles through a chemical bond formed by its terminal thiol group (-SH). The peptide nucleic acid binds to the other end of the polypeptide substrate sequence through a covalent bond, forming an integrated probe structure.

10. An electrochemical sensor for detecting cathepsin B, characterized in that: The electrochemical sensor for cathepsin B analysis comprises: a wobbling electrode as described in claim 4 for immobilizing the DNAwalker as described in claim 4; a substrate sequence as described in claim 4, labeled with methylene blue MB, for signal amplification; and an electrochemical detection unit for specifically detecting PNA probes released by the peptide-peptide nucleic acid PNA tandem probe as described in claim 1.

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