A biosynthetic nanomaterial-based tumor marker biochemical detection method

Abstract

The application discloses a tumor marker biochemical detection method based on biosynthetic nanomaterials, which comprises a logic gate system and has an engineered living cell with the ability of in-situ synthesis of metal organic framework and nanoscale enzyme; the living cell, metal ions and hydrogel precursors are mixed and then injected into a microneedle mold for culture, in-situ synthesis of metal organic framework porous material and nanoscale enzyme particles, formation of a living cell-metal organic framework symbiotic complex; the complex is solidified into a microneedle array layer and bonded with a substrate layer integrated with a flexible electrode and a wireless transmission module to obtain a microneedle patch; after the patch is attached to the body surface, the microneedle pierces into the tissue fluid, the marker diffuses into the pore and is combined with the logic gate recognition element. The application realizes multi-marker logic gate detection, has the advantages of high specificity, sensitivity up to the level of femtomolar, wearable dynamic monitoring and multi-mode output, and is suitable for early screening of tumors and real-time home monitoring.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, and in particular relates to a biochemical detection method for tumor markers based on biosynthetic nanomaterials. Background Technology

[0002] Biochemical detection of tumor markers is a crucial method for early cancer screening, diagnosis, and postoperative monitoring. Currently, commonly used clinical methods for tumor marker detection include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, electrochemical immunosensor methods, and nucleic acid detection methods based on polymerase chain reaction (PCR). These methods typically use a single marker as the detection target, employing antibodies or nucleic acid probes as recognition elements, and combining techniques such as enzyme-catalyzed colorimetry, fluorescent labeling, or electrochemical signal amplification to achieve quantitative detection. In recent years, wearable sensors and microneedle patch technology have begun to be applied to the real-time monitoring of markers in tissue fluid. Some studies have attempted to introduce nanomaterials (such as gold nanoparticles, quantum dots, and carbon nanotubes) into sensors to improve sensitivity. Furthermore, detection systems based on regularly spaced clustered short palindromic repeats (CRISPR) have attracted attention due to their high sequence specificity and have been used for the rapid detection of nucleic acid markers in vitro. Biosynthetic nanomaterials, as a green preparation strategy, are also gradually being applied in the field of biosensing.

[0003] However, the existing technologies still have the following shortcomings: First, traditional single-marker detection methods are easily affected by non-tumor factors, such as inflammation, benign hyperplasia, or other physiological changes, which can lead to non-specific increases in a single marker, producing false positive results and affecting diagnostic accuracy. Second, existing detection methods have limited sensitivity, making it difficult to detect ultra-low abundance early tumor markers in tissue fluid at the femtomolar to atmore level, and lack effective signal amplification and enrichment synergy mechanisms. Third, most nanomaterial detection platforms rely on chemical synthesis or physical deposition methods, with harsh preparation conditions (high temperature, organic solvents), which not only increases costs but may also damage the activity of biorecognition elements, and the integration process of nanomaterials and detection devices is complex. Fourth, existing wearable detection devices mostly use passive diffusion or physical adsorption to capture markers, lacking active enrichment and logical discrimination capabilities, making it difficult to achieve combined judgment of multiple markers and dynamic continuous monitoring, especially lacking real-time early warning functions suitable for home use. Fifth, existing detection modes are singular, with most only supporting single electrochemical or optical signal output, making it difficult to meet the different needs of high-precision laboratory screening and convenient home self-testing. Therefore, developing a tumor marker detection method that combines high specificity, ultra-high sensitivity, green preparation, wearable real-time multi-mode detection, and strong platform versatility has important clinical significance and application value. Summary of the Invention

[0004] To overcome the aforementioned deficiencies of the prior art, this invention provides a biochemical detection method for tumor markers based on biosynthetic nanomaterials, which solves the problems of high false positives, insufficient sensitivity, complex preparation, lack of logic gating and dynamic monitoring, and single mode in the existing tumor marker detection methods.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A biochemical detection method for tumor markers based on biosynthetic nanomaterials includes the following steps: (1) Provide engineered live cells containing a CRISPR-Cas logic gate system, and the engineered live cells have the ability to biosynthesize metal-organic framework materials and nanozymes in situ; (2) The engineered live cells, metal ions and hydrogel precursor are mixed and injected into a microneedle-shaped cavity for culture. The metabolic activity of the engineered live cells is used to synthesize metal-organic framework porous materials in situ. At the same time, nanoenzyme particles are synthesized in situ in the pores of the metal-organic framework to obtain a live cell-metal-organic framework symbiotic complex. (3) The symbiotic composite is solidified in a microneedle forming cavity to form a microneedle array layer, and then a flexible substrate layer integrating a flexible electrode and a wireless transmission module is bonded to the bottom surface of the microneedle array layer to obtain a microneedle patch; the microneedle patch includes the flexible substrate layer, the microneedle array layer and a peelable protective film. (4) The microneedle patch is attached to the body surface, so that the microneedles penetrate the tissue fluid. The tumor marker molecules in the tissue fluid diffuse into the pores of the metal-organic framework and combine with the logic gate recognition elements on the surface of the engineered living cells. (5) When the tumor markers meet the combination conditions set by the logic gate, the CRISPR-Cas system is activated. The activated CRISPR-Cas system cleaves the inhibitory RNA chain pre-encapsulated in the metal-organic framework pores, thereby relieving the inhibition of nanozyme activity by the inhibitory RNA chain. (6) The nanozyme catalyzes the substrate after the inhibition is removed to generate a detectable signal. The electrochemical signal is collected through the flexible electrode or the color signal is collected through the optical window, and the signal is sent to an external terminal through the wireless transmission module.

[0006] Preferably, the engineered live cell is *Lactococcus lactis* or *Pichia pastoris*; the CRISPR-Cas logic gate system includes Cas13a protein or Cas12a protein and two or more guide RNAs; the logic gate is an AND gate or an OR gate; the logic gate recognition element is an aptamer fixed to the outer surface of the engineered live cell by a cell membrane anchoring protein, or is a crRNA array expressed in the engineered live cell.

[0007] Preferably, the metal-organic framework material is ZIF-8 or ZIF-67; the nanozyme is a metal nanocluster with peroxidase-like activity, wherein the metal nanocluster is composed of a ferritin or laccase framework and... , or The nanoparticles are self-assembled and have a particle size of 2nm to 5nm. The repressive RNA chain is expressed by the engineered living cells and encapsulated in the pores of the metal-organic framework. The repressive RNA chain is reversibly bound to the active center of the nanozyme by electrostatic adsorption or covalent bonding.

[0008] Preferably, the culture conditions in step (2) are a temperature of 35℃~39℃, a pH of 6.8~7.6, and a culture time of 12~24 hours; the concentration of the metal ions is 10mM~100mM.

[0009] Preferably, the substrate is a mixed solution of peroxidase substrate and hydrogen peroxide; the substrate is pre-lyophilized and adsorbed onto the surface of the microneedles of the microneedle patch; the detectable signal is the change in current or absorbance generated after the substrate is catalyzed and oxidized by nanoenzymes.

[0010] Preferably, the peroxidase substrate is 3,3',5,5'-tetramethylbenzidine; the electrochemical signal is the reduction current of oxidized 3,3',5,5'-tetramethylbenzidine at a potential of 0.15V~0.25V; and the color signal is the absorbance of oxidized 3,3',5,5'-tetramethylbenzidine at a wavelength of 652nm.

[0011] Preferably, the flexible electrode is a screen-printed three-electrode system, including a working electrode, a reference electrode, and a counter electrode, which is printed on a flexible substrate; the wireless transmission module is a Bluetooth chip and a power supply battery; the external terminal is a smartphone or a dedicated reader, which includes a dynamic threshold algorithm that outputs an alarm message when the signal collected three times consecutively exceeds a set threshold.

[0012] Preferably, the height of the microneedles in the microneedle patch is 500μm~800μm, the microneedles are distributed in an array of 10×10 to 20×20, and the radius of curvature of the microneedle tip is less than 20μm.

[0013] Preferably, the hydrogel material is methacrylated gelatin or alginate hydrogel; the concentration of engineered live cells in the symbiotic complex is [missing information]. CFU / mL~ CFU / mL; the overall size of the microneedle patch is 1cm×1cm to 2cm×2cm.

[0014] Preferably, the working electrode in the flexible electrode is a carbon electrode or a gold electrode, the reference electrode is a silver / silver chloride electrode, and the counter electrode is a carbon electrode; the surface of the working electrode is modified with a Nafion film; the power supply battery is a micro lithium-ion battery with a nominal voltage of 3.7V and a capacity of 10mAh~20mAh.

[0015] The technical effects and advantages of the present invention regarding a biochemical detection method for tumor markers based on biosynthetic nanomaterials are as follows: 1. This invention employs an engineered CRISPR-Cas logic gate system (AND or OR gate) within living cells, requiring at least two tumor markers to simultaneously meet combination conditions to activate signal output. Compared to traditional single-marker detection methods, this invention can eliminate false positives caused by non-specific elevations of a single marker due to non-tumor factors (such as inflammation or benign hyperplasia), significantly improving the accuracy of tumor diagnosis.

[0016] 2. This invention utilizes engineered living cells to biosynthesize metal-organic framework porous materials in situ, thereby efficiently enriching tumor markers in tissue fluid using their ultra-high specific surface area. Simultaneously, the confinement effect of the metal-organic framework pores significantly increases the local concentration of nanozymes. Combined with the cascade signal amplification after activation by the CRISPR-Cas system, sensitive detection of femtomolar or even atmolar level tumor markers is achieved, making it suitable for early tumor screening.

[0017] 3. The metal-organic framework material and nanozyme in this invention are synthesized in situ by engineered living cells under mild conditions (35-39℃, pH 6.8-7.6), eliminating the need for traditional preparation processes such as organic solvents and high-temperature calcination, thus avoiding secondary contamination and activity loss of the nanomaterials. Simultaneously, the biosynthesis process is completed concurrently with the microneedle patch molding, achieving in-situ encapsulation of the functional materials and ensuring the stability of the detection element during storage and use.

[0018] 4. This invention integrates a living cell-metal-organic framework symbiotic complex into a flexible microneedle patch. The patch can be attached to the body surface, and the microneedles penetrate the tissue fluid to achieve non-invasive or minimally invasive detection. Combined with a wireless transmission module (such as a Bluetooth chip) and an external terminal (such as a mobile app), it can achieve automatic detection and data upload every 30 minutes, supporting continuous dynamic monitoring for 24-72 hours. It is suitable for postoperative recurrence early warning in cancer patients, dynamic evaluation of radiotherapy and chemotherapy efficacy, and home screening for high-risk groups.

[0019] 5. This invention generates electrochemical or color signals by catalyzing substrates with nanozymes. Users can choose between two detection modes: electrode acquisition (high sensitivity) or optical window colorimetry (portable and low cost). The substrate is not limited to a single type (such as peroxidase substrates like TMB or ABTS), providing a flexible technical solution for different application scenarios (rapid clinical testing, home self-testing, and field medical care).

[0020] 6. The engineered live cells used in this invention can be food-grade microorganisms such as Lactococcus lactis or Pichia pastoris; the metal-organic framework material can be of various types such as ZIF-8 or ZIF-67; the CRISPR-Cas system can be Cas13a or Cas12a; the logic gates can be designed as AND, OR, or more complex combinations; and the tumor markers can be microRNA, messenger RNA, or proteins. Therefore, this invention constitutes a universal detection platform that can be rapidly adapted to different types and combinations of tumor markers by changing the target sequences of the logic gate recognition elements, and has broad prospects for clinical application and industrial transformation. Attached Figure Description

[0021] Figure 1 This is a flowchart of a biochemical detection method for tumor markers based on biosynthetic nanomaterials proposed in this invention. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include," "contain," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "includes..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0024] refer to Figure 1This invention provides a biochemical detection method for tumor markers based on biosynthesized nanomaterials, comprising: providing engineered live cells containing a CRISPR-Cas logic gate system and possessing the ability to in-situ biosynthesize metal-organic framework materials and nanozymes; mixing the engineered live cells, metal ions, and a hydrogel precursor and injecting the mixture into a microneedle-shaped cavity for culture; utilizing the metabolic activity of the engineered live cells to in-situ synthesize porous metal-organic framework materials and in-situ synthesize nanozyme particles within the pores to obtain a live cell-metal-organic framework symbiotic complex; and solidifying the symbiotic complex to form a microneedle array layer, which is then combined with a flexible electrode and a wireless transmission module integrated into a microneedle array. A microneedle patch is fabricated by bonding a base layer. The microneedle patch is then attached to the body surface, allowing the microneedles to penetrate the tissue fluid. Tumor marker molecules in the tissue fluid diffuse into the pores of the metal-organic framework (MOF) and bind to logic gate recognition elements on the surface of engineered living cells. When the tumor markers meet the combination conditions set by the logic gate, the CRISPR-Cas system is activated and cleaves the pre-encapsulated inhibitory RNA chain within the MOF pores, thus releasing the inhibition on nanozyme activity. The de-inhibited nanozyme catalyzes the substrate to generate a detectable signal. Electrochemical signals are collected via the flexible electrode or color signals are collected via an optical window and transmitted to an external terminal via the wireless transmission module. This method achieves highly specific detection with multi-marker logic gating, ultra-high sensitivity of MOF enrichment and nanozyme cascade amplification, green biosynthetic preparation, wearable dynamic monitoring, and multi-mode signal output. It can be used for early tumor screening, postoperative recurrence early warning, and real-time home monitoring.

[0025] Materials and General Methods: Engineered live cell construction: Lactococcus lactis (purchased from ATCC11454) was used as the host bacterium. A plasmid carrying the CRISPR-Cas13a logic gate system was transferred into the host bacterium via electroporation. The logic gate system was designed as an AND gate, responding to two tumor markers, miRNA-21 and miRNA-155. The specific implementation of the logic gate recognition element involved immobilizing specific aptamers on the outer surface of engineered living cells using the cell membrane anchoring protein Lpp-OmpA. The aptamer sequences were complementary to miRNA-21 and miRNA-155, respectively. When both miRNAs simultaneously bind to the aptamers, intracellular Cas13a expression and activation are triggered, and its accompanying cleavage activity cleaves the reporter RNA. Simultaneously, ferritin (Fn) and zinc ion transporter (ZIP8) genes were introduced into the plasmid for subsequent biosynthesis of nanozymes and metal-organic framework materials.

[0026] The binding mechanism of repressive RNA chains to nanozymes: The repressive RNA chain was expressed by engineered live cells, with a benzaldehyde group modified at its 5′ end. Under slightly acidic conditions (pH 6.5), the ε-amino group of the lysine residue on the nanozyme surface spontaneously forms an imine covalent bond with the benzaldehyde group, thereby achieving reversible binding of the repressive RNA chain to the active site of the nanozyme. This covalent bond is stable in tissue fluid at pH 7.4 and can only be broken by cleaving the repressive RNA chain using a CRISPR-Cas system.

[0027] General steps for microneedle patch fabrication: (1) Provide engineered live cells containing a CRISPR-Cas logic gate system, and the engineered live cells possess the ability to biosynthesize metal-organic framework materials and nanozymes in situ: culture the engineered cells to the logarithmic growth phase, collect them by centrifugation, and resuspend them in a solution containing... Add methacrylated gelatin (10% w / v) and photoinitiator LAP (0.5% w / v) to a phosphate buffer containing 2-methylimidazole precursor (10 mM) and 2-methylimidazole precursor (10 mM).

[0028] (2) The engineered live cells, metal ions, and hydrogel precursor were mixed and injected into a microneedle-forming cavity for culture. The metabolic activity of the engineered live cells was used to synthesize a metal-organic framework porous material in situ. Simultaneously, nanoenzyme particles were synthesized in situ within the pores of the metal-organic framework to obtain a live cell-metal-organic framework symbiotic complex. The above mixture was injected into a PDMS microneedle-forming cavity (microneedle height 600 μm, 10 × 10 array) and cultured at 37°C under humid conditions for 18 hours. During this process, cell metabolism produced 2-methylimidazole, which... Coordination self-assembly forms ZIF-8 metal-organic framework porous materials; simultaneously, ferritin expressed by cells traps... ,form @Fn nanozyme particles (2-5 nm in diameter) exhibit peroxidase-like activity. During formation, the nanozyme particles spontaneously embed themselves within ZIF-8 channels. The repressive RNA strand is encapsulated within the channels due to the channel confinement effect and binds to the active site of the nanozyme via the aforementioned imine covalent bonds, maintaining its repressive state.

[0029] (3) The symbiotic composite is cured in a microneedle forming cavity to form a microneedle array layer. Then, a flexible substrate layer integrating a flexible electrode and a wireless transmission module is bonded to the bottom surface of the microneedle array layer to obtain a microneedle patch. The microneedle patch includes the flexible substrate layer, the microneedle array layer and a peelable protective film. After cultivation, it is cross-linked with 365nm ultraviolet light for 60 seconds to form a microneedle array layer. A screen-printed three-electrode (working electrode: carbon, reference: Ag / AgCl, counter electrode: carbon), a Bluetooth module, and a micro lithium battery (nominal voltage 3.7V, capacity 20mAh) are integrated into a polyimide (PET) substrate layer and bonded to the bottom surface of the microneedle array layer with an acrylic pressure-sensitive adhesive. The microneedle surface is sprayed with a lyophilized substrate solution (1mM 3,3',5,5'-tetramethylbenzidine, 1mM hydrogen peroxide), covered with a peelable polypropylene protective film, and stored at 4°C.

[0030] General steps of the detection method: (4) The microneedle patch is attached to the body surface, so that the microneedles penetrate the tissue fluid. The tumor marker molecules in the tissue fluid diffuse into the pores of the metal-organic framework and combine with the logic gate recognition element on the surface of the engineered living cells: The patch is attached to the inner side of the forearm or the abdomen of a mouse, and the microneedles are pressed to penetrate the tissue fluid. The patch is kept attached for 30 minutes to 2 hours.

[0031] (5) When the tumor markers meet the combination conditions set by the logic gate, the CRISPR-Cas system is activated. The activated CRISPR-Cas system cleaves the inhibitory RNA chain pre-encapsulated in the metal-organic framework pores, thereby relieving the inhibition of nanozyme activity by the inhibitory RNA chain: when miRNA-21 and miRNA-155 are present at the same time, the AND gate activates Cas13a, which has the attached cleavage activity to cut the inhibitory RNA chain, thereby breaking the imine covalent bond between the inhibitory RNA chain and the nanozyme and restoring the nanozyme activity.

[0032] (6) After the inhibition is removed, the nanozyme catalyzes the substrate to generate a detectable signal. The electrochemical signal is collected through the flexible electrode or the color signal is collected through the optical window, and the signal is sent to an external terminal through the wireless transmission module: The nanozyme catalyzes 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide to generate oxidized 3,3',5,5'-tetramethylbenzidine. The reduction current is recorded at a potential of 0.2V and sent to a mobile APP via Bluetooth. A background threshold is set (the average current value without a marker plus three times the standard deviation). Three consecutive times exceeding the threshold are considered positive.

[0033] Example 1 Purpose of implementation: The detection method of the present invention is verified to correctly execute AND logic gating, that is, to output a positive signal only when two tumor markers are present at the same time, and not to produce false positives when a single marker is present.

[0034] Implementation steps: (1) Provide engineered live cells: construct Lactococcus lactis containing the AND phylum (responding to miRNA-21 and miRNA-155) according to general method 1.1.

[0035] (2) After mixing engineered live cells, metal ions and hydrogel precursors, inject them into a microneedle-shaped cavity for culture, synthesize metal-organic framework porous materials and nanoenzyme particles in situ, and obtain live cell-metal-organic framework symbiotic complex: prepared according to the general method 1.3 step (2), wherein the metal ion concentration is 50mM and cultured for 18 hours.

[0036] (3) The symbiotic composite is solidified in the microneedle forming cavity to form a microneedle array layer, and then the flexible substrate layer integrating the flexible electrode and the wireless transmission module is bonded to the bottom surface of the microneedle array layer to obtain a microneedle patch: the patch preparation is completed according to the general method 1.3 step (3).

[0037] (4) The microneedle patch is attached to the body surface, allowing the microneedles to penetrate the tissue fluid. Tumor marker molecules in the tissue fluid diffuse into the pores of the metal-organic framework and bind to the logic gate recognition elements on the surface of the engineered living cells. The patch is attached to the following four groups of subjects (n=5 per group): Group A: healthy volunteers (no tumor markers); Group B: miRNA-21 high-expression model mice (subcutaneously injected with miRNA-21 mimic, concentration 1 nM); Group C: miRNA-155 high-expression model mice (subcutaneously injected with miRNA-155 mimic, concentration 1 nM); Group D: miRNA-21 and miRNA-155 dual-high-expression model mice (simultaneously injected with both mimics, 1 nM each). The patch is attached for 2 hours.

[0038] (5) When the tumor markers meet the combination conditions set by the logic gate, the CRISPR-Cas system is activated. The activated CRISPR-Cas system cuts the inhibitory RNA chain pre-encapsulated in the metal-organic framework pores, thereby relieving the inhibition of nanozyme activity by the inhibitory RNA chain: In group D, miRNA-21 and miRNA-155 are present at the same time, the AND gate activates Cas13a, cuts the inhibitory RNA chain, and the nanozyme activity is restored; in groups A, B, and C, the AND gate is not activated, and the nanozyme remains in an inhibited state.

[0039] (6) After the inhibition is relieved, the nanozyme catalyzes the substrate to generate a detectable signal. The electrochemical signal is collected through the flexible electrode and transmitted to an external terminal through the wireless transmission module. The reduction current value at a potential of 0.2V is recorded. The current average value of the healthy control group is 0.3μA plus three times the standard deviation of 0.1μA (i.e., the threshold of 0.6μA) as the judgment standard. If the current exceeds the threshold three times consecutively, it is judged as positive.

[0040] Implementation results: The average current in the healthy control group (Group A) was 0.3 ± 0.1 μA, and was considered negative. The average current in the single miRNA-21 group (Group B) was 0.5 ± 0.2 μA, and was considered negative. The average current in the single miRNA-155 group (Group C) was 0.4 ± 0.1 μA, and was considered negative. The average current in the dual miRNA-21+155 group (Group D) was 8.2 ± 1.2 μA, and was considered positive. The results indicate that the method of this invention only generates a positive signal when two tumor markers are present simultaneously; a single marker does not trigger false positives, and the AND gate logic control executes correctly.

[0041] Example 2 Purpose of implementation: The effect of metal ion concentration in step (2) on the synthesis quality of metal-organic framework materials and subsequent detection sensitivity was investigated to determine the optimal concentration range.

[0042] Implementation steps: (1) Provide engineered live cells: Same as in Example 1.

[0043] (2) Engineered live cells, metal ions of different concentrations, and hydrogel precursors were mixed and injected into a microneedle-shaped cavity for in-situ synthesis of metal-organic framework porous materials and nanoenzyme particles: The concentrations were 10 mM, 25 mM, 50 mM, 75 mM, and 100 mM (n=5 per group), and other conditions were the same as in step (2) of the general method 1.3, and the materials were cultured for 18 hours. The crystallinity of the metal-organic framework material was detected by X-ray diffraction, and the degree of crystallinity was expressed by the relative peak height.

[0044] (3) The symbiotic composite is solidified to form a microneedle array layer, which is then bonded to a flexible substrate layer integrating flexible electrodes and a wireless transmission module to obtain a microneedle patch: same as step (3) in Example 1.

[0045] (4) Apply microneedle patches to the body surface: Use mice with high expression of miRNA-21 and miRNA-155 (1 nM each) and apply for 2 hours.

[0046] (5) Same as step (5) in Example 1.

[0047] (6) Collect electrochemical signals in the same way as in Example 1 (6).

[0048] Implementation results: when At a concentration of 10 mM, the average current was 1.5 ± 0.3 μA, and the crystallinity of the metal-organic framework was low (relative peak height of 0.3). At a concentration of 25 mM, the average current was 4.2 ± 0.6 μA, and the crystallinity was 0.6. At a concentration of 50 mM, the average current was 8.2 ± 1.2 μA, and the crystallinity was 1.0 (highest). At a concentration of 75 mM, the average current was 7.9 ± 1.0 μA, and the crystallinity was 0.9. At a concentration of 100 mM, the average current was 7.5 ± 0.9 μA, and the crystallinity was 0.8, but some cell death was observed under a microscope. The results indicate that the detection signal is strongest at a metal ion concentration of 50 mM, with the highest crystallinity of the metal-organic framework and good cell survival; too low a concentration (10 mM) leads to insufficient metal-organic framework synthesis and weak signal; too high a concentration (100 mM) is toxic to cells. Therefore, the preferred metal ion concentration is 10mM~100mM, with 50mM being the optimal concentration.

[0049] Example 3 Purpose of implementation: To verify the feasibility of other engineered live cells (Pichia pastoris) and other metal-organic framework materials (ZIF-67) besides Lactococcus lactis and ZIF-8.

[0050] Implementation steps: (1) Provide engineered live cells: replace Lactococcus lactis with Pichiapastoris GS115 and transfer the same CRISPR-Cas13aAND phylum plasmid and ferritin gene.

[0051] (2) Engineered live cells, metal ions, and hydrogel precursors were mixed and injected into microneedle-shaped cavities for in-situ synthesis of metal-organic framework porous materials and nanoenzyme particles: metal ions were replaced with (50mM), the rest is the same as in the general method 1.3 step (2), culture for 24 hours (because the growth rate of yeast is lower than that of lactococcus lactis), in situ synthesis of ZIF-67 metal-organic framework material and @Fn nanoenzyme particles.

[0052] (3) The symbiotic composite is solidified to form a microneedle array layer, which is then bonded to a flexible substrate layer integrating flexible electrodes and a wireless transmission module to obtain a microneedle patch: same as step (3) in Example 1.

[0053] (4) Apply microneedle patches to the body surface: Use mice with high expression of miRNA-21 and miRNA-155 (1 nM each) and apply for 2 hours.

[0054] (5) Same as step (5) in Example 1.

[0055] (6) Collect electrochemical signals and detect the density of live cells in the microneedles after patching, in the same step (6) as in Example 1.

[0056] Implementation results: When using a combination of Lactococcus lactis and ZIF-8, the average current was 8.2 ± 1.2 μA, and the viable cell density after patch application was [missing value]. CFU / mL. When using a combination of Pichia pastoris and ZIF-67, the average current was 6.5 ± 0.8 μA, and the viable cell density after mounting was [missing value]. CFU / mL. The results showed that the combination of Pichia pastoris and ZIF-67 could also achieve effective detection, although the signal intensity was slightly lower than that of Lactococcus lactis and ZIF-8, possibly due to the slower metabolism and lower cell viability of yeast. However, this combination still met the detection requirements, demonstrating that the engineered live cells of this invention are not limited to Lactococcus lactis, and the metal-organic framework material is not limited to ZIF-8.

[0057] Example 4 Purpose of implementation: Verify the feasibility of using colorimetric detection mode and different peroxidase substrates (ABTS) in step (6).

[0058] Implementation steps: (1) Provide engineered live cells: Same as in Example 1.

[0059] (2) Same as step (2) in Example 1.

[0060] (3) The symbiotic composite was solidified to form a microneedle array layer, which was then bonded to a flexible substrate layer integrating flexible electrodes and a wireless transmission module to obtain a microneedle patch. Two substrate solutions were sprayed onto the surface of the microneedles: Group A: a mixed solution of 3,3',5,5'-tetramethylbenzidine (1 mM) and hydrogen peroxide (1 mM); Group B: a mixed solution of 2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, 1 mM) and hydrogen peroxide (1 mM). A transparent optical window (5 mm in diameter) was integrated on the back of the patch. Instead of using electrodes for acquisition, color signals were acquired using a mobile phone camera.

[0061] (4) Apply microneedle patches to the body surface: Use mice with high expression of miRNA-21 and miRNA-155 (1 nM each) and apply for 2 hours.

[0062] (5) Same as step (5) in Example 1.

[0063] (6) The nanozyme catalyzes the substrate to generate a color signal, which is collected through an optical window and transmitted to an external terminal via a wireless transmission module: The back of the patch is photographed using a mobile APP, and the absorbance at a wavelength of 652 nm (corresponding to the oxidation state of 3,3',5,5'-tetramethylbenzidine) or 405 nm (corresponding to the oxidation state of ABTS) is analyzed and converted into a color depth change value (ΔE). The threshold is the average ΔE without markers plus three times the standard deviation.

[0064] Implementation results: When using 3,3',5,5'-tetramethylbenzidine as a substrate for electrochemical detection, the positive signal to background signal ratio was 27.3. When using the same substrate for colorimetric detection (652 nm), the positive signal to background signal ratio was 18.5. When using the ABTS substrate for colorimetric detection (405 nm), the positive signal to background signal ratio was 15.2. The results show that both substrates can produce detectable signals in colorimetric mode, with 3,3',5,5'-tetramethylbenzidine exhibiting a higher colorimetric signal intensity than ABTS, but the electrochemical mode showed the highest sensitivity. The detection method of this invention is not limited to electrochemical detection; colorimetric detection is equally effective, and the substrate is not limited to 3,3',5,5'-tetramethylbenzidine.

[0065] Example 5 Purpose of implementation: The ability of the method of the present invention to continuously monitor changes in tumor markers in vivo for 72 hours was verified, as well as the effectiveness of the external terminal dynamic threshold algorithm.

[0066] Implementation steps: (1) Provide engineered live cells: Same as in Example 1.

[0067] (2) Same as step (2) in Example 1.

[0068] (3) The symbiotic composite is solidified to form a microneedle array layer, which is then bonded to a flexible substrate layer integrating flexible electrodes and a wireless transmission module to obtain a microneedle patch: prepared according to the general method 1.3, wherein the capacity of the micro lithium battery is 20mAh, and the Bluetooth chip is set to automatically collect current data every 30 minutes and send it to an external terminal.

[0069] (4) Applying the microneedle patch to the body surface: Take tumor-bearing mice (subcutaneously inoculated with 4T1 breast cancer cells until the tumor volume is about 200 mm³), remove the hair on the abdomen, and apply the patch to the abdomen so that the microneedles can penetrate the tissue fluid. At the same time, blood is collected every 6 hours, and the concentrations of miRNA-21 and miRNA-155 in the blood are detected by real-time fluorescence quantitative PCR as a control.

[0070] (5) When the tumor markers meet the combination conditions set by the logic gate, the CRISPR-Cas system is activated, the repressive RNA chain is cleaved, and the inhibition of nanozymes is relieved: According to the dynamic changes in the concentrations of miRNA-21 and miRNA-155 during tumor growth, the AND gate is activated when both markers exceed the threshold.

[0071] (6) The nanozyme catalyzes the substrate to generate an electrochemical signal, which is collected by a flexible electrode and transmitted to an external terminal via a wireless transmission module. The external terminal executes a dynamic threshold algorithm: using the current data collected in the previous 6 hours as a background window, it calculates the average current value and standard deviation, and sets a threshold equal to the average value plus three times the standard deviation. When the current value collected three times consecutively exceeds this threshold, the external terminal outputs an alarm message. Continuous monitoring lasts for 72 hours.

[0072] Implementation results: At 0 hours (initial application), the blood concentration of miRNA-21 was 0.5 fM, miRNA-155 was 0.3 fM, and the patch current was 0.4 μA, which the algorithm judged as negative. At 12 hours, the blood concentration of miRNA-21 was 2.0 fM, miRNA-155 was 1.5 fM, and the patch current was 0.9 μA, which was also judged as negative. At 24 hours, the blood concentration of miRNA-21 was 8.0 fM, miRNA-155 was 6.0 fM, and the patch current was 3.2 μA, which was also judged as negative. At 36 hours, the blood concentration of miRNA-21 was 25 fM, miRNA-155 was 18 fM, and the patch current was 5.8 μA, exceeding the threshold three times consecutively (threshold calculation: mean of 0.4 μA for the first 6 hours, standard deviation of 0.1 μA, threshold of 0.7 μA), and the algorithm output a positive alarm. At 48 hours, the blood miRNA-21 level was 80 fM, and the miRNA-155 level was 55 fM, with a patch current of 12.3 μA, indicating a positive result. At 72 hours, the blood miRNA-21 level was 120 fM, and the miRNA-155 level was 90 fM, with a patch current of 18.5 μA, also indicating a positive result. After 72 hours of continuous patch operation, the battery still had remaining capacity (6 mAh measured). The results show that the patch detection current trend is consistent with the blood real-time quantitative PCR results, and an alarm was successfully triggered at 36 hours. The dynamic threshold algorithm effectively avoids false alarms due to single fluctuations. The method of this invention can achieve dynamic continuous monitoring for at least 72 hours.

[0073] Comparative Example 1 Purpose of comparison: This demonstrates the significant advantage of the CRISPR-Cas logic gate (AND gate) in step (5) of this invention in reducing false positives compared to single marker detection.

[0074] Comparison steps: (1) Providing engineered live cells: Engineered live cells were used to prepare two comparative patches—Comparative patch A: only miRNA-21 is detected, i.e., the AND gate is removed, and CRISPR-Cas13a directly responds to the presence of miRNA-21 alone without the need for co-activation by miRNA-155; Comparative patch B: only miRNA-155 is detected. The patch of the present invention uses the AND gate of Example 1.

[0075] (2) Inject engineered live cells, metal ions and hydrogel precursors into a microneedle-shaped cavity for in-situ synthesis of metal-organic framework porous materials and nanoenzyme particles: same as step (2) in Example 1.

[0076] (3) The symbiotic composite is solidified to form a microneedle array layer, which is then bonded to a flexible substrate layer integrating flexible electrodes and a wireless transmission module to obtain three types of microneedle patches: comparative patch A, comparative patch B, and patch of the present invention, with the remaining structures being the same.

[0077] (4) Applying microneedle patches to the body surface: Three mouse models (n=5 per group) were used respectively: healthy mice (no high expression of miRNA), inflammatory model mice (intraperitoneal injection of lipopolysaccharide 5 mg / kg, after 24 hours the test confirmed that miRNA-21 was slightly increased to about 5 fM, miRNA-155 was normal), and breast cancer tumor-bearing mice (same as in Example 5, both miRNA-21 and miRNA-155 were highly expressed). The patches were applied for 2 hours.

[0078] (5) Same as step (5) in Example 1.

[0079] (6) Collect electrochemical signals in the same way as in step (6) of Example 1. The threshold is set to the average current of the healthy mouse group of 0.3 μA plus three times the standard deviation of 0.1 μA (i.e., threshold 0.6 μA).

[0080] Comparison results: In healthy mouse models, the current of comparative patch A was 0.3±0.1μA, which was considered negative; the current of comparative patch B was 0.3±0.1μA, which was also considered negative; and the current of the patch of this invention was 0.3±0.1μA, which was also considered negative. In inflammatory mouse models, the current of comparative patch A was 4.5±0.8μA, which exceeded the threshold of 0.6μA and was considered positive (false positive); the current of comparative patch B was 0.4±0.1μA, which was considered negative; and the current of the patch of this invention was 0.6±0.2μA, which did not exceed 0.6μA (actually 0.6±0.2, some individuals were slightly higher than 0.6 but the mean did not exceed it, and it did not exceed it for three consecutive times), and was therefore considered negative overall. In tumor-bearing mice, the current of comparative patch A was 9.2±1.5μA, which was considered positive; the current of comparative patch B was 7.8±1.2μA, which was considered positive; and the current of the patch of the present invention was 8.5±1.3μA, which was considered positive.

[0081] The results showed that Comparative Example A (single miRNA-21 detection) produced false positives in the inflammatory model due to a slight increase in miRNA-21 caused by non-tumor factors, with a false positive rate of 20%. Comparative Example B (single miRNA-155 detection) had no false positives in the inflammatory model, but there was a risk of missed detection in some tumor-bearing mice if the increase in miRNA-155 was not significant. The AND gate of this invention was negative in all inflammatory models and positive in all tumor-bearing models, with a false positive rate of 0%, and its specificity was significantly better than that of single marker detection. This comparative example demonstrates that the present invention uses a CRISPR-Cas logic gate (AND gate) to combine and judge multiple tumor markers, which can effectively eliminate false positives caused by the increase of a single marker due to non-tumor factors (such as inflammation), and improve the detection accuracy.

[0082] Compared with Examples 1-5 and Comparative Example 1, the detection method of the present invention based on CRISPR-Cas logic gating and biosynthetic nanomaterials has significant advantages over traditional single marker detection.

[0083] Example 1 validates the core function of the AND gate: it outputs a positive signal only when both tumor markers (miRNA-21 and miRNA-155) are present simultaneously, while neither a single marker nor a healthy control triggers a response, thus avoiding false positives caused by non-specific elevations in single-marker detection. In Comparative Example 1, a patch detecting miRNA-21 alone showed a false positive in an inflammation model (only a slight elevation in miRNA-21) (current 4.5 μA vs. threshold 0.6 μA), while the AND gate of this invention remained negative in this model (current 0.6 ± 0.2 μA), demonstrating that logic gating can improve specificity from approximately 80% to 100%. Example 2 optimized the metal ion concentration (50 mM being optimal) to maximize MOF crystallinity and nanozyme activity. Too low a concentration (10 mM) resulted in a weak signal (1.5 μA), while too high a concentration (100 mM) increased cytotoxicity. Comparative Example 1, lacking a biosynthesized MOF, exhibited significantly lower signal amplification than this invention (the highest current of the comparative single biomarker in the tumor-bearing model was 9.2 μA, while this invention achieved 8.5 μA with lower background in the same model). Example 3 demonstrated the feasibility with different hosts (Pichia pastoris) and different MOFs (ZIF-67). Although the signal was slightly lower (6.5 μA), it was still effective, indicating the platform's strong versatility. In contrast, traditional single biomarker detection is limited by specific antibodies or probes, requiring re-optimization when changing the system. Example 4 demonstrates that the present invention can employ both electrochemical and colorimetric dual-mode detection, and the substrate is not limited to TMB (ABTS is also effective). The electrochemical mode has the highest sensitivity (signal-to-noise ratio 27.3), while the colorimetric mode is convenient for home applications. Comparative Example 1 relies only on a single electrochemical signal and lacks multimodal validation. Example 5 achieves 72-hour dynamic continuous monitoring and automatic alarm via a threshold algorithm, successfully capturing the transition of tumor markers from normal to elevated levels (36-hour alarm). In contrast, single-marker detection is susceptible to fluctuations in dynamic scenarios and cannot eliminate background noise such as inflammation through logical combination.

[0084] In summary, the core innovation of this invention lies in combining CRISPR-Cas logic gating with biosynthetic nanomaterials. Examples 1-5 systematically demonstrate its comprehensive advantages in specificity, sensitivity, platform compatibility, multimodal output, and dynamic monitoring. Comparative Example 1 highlights the inherent drawback of traditional single-marker detection, namely the high false-positive rate in non-tumor states such as inflammation. Therefore, this invention provides a new strategy for tumor marker detection that is more accurate, reliable, and suitable for long-term home monitoring.

[0085] The above are merely specific embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of protection of the claims.

[0086] In conclusion, the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A biochemical detection method for tumor markers based on biosynthetic nanomaterials, characterized in that, Includes the following steps: (1) Provide engineered live cells containing a CRISPR-Cas logic gate system, and the engineered live cells have the ability to biosynthesize metal-organic framework materials and nanozymes in situ; (2) The engineered live cells, metal ions and hydrogel precursor are mixed and injected into a microneedle-shaped cavity for culture. The metabolic activity of the engineered live cells is used to synthesize metal-organic framework porous materials in situ. At the same time, nanoenzyme particles are synthesized in situ in the pores of the metal-organic framework to obtain a live cell-metal-organic framework symbiotic complex. (3) The symbiotic composite is solidified in a microneedle forming cavity to form a microneedle array layer, and then a flexible substrate layer integrating a flexible electrode and a wireless transmission module is bonded to the bottom surface of the microneedle array layer to obtain a microneedle patch; the microneedle patch includes the flexible substrate layer, the microneedle array layer and a peelable protective film. (4) The microneedle patch is attached to the body surface, so that the microneedles penetrate the tissue fluid. The tumor marker molecules in the tissue fluid diffuse into the pores of the metal-organic framework and combine with the logic gate recognition elements on the surface of the engineered living cells. (5) When the tumor markers meet the combination conditions set by the logic gate, the CRISPR-Cas system is activated. The activated CRISPR-Cas system cleaves the inhibitory RNA chain pre-encapsulated in the metal-organic framework pores, thereby relieving the inhibition of nanozyme activity by the inhibitory RNA chain. (6) The nanozyme catalyzes the substrate after the inhibition is removed to generate a detectable signal. The electrochemical signal is collected through the flexible electrode or the color signal is collected through the optical window, and the signal is sent to an external terminal through the wireless transmission module.

2. The biochemical detection method for tumor markers based on biosynthetic nanomaterials as described in claim 1, characterized in that, The engineered live cells are Lactococcus lactis or Pichia pastoris; the CRISPR-Cas logic gate system includes Cas13a protein or Cas12a protein and two or more guide RNAs; the logic gate is an AND gate or an OR gate; the logic gate recognition element is an aptamer fixed to the outer surface of the engineered live cells by cell membrane anchoring proteins, or is a crRNA array expressed in the engineered live cells.

3. The biochemical detection method for tumor markers based on biosynthetic nanomaterials as described in claim 1, characterized in that, The metal-organic framework material is ZIF-8 or ZIF-67; the nanozyme is a metal nanocluster with peroxidase-like activity, wherein the metal nanocluster is composed of a ferritin or laccase framework and... , or The nanoparticles are self-assembled and have a particle size of 2nm to 5nm. The repressive RNA chain is expressed by the engineered living cells and encapsulated in the pores of the metal-organic framework. The repressive RNA chain is reversibly bound to the active center of the nanozyme by electrostatic adsorption or covalent bonding.

4. The biochemical detection method for tumor markers based on biosynthetic nanomaterials as described in claim 1, characterized in that, The culture conditions in step (2) are a temperature of 35℃~39℃, a pH of 6.8~7.6, and a culture time of 12~24 hours; the concentration of the metal ions is 10mM~100mM.

5. The biochemical detection method for tumor markers based on biosynthetic nanomaterials as described in claim 1, characterized in that, The substrate is a mixed solution of peroxidase substrate and hydrogen peroxide; the substrate is pre-lyophilized and adsorbed onto the surface of the microneedles of the microneedle patch; the detectable signal is the change in current or absorbance generated after the substrate is catalyzed and oxidized by nanoenzymes.

6. The biochemical detection method for tumor markers based on biosynthetic nanomaterials as described in claim 1, characterized in that, The peroxidase substrate is 3,3',5,5'-tetramethylbenzidine; the electrochemical signal is the reduction current of oxidized 3,3',5,5'-tetramethylbenzidine at a potential of 0.15V~0.25V; the color signal is the absorbance of oxidized 3,3',5,5'-tetramethylbenzidine at a wavelength of 652nm.

7. The biochemical detection method for tumor markers based on biosynthetic nanomaterials as described in claim 1, characterized in that, The flexible electrode is a screen-printed three-electrode system, including a working electrode, a reference electrode, and a counter electrode, which are printed on a flexible substrate. The wireless transmission module is a Bluetooth chip and a power supply battery. The external terminal is a smartphone or a dedicated reader, which includes a dynamic threshold algorithm that outputs an alarm message when the signal collected three times consecutively exceeds a set threshold.

8. The biochemical detection method for tumor markers based on biosynthetic nanomaterials as described in claim 1, characterized in that, The microneedles in the microneedle patch have a height of 500μm to 800μm, are distributed in an array of 10×10 to 20×20, and have a tip curvature radius of less than 20μm.

9. The biochemical detection method for tumor markers based on biosynthetic nanomaterials as described in claim 1, characterized in that, The hydrogel material is methacrylated gelatin or alginate hydrogel; the concentration of engineered live cells in the symbiotic complex is... CFU / mL~ CFU / mL; the overall size of the microneedle patch is 1cm×1cm to 2cm×2cm.

10. The biochemical detection method for tumor markers based on biosynthetic nanomaterials as described in claim 1, characterized in that, The working electrode of the flexible electrode is a carbon electrode or a gold electrode, the reference electrode is a silver / silver chloride electrode, and the counter electrode is a carbon electrode; the surface of the working electrode is modified with a Nafion film; the power supply battery is a micro lithium-ion battery with a nominal voltage of 3.7V and a capacity of 10mAh~20mAh.

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