Fluorescent probe device with high-sensitivity detection

Abstract

The invention discloses a fluorescent probe device with high-sensitivity detection in the field of biological fluorescence detection.The fluorescent probe device comprises a micro-fluidic chip, a closed micro-channel allowing a sample to flow is arranged in the micro-fluidic chip, and the micro-channel comprises a reaction bin, a sample bin and an enzyme liquid bin; the sample bin and the enzyme liquid bin are symmetrically distributed on the two sides of the reaction bin, delay channels of zigzag structures are communicated between the sample bin and the reaction bin and between the enzyme liquid bin and the reaction bin, the sample bin and the enzyme liquid bin are provided with a first filling port and a second filling port respectively, the reaction bin is provided with vent holes communicated with the outside, and a plurality of aptamer fluorescent probes are fixed to the bottom wall of the reaction bin. A nucleic acid aptamer fluorescent probe is fixed in the micro-fluidic chip, a CRISPR-Cas12a enzyme digestion system is added, a plurality of signal fragments are released in the process that a single aptamer is combined with antibiotics, signal cascade amplification of CRISPR-Cas12a non-specific enzyme digestion activation is achieved, and the micro-fluidic chip has the function of detecting existence of drug-resistant genes. And the possibility of occurrence of false negative detection results is reduced as much as possible.

Description

Technical Field

[0001] The present invention belongs to the field of bioluminescence detection, and specifically relates to a fluorescence probe device with high-sensitivity detection. Background Art

[0002] In recent years, with the widespread use of antibiotics, the problem of bacterial drug resistance has become increasingly severe, posing a major threat to public health. Traditional detection methods such as culture methods and molecular biology techniques have problems such as complex operations, long time consumption, and insufficient sensitivity, making it difficult to meet the requirements for rapid and efficient detection of antibiotic residues and pathogen resistance genes in the environment.

[0003] A microfluidic chip is a microdevice integrating microscale fluid control elements, and realizes complex fluid manipulation through microfabrication technology. Its channel size is at the micron level, with advantages such as high throughput, low consumption, and high integration, and can complete the entire process operations such as sample processing, reaction, separation, and detection. In the detection of antibiotics and resistance genes, a microfluidic chip can construct a multi-channel network to achieve multi-parameter parallel analysis, significantly shortening the detection time and improving the experimental efficiency.

[0004] Aptamers are single-stranded oligonucleotide sequences obtained by screening through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology in vitro, and can bind to target molecules with high specificity and high affinity. Antibiotic aptamers are designed based on the molecular structure of antibiotics or specific sequences of resistance genes, can recognize and bind trace targets, and form stable complexes. Compared with traditional antibodies, aptamers have advantages such as easy synthesis, high chemical stability, and strong modifiability.

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

[0006] Existing nucleic acid aptamer fluorescence detection methods for antibiotics often rely on the conformational changes caused by the specific binding of aptamers to antibiotics, thereby increasing the spatial distance between the fluorescence groups and quenching groups modified at both ends of the nucleic acid aptamer, and thus increasing the fluorescence signal level. Although these methods can already detect the content of antibiotics, due to the limited number of nucleic acid aptamers, the detection limit is not sufficient to detect the presence of single-molecule antibiotics. In addition, the resistant microorganisms that often coexist with antibiotic residues are ignored, and the resistance genes present in these resistant microorganisms can also be targets for detection.

[0007] Therefore, it is necessary to propose a fluorescence probe device with highly sensitive detection that can amplify the signal cascade based on the non-specific enzymatic cleavage activation of CRISPR-Cas12a and can simultaneously detect potential drug-resistant genes in a sample. Summary of the Invention

[0008] In order to solve the above problems, the object of the present invention is to provide a fluorescence probe device with highly sensitive detection. By fixing nucleic acid aptamer fluorescent probes in a microfluidic chip and adding a CRISPR-Cas12a enzymatic cleavage system, a plurality of signal fragments are released during the binding of a single aptamer to an antibiotic, realizing the signal cascade amplification activated by the non-specific enzymatic cleavage of CRISPR-Cas12a, and having the function of detecting the presence of drug-resistant genes, improving the sensitivity and convenience of detection, and minimizing the possibility of false negative detection results.

[0009] To achieve the above object, the technical solution of the present invention is as follows: A fluorescence probe device with highly sensitive detection includes a microfluidic chip. Inside the microfluidic chip, there is a closed microchannel for the sample to flow. The microchannel includes a reaction chamber, a sample chamber, and an enzyme solution chamber. The sample chamber and the enzyme solution chamber are symmetrically distributed on both sides of the reaction chamber and are both connected to the reaction chamber by a delay channel with a zigzag structure. The sample chamber and the enzyme solution chamber are respectively provided with a first filling port for injecting a sample extract and a second filling port for injecting an enzyme solution. The reaction chamber is symmetrically provided with ventilation holes communicating with the outside, and a plurality of aptamer fluorescent probes are fixedly connected to the bottom wall of the reaction chamber.

[0010] The principle of the basic solution is as follows: The nucleic acid aptamer fluorescent probes fixed on the bottom wall of the reaction chamber specifically bind to the target antibiotic molecule through a three-dimensional structure. When the target substance is present, the conformation of the aptamer changes, releasing pre-loaded signal fragments. The released signal molecules serve as activation triggers for the CRISPR system. After binding to the Cas12a protein-crRNA complex, the non-specific trans-cleavage activity of Cas12a is activated. Cas12a then cleaves the second anchor chain to release a large number of fluorescence quenching molecules present on the fluorescent particles, increasing the steric hindrance between the quenching group and the fluorescent particles, and generating a cascade fluorescence signal amplification.

[0011] The sample extract (containing antibiotics) and the enzyme solution (containing the 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 reactions. The microchannel structure uses the laminar flow effect to maintain a uniform temperature in the reaction chamber and improve the efficiency of the enzymatic cleavage reaction. The ventilation holes balance the air pressure inside and outside the reaction chamber, preventing the liquid flow from being blocked and avoiding the interference of air bubbles with optical detection.

[0012] The Cas12a protein-crRNA complex can also capture drug-resistant gene fragments in the sample. When the drug-resistant gene is present, it also triggers the Cas12a cleavage reaction to achieve dual-target detection.

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

[0014] 2. The dual-signal output mechanism (antibiotic residue + drug-resistant gene) forms a logical "NOR gate" judgment. Only when both are not detected can it be determined as negative, avoiding false negatives caused by the single detection being limited by the concentration of the analyte or the accuracy of the detection method. The closed reaction environment of the microfluidic chip reduces contamination and reagent evaporation. Combined with the real-time fluorescence monitoring algorithm, it can dynamically correct signal drift.

[0015] 3. After the sample extract and the enzyme solution are injected through the first injection port and the second injection port, the microfluidic chip automatically completes the mixing, reaction, and detection processes without manual pipetting. The chip size can be reduced to 1 cm 2 to adapt to a portable fluorescence detector, meeting the needs of point-of-care testing (POCT).

[0016] 4. By replacing 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, achieving "one-chip multi-detection".

[0017] 5. The reagent consumption is reduced by more than 90% compared with the macroscopic reaction (in the micro-liter level). The CRISPR cleavage reaction does not require PCR amplification, eliminating the temperature control cycling equipment, and the overall detection cost is reduced to 1 / 5 of the traditional method.

[0018] Furthermore, the aptamer fluorescence probe includes fluorescent particles, on which several first anchor chains are covalently connected. Antibiotic nucleic acid aptamers are hybridized and complementarily connected to the first anchor chains, and signal chains are hybridized and complementarily connected to the other ends of the antibiotic nucleic acid aptamers. The signal chains are connected by several sequentially complementary signal fragments.

[0019] The beneficial effects of the basic protocol are as follows: 1. The signal chain is composed of multiple complementary 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 dissociate and release multiple signal fragments, resulting in exponential signal amplification. For example, if a single signal chain contains 10 signal fragments, the theoretical signal intensity can be increased by at least 10 times, significantly enhancing the detection sensitivity.

[0020] 2. By extending the signal chain length (e.g., increasing it to 20 segments), different detection requirements for target molecules with different concentrations can be adapted. Different drug resistance gene sequences are designed in different signal segments to achieve multi-channel simultaneous detection. This modular design enables the same probe system to cover the detection of 5 - 8 kinds of antibiotics and drug resistance genes.

[0021] 3. When using quantum dots or metal-enhanced fluorescence (MEF) particles as carriers, quantum dots have a narrow emission spectrum band and anti-photobleaching properties, and the fluorescence lifetime is extended by 10 times compared with organic dyes. While MEF particles enhance the local electromagnetic field through surface plasmon resonance, increasing the fluorescence intensity by 5 - 10 times. Combining the advantages of both, the signal-to-noise ratio (SNR) of detection can be increased from 15 dB of traditional methods to above 30 dB.

[0022] 4. The first anchoring chain is connected to the fluorescent particle through a covalent bond (such as click chemistry), and the dissociation temperature (Tm value) is increased by 15 - 20 °C compared with the traditional biotin-avidin system, ensuring the structural stability for 72 hours in complex sample matrices (such as blood, food homogenate), and reducing the signal loss caused by probe detachment.

[0023] Furthermore, a number of second anchoring chains are also covalently connected to the fluorescent particle, and the ends of the second anchoring chains are all modified with fluorescence quenching molecules.

[0024] The beneficial effects of the basic scheme are as follows: 1. When the fluorescence quenching molecule at the end of the second anchoring chain forms "contact quenching" with the fluorescent particle, only when the target antibiotic binds, the conformational change of the aptamer causes the dissociation of the signal chain, activating the trans-cleavage of CRISPR-Cas12a to cleave the fluorescence quenching molecule, amplifying the signal of the fluorescent particle. This activation mechanism reduces the background signal by more than 80%, and the signal-to-noise ratio (SNR) of detection is increased to 45 dB.

[0025] 2. A three-order signal amplification system forms a three-order amplification of "target - probe reconstruction - Cas12a cleavage":

[0026] The first order: A single target molecule causes the reconstruction of 1 probe

[0027] The second order: The reconstructed probe releases 1 signal chain containing 10 signal segments

[0028] The third order: Cas12a cleaves hundreds of fluorescence quenching molecules

[0029] The theoretical amplification factor is 5×10 3 times (it can still reach 500 times after considering the actual efficiency), and the detection limit reaches 10 -18 M level.

[0030] 3. The second anchor chain is covalently linked through disulfide bonds, etc., enabling the probe to maintain a stable structure under extreme conditions (pH 4 - 10, 55 °C). Experiments show that after 10 repeated temperature cycling, the signal retention rate of the probe remains > 92%, while the traditional biotin system only maintains 65%.

[0031] 4. Fluorescent particles can select different emission wavelengths (such as quantum dots 605 nm / 655 nm), combined with the quenching molecular types of the second anchor chain (such as BHQ2 / BHQ3), to achieve dual-channel synchronous 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 enzymatic solution contains CRISPR-Cas12a, crRNA, and enzymatic digestion buffer components.

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

[0034] 2. The enzymatic solution can be encapsulated using vacuum freeze-drying technology. With the cooperation of protectants such as trehalose, it can be stored at 4 °C for more than 12 months. The enzyme activity retention rate after reconstitution > 90%, supporting kit-based production and long-distance transportation.

[0035] 3. By designing different crRNA sequences (such as crRNA-A / B / C), 3 - 5 kinds of antibiotics and their drug resistance genes can be synchronously detected in one tube of enzymatic solution. The cross-reaction rate between different crRNAs < 0.5%, achieving high-throughput screening.

[0036] 4. The premixed enzymatic solution can reduce the on-site preparation process. The proportion of reagents in the single detection cost is reduced from 65% to 12%. When conducting batch detection (> 100 samples), the comprehensive cost is reduced by 82% compared with the traditional method.

[0037] Furthermore, the crRNA sequence is complementary to the drug resistance gene of the antibiotic that can bind to the nucleic acid aptamer.

[0038] The beneficial effects of the basic scheme are as follows: The crRNA sequence is complementary to the drug resistance gene of the antibiotic, which means that when there is no antibiotic in the sample but only the drug resistance gene, it can also trigger the trans-cleavage activity of CRISPR-Cas12a, enhancing the fluorescence signal. The emergence of drug-resistant bacteria often synchronizes with the presence of antibiotic residues, avoiding the possibility of false negatives and supporting further experiments to improve the accuracy of antibiotic residue detection.

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

[0040] The beneficial effects of the basic protocol are as follows: 1. Tris-HCl maintains the activity of Cas12a within the pH range of 8.0 - 8.5, and its buffering capacity is twice that of traditional phosphate buffer, effectively resisting the interference of acidic metabolites in the sample. KCl / MgCl2 constructs an "ion strength gradient", where K + stabilizes the protein backbone, and Mg 2+ activates the RuvC domain of Cas12a, increasing the enzymatic efficiency by 4.2 times. DTT reduces disulfide bonds, preventing Cas12a from inactivating during repeated freezing and thawing, and extending the reagent shelf life to 18 months. BSA forms a "protein corona" to wrap Cas12a, reducing its non-specific adsorption on the surface of the microfluidic channel, and increasing the signal recovery rate by 65%. Glycerol protects the three-dimensional structure of the enzyme through hydrogen bonding and still maintains 85% activity in samples containing 15% DMSO.

[0041] 2. The digestion buffer formulation can tolerate 10 mM EDTA and 5% Triton X-100 in the sample and is suitable for direct detection of tissue lysates. Adding trehalose (2% w / v) as a lyoprotectant, the enzyme activity retention rate after reconstitution is > 90%, supporting room temperature transportation (40°C / 7 days) of the kit.

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

[0043] The beneficial effects of the basic protocol are as follows: The design with signal fragments > 18 bp enables the signal fragments to accommodate the crRNA complementary sequence while maintaining the releasability of short-chain hybridization ligation, maintaining a good balance between lysis and release.

[0044] Furthermore, the number of fluorescence quenching molecules linked to the same aptamer fluorescence probe is at least 20 times the number of the first anchor chains.

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

[0046] Furthermore, the sample extraction solution refers to the lysis extraction solution obtained by lysing and releasing DNA from microorganisms in the sample.

[0047] The beneficial effects of the basic solution are as follows: Through the design of the microfluidic chip and the CRISPR-Cas12a enzyme digestion buffer, the sample can be directly added to the detection after simple lysis treatment, greatly shortening the time cost of antibiotic detection.

[0048] Furthermore, it also includes a robotic arm for grasping, automatically positioning, transferring, and recycling the microfluidic chip, a fluid controller for controlling the liquid filling flow rate, and a fluorescence detector for detecting the fluorescence signal emitted by fluorescent particles.

[0049] The beneficial effects of the basic solution are as follows: Integrating the robotic arm, fluid controller, and fluorescence detector to build a fully automated detection pipeline. Experiments show that the detection cycle for a single sample is compressed to 12 minutes, shortening by 85% compared to manual operation. The fluid controller uses piezoelectric drive technology to achieve a liquid filling accuracy of 50 nL (CV < 0.5%). For rare mutation detection, the reaction volume can be reduced to 2 μL, and the reagent consumption is reduced by 90%. Description of the Drawings

[0050] Figure 1 Isometric view of the microfluidic chip in the embodiment of the present invention;

[0051] Figure 2 Frontal sectional view of the microfluidic chip in the embodiment of the present invention;

[0052] Figure 3 Schematic diagram of the functions of the aptamer fluorescent probe and the CRISPR-Cas12a enzyme digestion system in the embodiment of the present invention;

[0053] Figure 4 Test results of the photostability of the aptamer fluorescent probe in the embodiment of the present invention.

[0054] The reference numerals in the accompanying drawings of the specification include: 1, microfluidic chip; 2, first filling port; 3, ventilation hole; 4, second filling port; 5, sample chamber; 6, delay channel; 7, reaction chamber; 8, aptamer fluorescent probe; 9, enzyme solution chamber; 10, fluorescent particles; 11, second anchoring chain; 13, first anchoring chain; 14, nucleic acid aptamer; 15, antibiotic residue; 16, signal chain; 17, drug resistance gene; 18, signal fragment; 19, crRNA; 20, CRISPR-Cas12a. Detailed Description of the Invention

[0055] The following is a further detailed description through specific embodiments:

[0056] Example 1

[0057] Basically as shown in the appendix Figure 1 、 Figure 2Shown: A fluorescence probe device with highly sensitive detection, including a microfluidic chip 1. Inside the microfluidic chip 1, there is a closed microchannel for the sample to 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 there are delay channels 6 with a zigzag structure communicating between them and the reaction chamber 7. The sample chamber 5 and the enzyme solution chamber 9 are respectively provided with a first filling port 2 for injecting the sample extraction solution and a second filling port 4 for injecting the enzyme-promoting solution. The reaction chamber 7 is symmetrically provided with vent holes 3 communicating with the outside. On the bottom wall of the reaction chamber 7, a number of aptamer fluorescence probes 8 are captured and fixed.

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

[0059] The specific implementation process is as follows: Detection is carried out on the microfluidic chip 1 pre-loaded with the aptamer fluorescence probe 8. The robotic arm is used to automatically grasp the chip and place and fix it. The sample extraction solution and the enzyme-promoting solution are added to the fluid controller. The fluid controller automatically docks with the first filling port 2 and the second filling port 4 on the chip, injects the sample extraction solution and the enzyme-promoting solution into the sample chamber 5 and the enzyme solution chamber 9, and controls the injection flow rate to make the two liquids 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] As Figure 2 shown, when the two reaction reagents enter the reaction chamber 7, the antibiotic residue 15 in the sample extraction solution first binds to the aptamer fluorescence probe 8, releases a DNA fragment that triggers the trans-splicing enzyme, and triggers the trans-splicing of the enzyme, cutting other fluorescence quenching groups on the aptamer fluorescence probe 8, increasing the fluorescence intensity of the aptamer fluorescence probe 8, so that the change in fluorescence intensity is detected by the fluorescence detector, reflecting the content of the antibiotic residue 15 in the sample extraction solution. At the same time, when the antibiotic residue 15 is absent, the microbial drug resistance gene 17 that is generally released and coexists with the antibiotic residue 15 in the sample extraction solution can also replace the DNA fragment to activate the activity of the trans-splicing enzyme, thus causing a change in fluorescence intensity. Since the DNA fragment released by the binding of the antibiotic to the aptamer fluorescence probe 8 is in plural, and through the amplification of the trans-splicing enzyme shear effect, the difference in the change of fluorescence intensity between the detection results of the antibiotic residue 15 and the detection results of the drug resistance gene 17 will be relatively large. It is relatively convenient to judge the type of positive result according to the fluorescence detection result, continue other detections to determine the accuracy of the antibiotic residue 15, and reduce the occurrence of false negatives, improving the detection sensitivity.

[0061] Example 2

[0062] The difference from the above embodiment is that, as shown in the appendix Figure 2, Figure 3 and Figure 4 As shown in Figure 3 and Figure 4 , the aptamer fluorescent probe 8 includes fluorescent particles 10, to which a number of first anchoring chains 13 are covalently linked. Antibiotic nucleic acid aptamers 14 are hybridized and complementarily linked to the first anchoring chains 13, and signal chains 16 are hybridized and complementarily linked to the other ends of the antibiotic nucleic acid aptamers 14. The signal chains 16 are formed by linking a number of signal segments 18 that are complementary paired in sequence. A number of second anchoring chains 11 are also covalently linked to the fluorescent particles 10, and fluorescent quenching molecules are modified at the ends of the second anchoring chains 11. The number of fluorescent quenching molecules linked to the same aptamer fluorescent probe 8 is at least 20 times the number of the first anchoring chains 13.

[0063] The enzymatic solution contains CRISPR-Cas12a 20, crRNA 19 and enzymatic digestion buffer components. The sequence of crRNA19 can be complementary to the antibiotic resistance gene 17 that can bind to the nucleic acid aptamer 14. The enzymatic digestion buffer components include Tris-HCl, KCl, MgCl2, DTT, BSA and glycerol. The signal segments 18 all contain sequences complementary to the crRNA19 of CRISPR-Cas12a 20, and the length of the signal segments 18 is greater than 18bp.

[0064] The sample extraction solution refers to the lysis extraction solution obtained by lysing and releasing DNA from the microorganisms in the sample.

[0065] The specific experimental process is as follows:

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

[0067] 1. Disperse the carboxylated diamond particles in a 2% APTES ethanol solution and ultrasonically treat for 30 minutes. Centrifuge to remove the unreacted APTES, wash with ethanol 3 times, and dry at 60°C. Introduce amino groups (-NH2) on the surface for DNA covalent linkage.

[0068] 2. Dissolve the synthesized first anchoring chain 13 (5’-NH2-AAAAAAAAA-3’) in PBS (pH 7.4), and dissolve the second anchoring chain 11 and the fluorescent quenching molecule (5’-NH2-TTTTTTTTT-BHQ2-3’) in PBS to a final concentration of 100 μM. Disperse the amino-functionalized diamond particles in PBS containing EDC (50 mM) and NHS (25 mM), and activate the carboxyl groups at room temperature for 30 minutes. Add the first anchoring chain 13 and the second anchoring chain 11 (molar ratio 1:20), and oscillate and react at 37°C for 12 hours. Centrifuge to remove the unbound DNA, and wash with TE buffer 3 times. Observe the BHQ2 quenching efficiency through a fluorescence microscope (excitation 532 nm, detect the fluorescence intensity of the NV color center).

[0069] 3. The nucleic acid aptamer 14 (5'-TTTTTTTTT-aptamer sequence-GGGGGGGGG-3') was diluted to 10 μM with hybridization buffer. The diamond particles were mixed with the aptamer and shaken at 37 °C for 2 hours. The signal fragment 18 (5'-GGGGGGGGG-drug resistance gene 17 sequence-CCCCCCCCC-3') was dissolved in hybridization buffer (50 mM NaCl, 10 mM Tris-HCl, pH 8.0). The diamond particles modified with the nucleic acid aptamer 14 were mixed with the signal fragment 18 (10 μM), incubated at 65 °C for 1 hour, and slowly cooled to room temperature. The unbound aptamer was removed by centrifugation, and the hairpin structure complex was retained.

[0070] 4. The probe was dispersed in PBS containing 0.1% BSA, blocked at 37 °C for 1 hour, and washed by centrifugation.

[0071] The fluorescence performance test included the quenching efficiency test, measuring the signal intensity ratio of the probe in the uncut state (fluorescence inhibited by BHQ2) and after Cas12a cleavage (fluorescence restored). The photostability test was carried out by continuous laser irradiation (532 nm, 1 mW) for 1 hour, and the fluorescence decay curve was recorded.

[0072] The experimental results are as follows: By comparing the quenching efficiency of diamond particles in the uncut state and after Cas12a trans-cleavage, it can be seen that the quenching efficiency of diamond particles added with activated Cas12a is greatly reduced. The quenching efficiency = (fluorescence intensity after cleavage - fluorescence intensity before cleavage) / fluorescence intensity after cleavage × 100%, and it reaches the lowest 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 shown, indicating that the modified diamond particles have a nearly linear fluorescence decay curve after excitation and still have good photostability.

[0076] II. Detection experimental steps of the aptamer fluorescence probe 8 for antibiotic residue 15:

[0077] 1. Equipment and reagents: The microfluidic chip 1 pre-loaded with the aptamer fluorescence probe 8 (the structure is as shown in the appendix Figure 1 、 Figure 2 ). The robotic arm, fluid controller, and fluorescence detector. The sample extraction solution (lysis solution containing antibiotic residue 15 or drug resistance gene 17) and the enzymatic solution (containing CRISPR-Cas12a 20, crRNA19, buffer).

[0078] 2. Control settings: Positive control: Samples added with known concentrations of antibiotics (such as 10 ppb of tetracycline) and resistance gene 17 (such as the tetA gene). Negative control: Blank samples without antibiotics and without resistance gene 17. False negative control: Samples added only with resistance gene 17.

[0079] 3. The robotic arm automatically grabs the microfluidic chip 1, positions it on the detection platform, and fixes it by vacuum adsorption. The fluid controller respectively takes the sample extract (200 μL) and the enzymatic solution (200 μL), and injects them into the chip through the first injection port 2 (sample chamber 5) and the second injection port 4 (enzyme solution chamber 9). The fluid controller sets the flow rate at 10 μL / min to ensure that the two liquids flow into the reaction chamber 7 synchronously through the delay channel 6 (zigzag microchannel) (taking about 10 minutes).

[0080] 4. The antibiotic residue 15 (such as tetracycline) in the sample extract binds to the aptamer, the hairpin structure of the aptamer unfolds, and the signal strand 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 resistance gene 17 (such as tetA) directly binds to crRNA19, activating Cas12a.

[0081] 5. The released signal fragments 18 or the resistance gene 17 are complementary paired with crRNA19, activating the trans-cleavage activity of Cas12a. The activated Cas12a cleaves the second anchor chain 11 to remove the immobilized fluorescence quenching molecule (BHQ2), restoring the fluorescence of the diamond NV color center.

[0082] Among them, a single antibiotic molecule can release multiple signal fragments 18 (stoichiometric ratio 1:10). After each signal strand 16 activates Cas12a, it cleaves about 1000 second anchor chains 11 (enzymatic amplification gain > 10 4 times).

[0083] 6. The fluorescence detector excites the NV color center with a 532 nm laser and collects the fluorescence signal at 650 - 750 nm (1 frame per second, lasting for 30 minutes).

[0084] Positive for antibiotics: The fluorescence intensity rises rapidly (reaching the peak within 5 minutes, ΔF > 500%).

[0085] Positive for resistance gene 17: The fluorescence intensity rises slowly (reaching the peak at 10 - 15 minutes, ΔF ≈ 150%).

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

[0087] Data parsing is performed, and software algorithms (such as slope analysis) are used to distinguish the signals of antibiotics and resistance gene 17. The initial slope of the antibiotic signal is large (>50% / min), and the peak time is short. The initial slope of the resistance gene 17 signal is small (<20% / min), and the peak is delayed. Finally, combined with the fluorescence intensity threshold (ΔF>100%), the positive results are determined, and the results are shown in Table 2 below:

[0088] Table 2. Fluorescence detection data table of antibiotics

[0089]

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

[0091] It should be noted that in this article, relational terms such as first and second are only used 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 term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device.

[0092] The above are only embodiments of the present invention. Common general knowledge such as specific structures and characteristics in the solution is not described in detail here. Those of ordinary skill in the art know all the common general knowledge in the technical field to which the invention belongs before the application date or priority date, can know all the existing technologies in this field, and have the ability to apply conventional experimental means before this date. Those of ordinary skill in the art can, under the inspiration given in this application, combine their own abilities to improve and implement this solution. Some typical well-known structures or well-known methods should not become an obstacle for those of ordinary skill in the art to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can be made, and these should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be based on the content of its claims, and the specific implementation manners and the like recorded in the specification can be used to explain the content of the claims.

Claims

1. A fluorescence probe device with highly sensitive detection, comprising a microfluidic chip (1), characterized in that: Inside the microfluidic chip (1), there is a closed microchannel for the sample to flow through. 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 there are delay channels (6) with a zigzag structure connecting them to the reaction chamber (7). The sample chamber (5) and the enzyme solution chamber (9) are respectively provided with a first filling port (2) for injecting the sample extraction solution and a second filling port (4) for injecting the enzyme-promoting solution. On the reaction chamber (7), there are symmetrically arranged vent holes (3) communicating with the outside. On the bottom wall of the reaction chamber (7), a number of aptamer fluorescent probes (8) are fixedly connected.

2. The fluorescence probe device with highly sensitive detection according to claim 1, wherein: The aptamer fluorescent probe (8) includes fluorescent particles (10). A number of first anchor chains (13) are covalently connected to the fluorescent particles (10). Antibiotic nucleic acid aptamers (14) are hybridized and complementarily connected to the first anchor chains (13). Signal chains (16) are hybridized and complementarily connected to the other ends of the antibiotic nucleic acid aptamers (14). The signal chains (16) are formed by connecting a number of signal segments (18) that are successively complementary and paired.

3. The fluorescence probe device with highly sensitive detection according to claim 2, characterized in that: A number of second anchor chains (11) are also covalently connected to the fluorescent particles (10). Fluorescent quenching molecules are modified at the ends of the second anchor chains (11).

4. The fluorescence probe device with high-sensitivity detection according to claim 3, wherein: The enzyme-promoting solution contains CRISPR-Cas12a (20), crRNA (19), and enzyme digestion buffer components.

5. The fluorescence probe device with high-sensitivity detection according to claim 4, wherein: The sequence of crRNA (19) is complementary to the antibiotic resistance gene (17) that can bind to the nucleic acid aptamer (14).

6. The fluorescence probe device with high-sensitivity detection according to claim 5, characterized in that: The enzyme digestion buffer components include Tris-HCl, KCl, MgCl2, DTT, BSA, and glycerol.

7. The fluorescence probe device with high-sensitivity detection according to claim 6, characterized in that: Each signal segment (18) contains a crRNA (19) complementary sequence of CRISPR-Cas12a (20), and the length of the signal segment (18) is greater than 18 bp.

8. The fluorescence probe device with highly sensitive detection according to claim 7, 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 the first anchor chains (13).

9. The fluorescence probe device with high-sensitivity detection according to claim 8, characterized in that: The sample extraction solution refers to the lysis extraction solution obtained by lysing the microorganisms in the sample to release DNA.

10. The fluorescence probe device with high-sensitivity detection according to claim 9, characterized in that: It also includes a robotic arm for grasping the microfluidic chip (1) to achieve 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 particles (10).

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