A fluorescent probe device with high sensitivity detection

Abstract

The application discloses a kind of fluorescent probe devices with high sensitivity detection in the field of biological fluorescence detection, including microfluidic chip, closed microfluid channel for test sample flow is equipped inside microfluidic chip, microfluid channel includes reaction bin, sample bin and enzyme liquid bin, sample bin and enzyme liquid bin are symmetrically distributed on the both sides of reaction bin and are connected with the delay channel of zigzag structure between reaction bin, first filling opening and second filling opening are respectively opened in sample bin and enzyme liquid bin, reaction bin is opened with the vent hole being communicated with outside, and a plurality of aptamer fluorescent probes are fixed on the bottom wall of reaction bin.The application fixes nucleic acid aptamer fluorescent probe in microfluidic chip, and adds CRISPR-Cas12a enzyme cutting system, releases multiple signal fragments in the process of single aptamer binding antibiotic, realizes signal cascade amplification of CRISPR-Cas12a non-specific enzyme cutting activation, and has the function of detecting the existence of drug-resistant gene, to minimize the possibility of false negative detection results.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological fluorescence detection, and specifically relates to a fluorescence probe device with high-sensitivity detection. BACKGROUND

[0002] In recent years, with the widespread use of antibiotics, the problem of bacterial drug resistance is becoming increasingly serious, posing a major threat to public health. Traditional detection methods such as culture method and molecular biology technology have problems such as complex operation, long time consumption, insufficient sensitivity, and are difficult to meet the demand for rapid and efficient detection of antibiotic residues and pathogen drug resistance genes in the environment.

[0003] Microfluidic chip is a micro device integrating micro-scale fluid control elements, which realizes complex fluid control through microfabrication technology. The channel size is in micrometer level, and it has advantages such as high throughput, low consumption and high integration, and can complete the whole process operation of sample processing, reaction, separation and detection. In the detection of antibiotics and drug resistance genes, microfluidic chip can construct a multi-channel network to realize multi-parameter parallel analysis, significantly shorten the detection time and improve the experimental efficiency.

[0004] Nucleic acid aptamer is a single-stranded oligonucleotide sequence obtained by in vitro exponential enrichment ligand system evolution technology (SELEX), which can specifically and highly bind to target molecules. Antibiotic nucleic acid aptamer is designed according to the structure of antibiotic molecules or specific sequences of drug resistance genes, which can recognize and bind to trace targets to form stable complexes. Compared with traditional antibodies, nucleic acid aptamer has advantages such as easy synthesis, high chemical stability and strong modification.

[0005] Fluorescent probe is a sensing molecule that combines recognition groups with fluorescent signal reporter groups, and when it binds to the target, the fluorescence intensity or wavelength changes characteristically. This technology has advantages such as high sensitivity, good selectivity and real-time visualization. In microfluidic chip, fluorescent probe can be fixed on the channel surface or embedded in the detection area through covalent coupling or physical adsorption. When the sample containing antibiotics or drug resistance genes flows through, nucleic acid aptamer captures the target and triggers the change of fluorescent signal, and quantitative determination is realized through integrated optical detector.

[0006] The existing nucleic acid aptamer fluorescence detection method for antibiotics often causes conformational change through specific binding of aptamer to antibiotics, thereby increasing the spatial distance between the fluorescent group and the quenching group modified at both ends of the nucleic acid aptamer, thereby improving the fluorescence signal level. Although these methods can detect the content of antibiotics, the detection limit is not enough to detect the existence of a single molecule of antibiotic due to the limitation of the number of nucleic acid aptamers, and in addition, the drug-resistant microorganisms often accompanied by antibiotic residues are ignored. The drug resistance genes existing in these drug-resistant microorganisms can also be the target of detection.

[0007] To this end, it is necessary to propose a fluorescent probe device with high sensitivity detection which can amplify the signal cascade based on the non-specific enzyme cutting activation of CRISPR-Cas12a and simultaneously detect potential drug-resistant genes in the sample. SUMMARY

[0008] In order to solve the above problems, the purpose of the present application is to provide a fluorescent probe device with high sensitivity detection, by fixing the nucleic acid aptamer fluorescent probe in the microfluidic chip, and adding the CRISPR-Cas12a enzyme cutting system, releasing multiple signal fragments in the process of single aptamer binding antibiotic, realizing the signal cascade amplification based on the non-specific enzyme cutting activation of CRISPR-Cas12a, and having the function of detecting the existence of drug-resistant genes, improving the sensitivity and convenience of detection, and as far as possible reducing the possibility of false negative detection results.

[0009] In order to achieve the above purpose, the technical scheme of the present application is as follows: a fluorescent probe device with high sensitivity detection, comprising a microfluidic chip, the microfluidic chip is internally provided with a closed microchannel for sample flow, the microchannel comprises a reaction chamber, a sample chamber and an enzyme liquid chamber, the sample chamber and the enzyme liquid chamber are symmetrically distributed on both sides of the reaction chamber and are communicated with the reaction chamber through a delay channel with a zigzag structure, the sample chamber and the enzyme liquid chamber are respectively provided with a first filling port for injecting sample extraction liquid and a second filling port for injecting enzyme liquid, the reaction chamber is symmetrically provided with a vent hole communicated with the outside, and a plurality of aptamer fluorescent probes are fixedly connected on the bottom wall of the reaction chamber.

[0010] The principle of the basic scheme is: the nucleic acid aptamer fluorescent probe fixed on the bottom wall of the reaction chamber specifically binds to the target antibiotic molecule through the three-dimensional structure. When the target exists, the aptamer conformation changes, and the preloaded signal fragment is released. The released signal molecule acts as an activation trigger of the CRISPR system, and after being combined with the Cas12a protein-crRNA complex, the non-specific transcleavage activity of Cas12a is activated. Cas12a then cuts the second anchor strand to release a large number of fluorescence quenching molecules existing on the fluorescent particles, increases the steric hindrance of the quenching group and the fluorescent particles, and produces cascade fluorescence signal amplification.

[0011] The sample extraction liquid (containing antibiotic) and the enzyme liquid (containing Cas12a system) slowly flow into the reaction chamber through the zigzag channel, ensuring that the two are fully mixed in the reaction chamber and avoiding premature reaction. The microchannel structure uses the laminar flow effect to maintain the uniform temperature in the reaction chamber, improving the enzyme cutting reaction efficiency. The vent hole balances the air pressure inside and outside the reaction chamber, preventing liquid flow from being blocked, while avoiding bubbles interfering with optical detection.

[0012] The Cas12a protein-crRNA complex can also capture the drug-resistant gene fragments in the sample. When the drug-resistant gene exists, it also triggers the Cas12a cleavage reaction, realizing double target detection.

[0013] The beneficial effects of the basic scheme are: 1. The trans-enzyme cleavage activity of CRISPR-Cas12a can cyclically cleave hundreds of fluorescence quenching molecules, converting a single target event into a large amount of fluorescence signal, with a detection limit as low as aM (10 -18 M) level, which is 3-4 orders of magnitude higher than traditional ELISA or qPCR.

[0014] 2. The double-signal output mechanism (antibiotic residues + drug-resistant genes) forms a logical "NOT gate" judgment, which is only determined as negative when both are not detected, avoiding single detection being limited by the concentration of the detected substance or the accuracy of the detection method, causing missed detection. The closed reaction environment of the microfluidic chip reduces pollution and reagent evaporation, combined with real-time fluorescence monitoring algorithm, which can dynamically correct signal drift.

[0015] 3. After the sample extraction solution and the enzymatic solution are injected through the first filling port and the second filling port, the microfluidic chip automatically completes the mixing, reaction, and detection process without manual pipetting. The size of the chip can be reduced to 1cm 2 , which is suitable for portable fluorescence detectors and meets the needs of on-site rapid detection (POCT).

[0016] 4. By changing the aptamer sequence, signal fragment sequence, and corresponding crRNA design, the same chip can detect different types of antibiotics (such as tetracycline, sulfonamides) and their drug-resistant genes, realizing "one chip multiple detection".

[0017] 5. The reagent consumption is reduced by more than 90% (microliter level) compared to macroscopic reactions. The CRISPR enzyme cleavage reaction does not require PCR amplification, and the temperature control cycle equipment is omitted, reducing the overall detection cost to 1 / 5 of the traditional method.

[0018] Further, the aptamer fluorescent probe comprises fluorescent particles, and a plurality of first anchor chains are covalently connected to the fluorescent particles, the antibiotic aptamer is hybridized and complementarily connected to the first anchor chains, and the other end of the antibiotic aptamer is hybridized and complementarily connected to a signal chain, and the signal chain is connected by a plurality of signal fragments which are sequentially complementary and paired.

[0019] The beneficial effects of the basic scheme are: 1. The signal chain is composed of a plurality of complementary and paired signal fragments in series, forming a "molecular wire" structure. When the nucleic acid aptamer binds to the antibiotic, the conformational change causes the signal chain to detach and release a plurality of signal fragments, resulting in exponential signal amplification. For example, if a single signal chain contains 10 signal fragments, the theoretical signal strength can be at least 10 times higher, significantly enhancing the detection sensitivity.

[0020] 2. By extending the signal chain length (e.g., increasing to 20 fragments), different drug resistance gene sequences can be designed in different signal fragments to meet the detection requirements of different target concentrations, enabling simultaneous detection across multiple channels. This modular design allows the same probe system to cover the detection of 5-8 antibiotics and drug resistance genes.

[0021] 3. When quantum dots or metal-enhanced fluorescence (MEF) particles are used as carriers, quantum dots have narrow emission bands and anti-photobleaching properties, and their fluorescence lifetime is 10 times longer than that of organic dyes. MEF particles enhance the local electromagnetic field through surface plasmon resonance, which increases the fluorescence intensity by 5-10 times. Combining the advantages of both, the detection signal-to-noise ratio (SNR) can be improved from 15dB in traditional methods to more than 30dB.

[0022] 4. The first anchoring chain is connected to the fluorescent particles via covalent bonds (such as click chemistry), and the dissociation temperature (Tm value) is 15-20℃ higher than that of the traditional biotin-avidin system, ensuring structural stability for 72 hours in complex sample matrices (such as blood and food homogenates) and reducing signal loss caused by probe detachment.

[0023] Furthermore, several second anchoring chains are covalently linked to the fluorescent particles, and the ends of the second anchoring chains are all modified with fluorescence quenching molecules.

[0024] The beneficial effects of the basic scheme are: 1. When the fluorescence quenching molecule at the end of the second anchoring chain forms "contact quenching" with the fluorescent particles, the aptamer conformation changes only when the target antibiotic binds, leading to the dissociation of the signal chain. This activates the trans-enzyme cleavage of CRISPR-Cas12a to cleave the fluorescence quenching molecule and amplify the fluorescent particle signal. This activation mechanism reduces the background signal by more than 80% and improves the detection signal-to-noise ratio (SNR) to 45dB.

[0025] 2. A third-order signal amplification system forms a "target-probe reconstruction-Cas12a cutting" third-order amplification:

[0026] First stage: A single target molecule leads to the reconfiguration of one probe.

[0027] Second stage: The reconstruction probe releases a signal chain containing 10 signal segments.

[0028] Third stage: Cas12a cleaves hundreds of fluorescence quenching molecules.

[0029] The theoretical magnification is 5×10. 3 The detection limit is 10 times higher (up to 500 times higher even after considering actual efficiency). -18 M level.

[0030] 3. The second anchoring chain is covalently linked through disulfide bonds, enabling the probe to maintain structural stability under extreme conditions (pH 4-10, 55℃). Experiments show that after 10 repeated temperature cycles, the probe signal retention rate is still >92%, while the traditional biotin system only maintains 65%.

[0031] 4. Fluorescent particles can be selected with different emission wavelengths (e.g., quantum dots 605nm / 655nm), combined with the quenching molecule type of the second anchoring chain (e.g., BHQ2 / BHQ3), to achieve simultaneous dual-channel detection. This design supports the integration of drug resistance gene detection and antibiotic residue quantification on the same chip, with a data correlation of 0.987.

[0032] Furthermore, the enzyme catalytic solution contains CRISPR-Cas12a, crRNA, and enzyme digestion buffer components.

[0033] The beneficial effects of the basic scheme are: 1. crRNA strictly limits the cleavage site of Cas12a (such as the drug resistance gene sequence in the signal fragment) through base complementarity, forming a "sequence-specific molecular trans scissors". Experiments show that the cleavage specificity guided by crRNA is 10% higher than that of the system without guide RNA. 5 This ensures the activation of the trans-cutting activity and improves detection sensitivity.

[0034] 2. The enzyme catalytic solution can be packaged using vacuum freeze-drying technology and protected with preservatives such as trehalose, allowing it to be stored at 4°C for more than 12 months. After reconstitution, the enzyme activity retention rate is >90%, supporting kit-based production and long-distance transportation.

[0035] 3. By designing different crRNA sequences (such as crRNA-A / B / C), 3-5 antibiotics and their resistance genes can be detected simultaneously in a single enzymatic solution. The cross-reactivity rate between different crRNAs is <0.5%, achieving high-throughput screening.

[0036] 4. Premixed enzyme solutions reduce on-site preparation steps, reducing the reagent percentage in a single test cost from 65% to 12%. For batch testing (>100 samples), the overall cost is reduced by 82% compared to traditional methods.

[0037] Furthermore, the crRNA sequence can complement antibiotic resistance genes that bind to nucleic acid aptamers.

[0038] The beneficial effects of the basic protocol are: the crRNA sequence can complement antibiotic resistance genes, which means that even when the sample does not contain antibiotics but only contains resistance genes, it can still trigger the trans-cleavage activity of CRISPR-Cas12a and enhance the fluorescence signal. The emergence of drug-resistant bacteria is often synchronous with antibiotic residues, avoiding the possibility of false negatives and supporting further experiments to improve the accuracy of antibiotic residue detection.

[0039] Furthermore, the enzyme cleavage buffer components include Tris-HCl, KCl, MgCl2, DTT, BSA, and glycerol.

[0040] The beneficial effects of the basic protocol are: 1. Tris-HCl maintains Cas12a activity within a pH range of 8.0-8.5, and its buffering capacity is twice that of traditional phosphate buffer, effectively resisting interference from acidic metabolites in the sample. KCl / MgCl2 constructs an "ionic strength gradient," K... + Stabilizing the protein backbone, Mg 2+ Activation of the RuvC domain of Cas12a increased enzymatic efficiency by 4.2 times. DTT reduced disulfide bonds, preventing Cas12a from becoming inactive during repeated freeze-thaw cycles and extending the reagent's shelf life to 18 months. BSA formed a "protein crown" encapsulating Cas12a, reducing its non-specific adsorption on the surface of microfluidic channels and improving signal recovery by 65%. Glycerol protected the enzyme's three-dimensional structure through hydrogen bonding, maintaining 85% activity even in samples containing 15% DMSO.

[0041] 2. The enzyme digestion buffer formulation is tolerant to 10mM EDTA and 5% Triton X-100 in samples, suitable for direct detection of tissue lysis buffer. Trehalose (2% w / v) is added as a lyophilization protectant, ensuring enzyme activity retention >90% after reconstitution, supporting room temperature transport of the kit (40℃ / 7 days).

[0042] Furthermore, all signal fragments contained the CRISPR-Cas12a crRNA complementary sequence, and the length of the signal fragments was greater than 18 bp.

[0043] The beneficial effect of the basic scheme is that the design of the signal fragment >18bp allows the signal fragment to accommodate the crRNA complementary sequence while maintaining the release of short-chain hybridization linkage, thus maintaining a good balance between cleavage and release.

[0044] Furthermore, the number of fluorescence quenching molecules attached to the same aptamer fluorescent probe is at least 20 times the number of the first anchoring strand.

[0045] The beneficial effects of the basic scheme are: the high-density quenching molecules form an "optical dark field," resulting in a non-specific fluorescence quenching rate of 99.8%. Even in samples containing 10% serum, the signal-to-noise ratio remains at 45:1.

[0046] Furthermore, sample extract refers to the lysed extract obtained by lysing the microorganisms in the sample to release DNA.

[0047] The beneficial effects of the basic approach are: through the design of microfluidic chips and CRISPR-Cas12a enzyme digestion buffer, samples can be directly added to the detection after simple lysis treatment, which greatly reduces the time cost of antibiotic detection.

[0048] Furthermore, it also includes a robotic arm for automatically positioning, transferring, and retrieving microfluidic chips, a fluid controller for controlling the liquid dispensing flow rate, and a fluorescence detector for detecting the fluorescence signals emitted by fluorescent particles.

[0049] The basic solution offers the following advantages: it integrates a robotic arm, a fluid controller, and a fluorescence detector to create a fully automated testing line. Experiments show that the testing cycle for a single sample is reduced to 12 minutes, an 85% reduction compared to manual operation. The fluid controller utilizes piezoelectric actuation technology to achieve a liquid dispensing accuracy of 50 nL (CV < 0.5%). For rare mutation detection, the reaction volume can be reduced to 2 μL, and reagent consumption is reduced by 90%. Attached Figure Description

[0050] Figure 1 This is an isometric view of the microfluidic chip in an embodiment of the present invention;

[0051] Figure 2 This is a top cross-sectional view of the microfluidic chip in an embodiment of the present invention;

[0052] Figure 3 This is a schematic diagram illustrating the role of the aptamer fluorescent probe and the CRISPR-Cas12a enzyme digestion system in an embodiment of the present invention.

[0053] Figure 4 The results show the photostability test results of the aptamer fluorescent probe in this embodiment of the invention.

[0054] The reference numerals in the accompanying drawings of the instruction manual include: 1. Microfluidic chip; 2. First injection port; 3. Vent; 4. Second injection port; 5. Sample chamber; 6. Delay channel; 7. Reaction chamber; 8. Aptamer fluorescent probe; 9. Enzyme solution chamber; 10. Fluorescent particle; 11. Second anchoring strand; 13. First anchoring strand; 14. Nucleic acid aptamer; 15. Antibiotic residue; 16. Signal strand; 17. Drug resistance gene; 18. Signal fragment; 19. crRNA; 20. CRISPR-Cas12a. Detailed Implementation

[0055] The following detailed description illustrates the specific implementation method:

[0056] Example 1

[0057] The basics are as follows: Figure 1 , Figure 2As shown: A fluorescent probe device with high sensitivity detection includes a microfluidic chip 1. The microfluidic chip 1 has a closed microchannel for sample flow. The microchannel includes a reaction chamber 7, a sample chamber 5, and an enzyme solution chamber 9. The sample chamber 5 and the enzyme solution chamber 9 are symmetrically distributed on both sides of the reaction chamber 7 and are connected to the reaction chamber 7 by a tortuous delay channel 6. The sample chamber 5 and the enzyme solution chamber 9 are respectively opened with a first injection port 2 for injecting sample extract and a second injection port 4 for injecting enzyme solution. The reaction chamber 7 has symmetrically opened vent holes 3 communicating with the outside. Several aptamer fluorescent probes 8 are captured and fixed on the bottom wall of the reaction chamber 7.

[0058] It also includes a robotic arm for automatically positioning, transferring and retrieving the microfluidic chip 1, a fluid controller for controlling the liquid dispensing flow rate, and a fluorescence detector for detecting the fluorescence signal emitted by the aptamer fluorescent probe 8.

[0059] The specific implementation process is as follows: Detection is performed on the microfluidic chip 1 preloaded with aptamer fluorescent probe 8. The chip is automatically picked up, placed and fixed by a robotic arm. Sample extraction solution and enzyme catalytic solution are added to the fluid controller. The fluid controller automatically connects to the first injection port 2 and the second injection port 4 on the chip to inject the sample extraction solution and enzyme catalytic solution into the sample chamber 5 and the enzyme solution chamber 9. The injection flow rate is controlled to allow the two liquids to flow into the reaction chamber 7 through the delay channel 6. The closed reaction environment of the microfluidic chip 1 reduces contamination and reagent evaporation.

[0060] like Figure 2 As shown, when the two reaction reagents enter reaction chamber 7, antibiotic residue 15 in the sample extract first binds to the aptamer fluorescent probe 8, releasing a DNA fragment that triggers trans-cleavage enzymes. This triggers trans-cleavage of the enzymes, cleaving other fluorescent quenching groups on the aptamer fluorescent probe 8, increasing the fluorescence intensity of the aptamer fluorescent probe 8. This change in fluorescence intensity is detected by a fluorescence detector, reflecting the content of antibiotic residue 15 in the sample extract. Simultaneously, when antibiotic residue 15 is absent, the antimicrobial resistance gene 17, which is generally associated with antibiotic residue 15 and released from the lysed sample extract, can also replace the DNA fragment to activate the trans-cleavage enzyme, thus changing the fluorescence intensity. Because the DNA fragments released by the antibiotic binding to the aptamer fluorescent probe 8 are multiple and amplified by the trans-cleavage enzyme effect, the difference in fluorescence intensity between the antibiotic residue 15 detection result and the antimicrobial resistance gene 17 detection result will be significant. This allows for convenient identification of the positive result type based on the fluorescence detection result, enabling further testing to confirm the accuracy of antibiotic residue 15 detection, reducing false negatives, and improving detection sensitivity.

[0061] Example 2

[0062] The difference from the above embodiments is that, as shown in the appendix Figure 2, Figure 3 and Figure 4 As shown: The aptamer fluorescent probe 8 includes fluorescent particles 10, on which several first anchoring strands 13 are covalently linked. Each of the first anchoring strands 13 is hybridized and complementary to an antibiotic nucleic acid aptamer 14. The other end of each antibiotic nucleic acid aptamer 14 is hybridized and complementary to a signal strand 16. The signal strand 16 is composed of several sequentially complementary signal fragments 18. Several second anchoring strands 11 are also covalently linked to the fluorescent particles 10. The ends of the second anchoring strands 11 are modified with fluorescence quenching molecules. The number of fluorescence quenching molecules linked to the same aptamer fluorescent probe 8 is at least 20 times the number of the first anchoring strands 13.

[0063] The enzyme catalytic solution contains CRISPR-Cas12a 20, crRNA 19, and enzyme digestion buffer. The crRNA 19 sequence is complementary to the antibiotic resistance gene 17 that binds to nucleic acid aptamer 14. The enzyme digestion buffer includes Tris-HCl, KCl, MgCl2, DTT, BSA, and glycerol. The signal fragment 18 contains the crRNA 19 complementary sequence of CRISPR-Cas12a 20, and the length of the signal fragment 18 is greater than 18 bp.

[0064] Sample extract refers to the lysed extract obtained by lysing microorganisms in a sample to release their DNA.

[0065] The specific experimental procedure is as follows:

[0066] I. Preparation steps of aptamer fluorescent probe 8:

[0067] 1. Disperse carboxylated diamond particles in a 2% APTES ethanol solution and sonicate for 30 minutes. Centrifuge to remove unreacted APTES, wash three times with ethanol, and dry at 60°C. Introduce amino groups (-NH2) on the surface for DNA covalent ligation.

[0068] 2. The synthesized first anchoring strand 13 (5'-NH2-AAAAAAAAA-3') was dissolved in PBS (pH 7.4), and the second anchoring strand 11 and the fluorescence quencher molecule (5'-NH2-TTTTTTTTT-BHQ2-3') were dissolved in PBS to a final concentration of 100 μM. Aminated diamond particles were dispersed in PBS containing EDC (50 mM) and NHS (25 mM), and the carboxyl groups were activated at room temperature for 30 minutes. The first anchoring strand 13 and the second anchoring strand 11 (molar ratio 1:20) were added, and the reaction was carried out at 37°C with shaking for 12 hours. Unbound DNA was removed by centrifugation, and the sample was washed three times with TE buffer. The quenching efficiency of BHQ2 was observed using a fluorescence microscope (excitation at 532 nm, detection of NV center fluorescence intensity).

[0069] 3. Aptamer 14 (5'-TTTTTTTTTT-aptamer sequence-GGGGGGGGG-3') was diluted to 10 μM with hybridization buffer. Diamond particles were mixed with the aptamer and incubated at 37°C with shaking for 2 hours. Signal fragment 18 (5'-GGGGGGGG-drug resistance gene 17 sequence-CCCCCCCCC-3') was dissolved in hybridization buffer (50 mM NaCl, 10 mM Tris-HCl, pH 8.0). Diamond particles modified with aptamer 14 were mixed with signal fragment 18 (10 μM) and incubated at 65°C for 1 hour, then slowly cooled to room temperature. Unbound aptamers were removed by centrifugation, retaining the hairpin complex.

[0070] 4. Disperse the probe in PBS containing 0.1% BSA, block at 37°C for 1 hour, and then centrifuge and wash.

[0071] Fluorescence performance testing included quenching efficiency testing, measuring the signal intensity ratio of the probe in the uncut state (fluorescence suppressed by BHQ2) to that after Cas12a cutting (fluorescence recovery). Photostability testing involved continuous laser irradiation (532nm, 1mW) for 1 hour, with fluorescence decay curves recorded.

[0072] The experimental results are as follows: Comparing the quenching efficiency of diamond particles in the uncut state and after Cas12a reverse cutting, it can be seen that the quenching efficiency of diamond particles with added activated Cas12a is greatly reduced. The quenching efficiency = (fluorescence intensity after cutting - fluorescence intensity before cutting) / fluorescence intensity after cutting × 100%, and reaches its lowest point after the Cas12a concentration reaches 1 mg / ml, as shown in Table 1 below:

[0073] Table 1. Quenching Efficiency Test Results

[0074]

[0075] The photostability test is as follows: Figure 4 As shown, the fluorescence decay curve of the modified diamond particles after excitation is almost a straight line, indicating that they still have good photostability.

[0076] II. Experimental steps for detecting antibiotic residue 15 using aptamer fluorescent probe 8:

[0077] 1. Equipment and reagents: Microfluidic chip 1 preloaded with aptamer fluorescent probe 8 (structure as shown in the attached diagram) Figure 1 , Figure 2 Robotic arm, fluid controller, and fluorescence detector. Sample extraction solution (containing lysate of antibiotic residue 15 or drug resistance gene 17) and enzyme reaction solution (containing CRISPR-Cas12a 20, crRNA19, and buffer).

[0078] 2. Control Setup: Positive Control: Samples with known concentrations of antibiotics (e.g., tetracycline 10 ppb) and resistance gene 17 (e.g., tetA gene) added. Negative Control: Blank samples without antibiotics and resistance gene 17. False Negative Control: Samples with only resistance gene 17 added.

[0079] 3. The robotic arm automatically grasps the microfluidic chip 1 and positions it on the detection platform, fixing it via vacuum adsorption. The fluid controller dispenses 200 μL of sample extract and 200 μL of enzyme reaction solution into the chip through the first inlet 2 (sample chamber 5) and the second inlet 4 (enzyme chamber 9). The fluid controller is set to a flow rate of 10 μL / min to ensure that the two liquids flow synchronously into the reaction chamber 7 through the delay channel 6 (zigzag microchannel) (taking approximately 10 minutes).

[0080] 4. Antibiotic residues 15 (such as tetracycline) in the sample extract bind to the aptamer, the aptamer hairpin structure unfolds, and the signal chain 16 (containing the complementary sequence of crRNA19) is released from the aptamer and dissociates into multiple signal fragments 18. In the false negative control, the drug resistance gene 17 (such as tetA) binds directly to crRNA19, activating Cas12a.

[0081] 5. The released signal fragment 18 or drug resistance gene 17 pairs complementary with crRNA 19, activating the trans-cleavage activity of Cas12a. The activated Cas12a cleaves the second anchoring strand 11 to remove the fixed fluorescence quenching molecule (BHQ2), restoring the fluorescence of the diamond NV center.

[0082] A single antibiotic molecule can release multiple signal fragments 18 (stoichiometric ratio 1:10). Each signal chain 16 activates Cas12a and cleaves approximately 1000 second anchoring chains 11 (enzyme amplification gain > 10). 4 times).

[0083] 6. The fluorescence detector uses a 532nm laser to excite the NV color center and collect fluorescence signals in the 650-750nm range (1 frame per second, for 30 minutes).

[0084] Antibiotic positive: Fluorescence intensity rises rapidly (peaks within 5 minutes, ΔF > 500%).

[0085] Drug resistance gene 17 positive: fluorescence intensity rises slowly (peaks in 10-15 minutes, ΔF≈150%).

[0086] Double negative: Fluorescence intensity change <3% (background noise level).

[0087] Data analysis was performed, and software algorithms (such as slope analysis) were used to distinguish between antibiotic signals and resistance gene 17 signals. Antibiotic signals had a large initial slope (>50% / min) and a short peak duration. Resistance gene 17 signals had a small initial slope (<20% / min) and a delayed peak. Finally, the positive results were determined by combining the fluorescence intensity threshold (ΔF>100%). The results are shown in Table 2 below:

[0088] Table 2. Fluorescence Detection Data of Antibiotics

[0089]

[0090] During the detection of antibiotic-positive samples, the binding of antibiotics to nucleic acid aptamer 14 releases multiple signal fragments 18, resulting in a greater slope of fluorescence intensity curve than the slope of fluorescence intensity increase caused by the trans-cleavage activity of Cas12a activated by the resistance gene 17. The experimental results of the other parts mentioned above are also consistent with the designed experimental results, indicating that sensitive and rapid fluorescence detection of antibiotics and their resistance gene 17 can be performed, greatly reducing the occurrence of false negatives.

[0091] 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, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0092] The above descriptions are merely embodiments of the present invention. Commonly known structures and characteristics are not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the application date or priority date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can, under the guidance of this application, improve and implement this solution in combination with their own capabilities. Some typical known structures or methods should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A fluorescent probe device with high sensitivity detection, comprising a microfluidic chip (1), characterized in that: The microfluidic chip (1) is internally provided with a closed microflow channel for sample flow, the microflow channel comprises a reaction bin (7), a sample bin (5) and an enzyme liquid bin (9), the sample bin (5) and the enzyme liquid bin (9) are symmetrically distributed on both sides of the reaction bin (7) and are communicated with the reaction bin (7) through the delay channel (6) with a zigzag structure, the sample bin (5) and the enzyme liquid bin (9) are respectively provided with a first filling port (2) for injecting sample extraction liquid and a second filling port (4) for injecting enzyme liquid, the reaction bin (7) is symmetrically provided with a vent hole (3) communicated with the outside, and a plurality of aptamer fluorescent probes (8) are fixedly connected to the bottom wall of the reaction bin (7); The aptamer fluorescent probe (8) comprises a fluorescent particle (10), a plurality of first anchor chains (13) are covalently connected to the fluorescent particle (10), an antibiotic nucleic acid aptamer (14) is hybridized and complementarily connected to the first anchor chain (13), a signal chain (16) is hybridized and complementarily connected to the other end of the antibiotic nucleic acid aptamer (14), and the signal chain (16) is connected by a plurality of signal segments (18) which are complementarily paired in sequence; A plurality of second anchor chains (11) are covalently connected to the fluorescent particle (10), and a fluorescent quenching molecule is modified at the tail end of the second anchor chain (11); the enzyme liquid contains CRISPR-Cas12a (20), crRNA (19) and enzyme cutting buffer components, and the sequence of the crRNA (19) can be complementary to the antibiotic-resistant gene (17) of the antibiotic nucleic acid aptamer (14); The signal segment (18) contains the crRNA (19) complementary sequence of the CRISPR-Cas12a (20), and the length of the signal segment (18) is greater than 18 bp.

2. The fluorescent probe device with high sensitivity detection according to claim 1, characterized in that: The enzyme cutting buffer components include Tris-HCl, KCl, MgCl2, DTT, BSA and glycerol.

3. The fluorescent probe device with high sensitivity detection according to claim 2, characterized in that: The number of fluorescent quenching molecules connected to the same aptamer fluorescent probe (8) is at least 20 times the number of first anchor chains (13).

4. The fluorescent probe device with high sensitivity detection according to claim 3, characterized in that: The sample extraction liquid refers to the lysis extraction liquid obtained by lysing the microorganism in the sample.

5. The fluorescent probe device with high sensitivity detection according to claim 4, characterized in that: Further comprising a mechanical arm for grabbing the microfluidic chip (1) to realize automatic positioning, transfer and recovery, a fluid controller for controlling the liquid filling flow rate and a fluorescence detector for detecting the fluorescence signal emitted by the fluorescent particle (10).

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