A pressure-driven multi-channel microfluidic chip based on photonic crystal signal enhancement and its detection method

Abstract

The present invention relates to the field of biological detection technology, specifically a pressure-driven multi-channel microfluidic chip based on photonic crystal signal enhancement and a detection method thereof, the present invention integrates a pressure-driven eight-channel microfluidic chip with a CRISPR / Cas12a system, and realizes simultaneous ultra-sensitive and quantitative detection of six fungal toxins, and uses quantum dots (QDs) as FRET donors through fluorescence resonance energy transfer FRET technology, and a fluorescence enhancement technology based on photonic crystals (PC) is used to further improve the detection sensitivity. The photonic crystal improves the collection efficiency of the emitted fluorescent photons by periodically and orderly arranging the photon bands of the colloidal crystals to reflect the fluorescence overlapping with the diffraction wavelength of the photonic crystals.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to a pressure-driven multi-channel microfluidic chip based on photonic crystal signal enhancement and a detection method thereof. Background Art

[0002] Mycotoxins are considered to be one of the most important causes of foodborne diseases and have received long-term attention from the world. Among them, aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZEN), fumonisin B1 (FB1), T-2 mycotoxin (T-2), and deoxynivalenol (DON) are the most common toxins. With the improvement of public health awareness, some organizations have set maximum residue limits (MRLS) for mycotoxins in food to avoid high-risk behaviors. However, most mycotoxins exist in trace amounts, which poses a serious challenge to food safety. In addition, the synergistic or antagonistic effects of multiple mycotoxins coexisting in the same food increase their toxicity to humans. Faced with the development trend of globalization of logistics, traditional mycotoxin analysis methods such as high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS) are limited in practical application due to high cost, time-consuming procedures, and the requirement of professional skills. Therefore, it is urgent to establish a sensitive, rapid, and accurate high-throughput detection method for multiple mycotoxins to meet the current new needs.

[0003] So far, some biosensors have been reported for the in situ detection of mycotoxins. The specific molecular recognition elements are mainly antibodies and aptamers. Compared with antibodies, aptamers have the advantages of easy synthesis, low cost and good thermal stability, making them ideal recognition elements for constructing biosensors. A variety of mycotoxins aptamers have been screened through systematic evolution of exponential enrichment ligands. Due to the stable signal output and programmability of aptamers, converting aptamers into fluorescent signals has a wider application prospect in biosensors. However, it is mainly used in competitive immunoassays for mycotoxins, and therefore, the output fluorescence intensity is negatively correlated with the analyte concentration, or positively correlated through secondary signal processing, which sacrifices the detection accuracy.

[0004] The CRISPR / Cas system makes it possible to establish a direct linear positive correlation between mycotoxins and fluorescent signals. The CRISPR-Cas system can achieve binding with matching RNA or DNA by designing specific guide RNA chains, making it an ideal molecular detection tool based on this feature. The CRISPR / Cas12a system is based on the Cas12a enzyme. When the functional DNA encounters the target analyte, Cas12a indiscriminately cuts the surrounding single-stranded DNA (ssDNA) probe. In addition, Cas12a has a low intrinsic tolerance to crRNA target DNA mismatches and requires a high degree of complementarity. The sensor is not restricted by the interface space when capturing the target molecule, which significantly improves the recognition efficiency. However, immune recognition on the solid phase interface is limited by the specific surface area, the slow diffusion process between the target molecule and the interface, and the nonspecific interaction between the target molecule and the solid phase interface. The nonspecific side branch cutting activity of the Cas12a enzyme limits its application in multi-target detection systems. Therefore, in order to expand the throughput detection strategy of the CRISPR / Cas12a system, combining it with a multiplexed microfluidic chip is a feasible solution. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the nonspecific side branch cleavage activity of Cas12a enzyme limits its application in multi-target detection system, resulting in low detection flux of CRISPR / Cas12a system; at the same time, the signal intensity of the label related to the molecular recognition event in the CRISPR / Cas12a system is low, resulting in low detection sensitivity.

[0006] In order to achieve high-throughput ultrasensitive detection of fungal toxins, the present invention constructs a functional DNA-guided transition state CRISPR / Cas12a microfluidic biosensor (FTMB). The signal transduction CRISPR / Cas12a strategy in FTMB uses DNA sequences and activators with specific recognition functions to form a trigger switch, and at the same time, by adjusting the composition ratio of crRNA and activators, a transition state CRISPR / Cas12a system is constructed to achieve high response to low concentrations of target fungal toxins. On the other hand, the signal enhancement of FTMB effectively integrates the signal output of quantum dots (QDs) and the fluorescence enhancement effect of photonic crystals (PC).

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The first technical solution of the present invention is:

[0009] A CRISPR / Cas12a-based mycotoxin detection system, the mycotoxin detection system is used to detect any one or a combination of aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZEN), fumonisin B1 (FB1), T-2 mycotoxin (T-2) or deoxynivalenol (DON);

[0010] The detection system includes the following substances for each mycotoxin:

[0011] 1) A complex consisting of aptamer-1, activator, and aptamer-2;

[0012] 2) crRNA;

[0013] 3) Probe;

[0014] Among them, for AFB1, the sequence of crRNA is: SEQ ID NO: 1, and the sequence of aptamer-1 is: SEQ ID NO:

[0015] 2, the sequence of the activator is: SEQ ID NO: 3, the sequence of aptamer-2 is: SEQ ID NO: 4;

[0016] For OTA, the sequence of crRNA is: SEQ ID NO: 5, the sequence of aptamer-1 is: SEQ ID NO: 6, the sequence of the activator is: SEQ ID NO: 7, and the sequence of aptamer-2 is: SEQ ID NO: 8;

[0017] For ZEN, the sequence of crRNA is: SEQ ID NO: 9, the sequence of aptamer-1 is: SEQ ID NO: 10, the sequence of the activator is: SEQ ID NO: 11, and the sequence of aptamer-2 is: SEQ ID NO: 12;

[0018] For FB1, the sequence of crRNA is: SEQ ID NO: 13, the sequence of aptamer-1 is: SEQ ID NO: 14, the sequence of the activator is: SEQ ID NO: 15, and the sequence of aptamer-2 is: SEQ ID NO: 16;

[0019] For T-2, the sequence of crRNA is: SEQ ID NO: 17, the sequence of aptamer-1 is: SEQ ID NO: 18, the sequence of the activator is: SEQ ID NO: 19, and the sequence of aptamer-2 is: SEQ ID NO: 20;

[0020] For DON, the sequence of crRNA is: SEQ ID NO: 21, the sequence of aptamer-1 is: SEQ ID NO: 22, the sequence of the activator is: SEQ ID NO: 23, and the sequence of aptamer-2 is: SEQ ID NO: 24.

[0021] Preferably, both ends of the probe are connected by a quencher group and a fluorescent group, respectively.

[0022] Preferably, the probe sequence is shown in SEQ ID NO:26.

[0023] Preferably, the probe is connected to the surface of quantum dots; the quantum dots are CdSe / ZnS quantum dots.

[0024] Preferably, the preparation method of the probe is:

[0025] S1: Weigh EDC and NHS and dissolve them in PB buffer, add quantum dots, activate, centrifuge, wash, and resuspend to obtain a suspension;

[0026] S2: adding ssDNA reporter molecules to the suspension, and gradient mixing under ultrasonic conditions to a final concentration to obtain a mixed solution;

[0027] S3: adding additional EDC to the mixed solution, incubating, centrifuging and collecting the product, which is the QD probe.

[0028] Preferably, the concentrations of EDC and NHS are 1-5 mg / ml; the activation time is 10-50 min; the washing and resuspension solution is PBS buffer; the gradient concentration of the gradient mixing is 150-250 nM, and the final concentration is 0.5-1.5 μM; the concentration of the additional EDC is 10-30 mg / mL; and the incubation time is 1-3 h.

[0029] Preferably, the complex is composed of aptamer-1, activator, and aptamer-2 in a molar ratio of 1-5:1:1-5, preferably 2:1:2.

[0030] Preferably, the complex is prepared by mixing the aptamer-1, activator and aptamer-2 of each fungal toxin, heating the mixture, and then naturally cooling it to room temperature.

[0031] Preferably, the detection system also includes a Cas12a enzyme.

[0032] The second technical solution of the present invention is:

[0033] A method for detecting mycotoxins comprises the following steps:

[0034] Step 1, dispersing the solution of the complex for each fungal toxin, the crRNA solution, the probe solution, and the Cas12a enzyme solution in ultrapure water, and lyophilizing to obtain microspheres;

[0035] Step 2, the microspheres react with the standard solution or the sample solution, and the fluorescence intensity released by the probe is recorded;

[0036] Step 3, obtaining a standard curve based on the standard solution, and calculating the concentration of the fungal toxin in the sample solution.

[0037] In step 1, the concentration of the complex solution is 4-20nM, the concentration of the crRNA solution is 0.5-5μM, the concentration of the probe solution is 0.5-5μM, and the concentration of the Cas12a enzyme solution is 1-20μM; the volume ratio of each solution is 1.5:0.5-5:0.5-5:0.5-4, respectively.

[0038] In the step 2, the fluorescence intensity is also amplified by polystyrene (PS) microspheres, and the diameter of the polystyrene (PS) microspheres is in the range of 250-550 nm.

[0039] The third technical solution of the present invention is:

[0040] A microfluidic chip, comprising:

[0041] The sample loading port is located at the upper layer;

[0042] At least one reaction chamber is located in the middle layer, and the reaction chamber is connected to the sample injection port through a flow channel; a detection reagent is pre-installed in the reaction chamber, and the detection reagent responds to the content of the substance to be detected based on fluorescence;

[0043] A light-transmitting lower layer, and a signal amplification layer is provided between the lower layer and the reaction chamber;

[0044] An exhaust channel is also provided at the bottom of the reaction chamber, and the diameter of the exhaust channel is smaller than the diameter of the reaction chamber. When the injection pressure of the sample solution is less than a threshold value, the sample solution cannot be discharged from the exhaust channel due to capillary force, and when the injection pressure of the sample solution is greater than the threshold value, the sample solution can be discharged.

[0045] The outlet end of the exhaust passage is located at the upper layer.

[0046] The signal amplification layer is composed of carboxyl-modified PS microspheres, which are prepared into a suspension, dripped into the surface of the bottom layer and left to stand to form a film.

[0047] The conditions for static film formation are: temperature of 30-60℃ and time of 10-15h.

[0048] There are multiple reaction chambers to form a reaction chamber array.

[0049] The detection reagent is the microsphere obtained after freeze-drying.

[0050] The fourth technical solution of the present invention is:

[0051] The above-mentioned detection method of the microfluidic chip comprises the following steps:

[0052] Step a, adding the sample solution from the sample adding port, and entering the reaction chamber through the flow channel, and reacting with the detection reagent to generate fluorescence;

[0053] Step b, after the fluorescence is amplified by the signal amplification layer, the fluorescence is collected from the lower layer, and the concentration of the target substance in the sample solution is measured according to the light intensity;

[0054] Step c: after the detection is completed, the sample addition port is pressurized to discharge the reaction system.

[0055] In the step a, after the sample solution is added, mineral oil is continuously added to seal the reaction chamber.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] (1) In the present invention, a DNA-guided transition state CRISPR / Cas12a system based on fungal toxin aptamers is used for signal transduction, and the universal quantum dots of the CRISPR / Cas12a system are enhanced with PC for signal enhancement, so that the fungal toxin recognition event can be positively correlated and converted into a fluorescent signal. On this basis, the pressure-driven eight-channel microfluidic chip is further integrated with them to achieve simultaneous ultrasensitive and quantitative detection of six fungal toxins. The high signal transduction and enhancement properties of this functional DNA-guided transition state CRISPR / Cas12a microfluidic biosensor (named FTMB) enable it to more accurately detect fungal toxins in complex biological backgrounds.

[0058] (2) The signal intensity of the label associated with the molecular recognition event is enhanced, thereby improving the sensitivity. The present invention uses quantum dots (QDs) as FRET donors in the fluorescence resonance energy transfer (FRET) technology; and the fluorescence enhancement technology based on photonic crystals (PC) is used to further improve the detection sensitivity. Photonic crystals improve the collection efficiency of emitted fluorescent photons by forming photon bands through the periodic and orderly arrangement of colloidal crystals to reflect fluorescence that overlaps with the diffraction wavelength of the photonic crystals. The adjustability of the PC Bragg reflection effect enables it to match fluorescence from different labeling material bands.

[0059] (3) The construction of universal quantum dots for the CRISPR / Cas12a system and PC membranes matching the photonic band gap produced a significant signal enhancement of 45.6 times. FTMB exhibited a wide analytical range, low detection limit, short detection cycle, high specificity, good precision, and satisfactory actual sample analysis capabilities, providing a new and reliable solution for the rapid detection of a variety of small molecules in the fields of clinical diagnosis and food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 : Schematic diagram of the present invention.

[0061] Figure 2 :The feasibility verification process of CRISPR / Cas12a signal transduction and PCs signal enhancement mechanism. (A) Polyacrylamide gel electrophoresis; (B) Real-time fluorescence curves of the CRISPR / Cas12a system activated by trigger switches corresponding to six fungal toxins; (C) The effect of different ratios of aptamers and activators on the locking effect; (D) Evaluation of different concentrations of reporter molecules on quenching quantum dot fluorescence; (E) TEM of PC composed of PS microspheres; (F) Fluorescence images of PDMS and PC loaded with quantum dots and FAM; (G) Enhancement factors of PC with different particle sizes for quantum dots and FAM.

[0062] Figure 3 :Establishment of a transition-state CRISPR / Cas12a system for mycotoxin detection. (A) Cas12a enzyme: (1) conversion between different forms, (2) different reaction states (closed, transition and open states), (3) effect of different crRNAs on cleavage efficiency; (B) effect of activation complex concentration on trans-cleavage activity of Cas12a enzyme; (C) effect of activator-crRNA ratio on trans-cleavage activity of Cas12a enzyme; (D) effect of activator concentration on trans-cleavage activity of Cas12a enzyme; (E) performance of the three states of the CRISPR / Cas12a system when incubated with on-target and off-target mycotoxins.

[0063] Figure 4 : Cleavage kinetics of the CRISPR / Cas12a system in three states designed for six fungal toxins.

[0064] Figure 5 :The composition and entire operation process of the pressure-driven microfluidic chip, (A) the structural layer composition of the chip; (B) the initial and final states of the fluid test in the chip (sample solution with added red dye); (C) the entire process of the sample flowing in the chip.

[0065] Figure 6 : Optimization of QD probe concentration.

[0066] Figure 7 : Optimization of crRNA concentration.

[0067] Figure 8 : Optimization of trigger switch concentration.

[0068] Fig. 9 : Performance analysis of FTMB. (A) Standard curves of six mycotoxins at different concentrations and their corresponding fluorescence images; (B) Specificity evaluation of single and multiple mycotoxins.

[0069] Fig.10 : Profile of the flow process of the sample solution in the chip. DETAILED DESCRIPTION

[0070] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0071] like Figure 1 As shown, the FTMB procedure consists of four parts: (1) automatic sample distribution through the fractal branching eight channels of the microfluidic chip; (2) programmed separation of activators and functional DNA, regarded as a trigger switch, in response to the induction of the target fungal toxin; (3) controlled release of fluorescence by quantum dots quenched by BHQ1 containing reporter genes through the side branch cleavage activity of the activated Cas12a enzyme; and (4) signal amplification and recording in a reaction chamber with PC.

[0072] The working principle of the detection system of the present invention: (A) The signal transduction and recognition enhancement of the target fungal toxin by the transition state CRISPR / Cas12a system guided by functional DNA: the target fungal toxin induces the separation of the corresponding trigger switch; the functional DNA is combined with the target fungal toxin to release the activator of the CRISPR / Cas12a system; the released activator binds to the crRNA to promote the transition of the CRISPR / Cas12a system to the activated state; the surface of the CdSe / ZnS quantum dots is modified with a reporter molecule with a quenching group BHQ1 as a probe of the CRISPR / Cas12a system; the activated CRISPR / Cas12a system shears the reporter molecule on the surface of the quantum dots to restore fluorescence; the PC under the reaction chamber enhances the fluorescence signal, and the fluorescence signal is finally collected and read. (B) The operation process of the pressure-driven eight-channel microfluidic chip: the sample solution enters the chip through a manual syringe and is evenly distributed; the reaction chamber is sealed with mineral oil to avoid cross contamination; the CRISPR / Cas12a system reacts at room temperature, and the generated fluorescence signal is collected by a smartphone; the color model is analyzed by the picture taken by the smartphone to obtain the concentration of six fungal toxins.

[0073] The fungal toxins detected in the following examples include AFB1, OTA, ZEN, FB1, T-2 and DON. In addition, carboxyl PEG-modified CdSe / ZnS quantum dots (emission wavelength of 525 nm) and PS microspheres with sizes of 250 nm, 350 nm, 450 nm and 550 nm were used.

[0074] Design and preparation of trigger switches: According to the six toxins (AFB1, OTA, ZEN, FB1, T-2, and DON), the corresponding sequences of modified aptamers, Cas12a-guide RNA (crRNA), and activators were designed as shown in Table 1.

[0075] Table 1 Nucleotide sequences

[0076]

[0077]

[0078] To ensure that all types of activators were completely locked, the molar ratio of aptamer-1, activator and aptamer-2 for each fungal toxin was set to 2:1:2. The mixture was heated at 90°C for 10 min and then naturally cooled to room temperature to obtain a trigger switch. The final concentration of the trigger switch for the fungal toxin was optimized in the range of 4-20 nM.

[0079] Synthesis of QD probes in CRISPR / Cas12a system:

[0080] The QD probe was prepared by an EDC and NHS-mediated amidation reaction. Specifically, 1.5 mg of EDC and NHS were dissolved in 1.0 mL of PB buffer, and 3 mg of CdSe / ZnS quantum dots were added and reacted in a vertical mixer at room temperature for 30 minutes to activate the carboxyl groups on the QDs. After activation, the solution was transferred to a 1.5 mL EP tube and centrifuged at 12000 rpm for 5 minutes. The activated QD was washed 3 times with PBS buffer to remove unreacted EDC and NHS, and then resuspended in PBS buffer solution. Then, ssDNA reporter molecules with quenching groups were added and ultrasonically mixed to a final concentration of 1 μM with a gradient of 200 nM. Subsequently, additional EDC at a concentration of 20 mg / mL was provided to the activation reaction to accelerate the coupling efficiency, and the incubation was continued for 2 hours. After removing the residual ssDNA reporter molecules by centrifugation (6000 rpm, 5 min), the product was collected, i.e., the QD probe, and the quenching efficiency of the collected product was tested.

[0081] Construction of CRISPR / Cas12a system:

[0082] A 25 μL volume of CRISPR / Cas12a solution was prepared for each fungal toxin. The ingredients include 1.5 μL LbaCas12a enzyme (5 μM), 2.5 μL crRNA (1 μM), 1.5 μL trigger switch solution (10 nM) for each fungal toxin, 2.5 μL QD probe or FAM-labeled probe (2 μM), and 5 μL NEBuffer2.1 and 10 μL ultrapure water. According to the above system, the feasibility of the CRISPR / Cas12a system was tested with a target fungal toxin solution at a concentration of 1000 ng / mL and a FAM-labeled probe, and the reaction results were analyzed by gel electrophoresis and real-time fluorescence curves. The CRISPR / Cas12a system was integrated into the chip by freeze-drying technology, and 2 μL 20% (V / V) trehalose solution was added to the 25 μL CRISPR / Cas12a system prepared above as a protective agent. The CRISPR / Cas12a solutions for different fungal toxins were dropped into liquid nitrogen and quickly frozen into microspheres.

[0083] Preparation of microfluidic chip:

[0084] The microfluidic chip used in this study consists of three layers: an upper layer with flow channels, a middle layer with reaction chamber arrays, and a bottom layer with PC membrane and PDMS layer. Considering the limitation of flow channel height, the three-layer structure was prepared by different processes, the mold of the upper layer was prepared by soft lithography, and the middle and bottom PDMS layers were made using metal molds. The PC membrane at the bottom of the reaction chamber was prepared by solvent evaporation method: carboxyl-modified PS microspheres of different sizes were centrifuged at high and low speeds to improve their dispersibility, and then diluted with ultrapure water at a ratio of 1:4 (v / v), and then 10μL of PS microspheres were vertically dropped to the center of the bottom reaction chamber by a pipette gun, and the bottom layer containing PS microspheres was placed at 40℃ for 12 hours to evaporate the water. CRISPR / Cas12a system microspheres for different mycotoxin detection, negative and positive controls were added to the reaction chamber for freeze drying, and the three PDMS layers were treated by oxygen plasma and then combined in sequence. The prepared microfluidic chip was wrapped with blue film and placed at -20℃ for further use. To test the enhancement effect of PC on probe fluorescence, 2 μL of 1 ng / mL AFB1 was used to activate the CRISPR / Cas12a system corresponding to AFB1.

[0085] How the chip works:

[0086] The normal operation of the fluid in the chip mainly depends on the consistency of the external driving force and the reverse driving force caused by the air compression in the eight reaction chambers. The exhaust channel is designed at the lower end of each reaction chamber to ensure that the fluid can be smoothly distributed to the 8 reaction chambers. Fig.10 A cross-sectional view of a single reaction chamber with corresponding flow channels and exhaust channels is shown. The fluid enters the reaction chamber through the upper flow channel, causing the gas in the reaction chamber to be compressed. Under the resistance of the compressed gas, the fluid will first form a column at the top of the chamber and then load by increasing the volume. Due to the existence of the exhaust channel, the gas in the chamber can be stably removed to maintain a low level of pressure intensity. When the gas pressure in the chamber reaches the threshold of the exhaust channel, the gas will be discharged from the chip through the bottom exhaust channel. Due to the capillary effect of the exhaust channel size and the hydrophobicity of PDMS, the liquid will not enter the exhaust channel before receiving a greater pressure. When all the sample solutions enter the reaction chamber, the mineral oil added subsequently seals the reaction chamber along the upper channel. It should be noted that if the exhaust channel is designed at the top of the chip, the solution will directly break through the capillary resistance and flow from the exhaust channel to the outside of the chip without entering the reaction chamber. At the microscopic scale, gravity can be ignored, and the fluid is mainly affected by the gas pressure of the reaction chamber below. The fluid first forms a column above the reaction chamber to prevent the gas in the reaction chamber from being discharged through the exhaust channel. The above reaction chamber diameter, capillary diameter and reaction liquid addition pressure can be determined according to parameters such as the surface tension of the reaction liquid.

[0087] Read the output signal:

[0088] In the feasibility analysis of the trigger switch, agarose gel electrophoresis and qPCR instruments (reaction conditions were 37°C) were used to analyze the regulation of the trigger switch on the CRISPR / Cas12a system, and a fluorescence spectrophotometer was used to study the optimal ratio of aptamers and activators and the effect of the concentration of reporters on the quenching of quantum dot fluorescence. In order to read the signal on the chip, we developed a simple reading device and a corresponding application in a smartphone. The reading device consists of a blue LED with a λ = 380nm and a black box with a smartphone holder obtained by 3D printing. The main function of the device is to provide a dark environment and an excitation light source for FTMB. Fluorescence intensity can also be collected by other optical detectors, which are not limited in this patent.

[0089] Data Analysis:

[0090] Read the green channel value in the RGB of the captured image. Extract the green channel signal value (G) of the image and analyze these values ​​by one-way analysis of variance and Tukey's multiple comparisons.

[0091] For the quantification of mycotoxins, standard curves were constructed by serial dilution of standard solutions. Mycotoxin stock solutions were prepared by dissolving 1 mg of mycotoxins (AFB1, OTA, ZEN, FB1, T-2, and DON) in 1 mL of acetonitrile and stored in amber glass bottles at -20 °C. Tenfold standard solutions of AFB1, OTA, ZEN, FB1, T-2, and DON were prepared by diluting the stock solutions in acetonitrile-water solution (0.1, v / v). To ensure that the selected concentrations covered the maximum residue limit range of mycotoxins, the dilutions of the standard solutions were 0, 1.0 × 10 -5 , 1.0×10 -4 , 1.0×10 -3 , 1.0×10 -2 , 1.0×10 -1, 1, 10 and 50 ng / mL, and a standard curve between the fluorescence signal and the mycotoxin concentration was plotted. The LOD of each mycotoxin was calculated by 3 times the standard deviation, which was obtained from the blank or samples without any mycotoxin added (n=20), divided by the absolute slope of each mycotoxin on the standard curve. The specificity of FTMB for different mycotoxins was evaluated by detecting AFB1, OTA, ZEN, FB1 and T-2 at a concentration of 50 μg / L, using PBS buffer as a negative control. All experiments were performed in triplicate, and authentic samples were detected by FTMB and HPLC. After grinding the three samples, the standard solutions of AFB1, OTA, ZEN, FB1, T-2 and DON were added to the corn, peanut and wheat sample powders, respectively, and after drying the above samples at room temperature for 6 hours, 10 mL of methanol-water mixture (6:4, v / v) was added. Then, the spiked samples were shaken on a shaker for 2 h and centrifuged at 8000 rpm for 10 min. Finally, the supernatant was filtered with a 0.22 μM syringe filter, and then the supernatant was diluted to 0.05, 1, and 5 ng / mL with Tris buffer to obtain corn, peanut, and wheat samples, which were stored at 4 °C for subsequent operational tests.

[0092] In the construction of the detection system, the recognition of the target mycotoxin is a key factor. As a trigger switch, the functional DNA must meet two conditions: locking the activator and recognizing the target molecule. Therefore, we carefully designed the functional DNA based on the previously reported aptamer, which can completely lock the activator without affecting the recognition site (Table S1). The present invention confirms that the complex is stable at room temperature, and they bind to the target, resulting in conformational changes, thereby releasing the activator. Therefore, the mycotoxin recognition event is successfully transduced and triggers the start of the Cas12a enzyme to perform a cascade reaction. The activated Cas12a enzyme will quickly cut the reporter molecule (NH2-TTATT-BHQ1) modified on the surface of the quantum dot, so that the quenching group BHQ1 is away from the emission zone, and the fluorescence of the quantum dot is restored. The fluorescence recovery efficiency of the quantum dot is closely related to the released activator, which is limited by the amount of the activated Cas12a enzyme and the cutting efficiency. PC is constructed at the bottom of the reaction chamber to achieve the enhancement of the fluorescence signal, which helps to reduce the detection threshold level and expand the dynamic detection range.

[0093] Feasibility verification of trigger switch:

[0094] To verify the utility of the trigger switch for six different fungal toxins, the functional DNA-guided CRISPR / Cas12a system was applied to the degradation of ssDNA (19 nt) and FAM-labeled probes (FAM-TATT-BHQ1), respectively. Figure 2As shown in the A region, ssDNA remains intact in the reaction system without the corresponding activator (lanes 1-2). When a trigger switch is used, the cutting activity of CRISPR / Cas12a is still limited (lanes 3-8). However, it can be triggered in the presence of target fungal toxins (100 ng / mL), so that ssDNA is cut into random short fragments (lanes 9-14). This degradation phenomenon indicates that the six functional DNAs can act as trigger switches to initiate subsequent cascade reactions. On the other hand, the trigger switch based on functional DNA was also tested in the CRISPR / Cas12a fluorescence detection system, and the real-time fluorescence curve showed that in the absence of a trigger switch or target fungal toxin, the fluorescence signal hardly accumulated, while in the presence of a trigger switch and target fungal toxin, the fluorescence signal increased rapidly ( Figure 2 According to the fluorescence intensity value, the optimal ratio of aptamer to activator is 2:1 ( Figure 2 C). These results suggest that functional DNA can act as a trigger switch to recognize the target fungal toxin.

[0095] Quantum Dots for CRISPR / Cas12a Systems:

[0096] In order to further improve the detection sensitivity and anti-interference ability of FTMB, quantum dots were selected as donor materials to construct probes for the CRISPR / Cas12a system. Compared with FAM-labeled probes, the narrow emission spectrum and high quantum yield of quantum dots help to highly match the photonic band gap of PC. Considering that the green band is the most common detection output signal, quantum dots with a green emission wavelength of 520nm were selected. Based on FRET, the reporter molecules were modified on the surface of the quantum dots to achieve fluorescence quenching, and the concentration of the reporter gene was further detected to obtain the best quenching efficiency. Figure 2 As shown in D (TEM of QDs is shown in the upper right corner), the quenching efficiency increases with increasing reporter gene concentration until complete quenching is achieved at 6 nmol.

[0097] Fluorescence enhancement effect of PC:

[0098] PC with polystyrene (PS) microspheres of different particle sizes were constructed to study the optimal photonic band gap matching with quantum dots. PS microsphere solutions evaporated on the hydrophobic polydimethylsiloxane (PDMS) surface and self-assembled into tightly packed, highly ordered periodic nanostructures ( Figure 2 E). Figure 2The F showed that four PCs composed of PS microspheres of different sizes (diameters of 250nm, 350nm, 450nm and 550nm) enhanced the fluorescence of quantum dots and FAM. The emission spectrum range of quantum dots and the existence of different photonic band gaps in PCs determined different signal enhancement effects. The narrow emission wavelength range of quantum dots can better enhance the fluorescence signal. According to quantitative measurements, the enhancement factor of PC composed of 250nm PS microspheres for quantum dots reached 45.6, which is much higher than the enhancement factor of PC composed of 250nm, 350nm, 450nm and 550nm PS microspheres for FAM ( Figure 2 G). These results indicate that PCs constructed from 250 nm PS microspheres are ideal for QDs signal amplification in the CRISPR / Cas12a system.

[0099] Design of transition state CRISPR / Cas12a system:

[0100] The dynamic equilibrium system with different forms of Cas12a enzyme (i.e., inactive, intermediate, and activated) can be easily altered using a small amount of activator to activate more Cas12a enzyme ( Figure 3 A(1)). However, the cutting efficiency of the CRISPR / Cas12a system will only be significantly improved when the activator concentration exceeds a certain threshold. In the closed state, the Cas12a enzyme requires a large amount of activator to initiate the cutting activity. In the open state, most Cas12a enzymes are fully activated by binding to excess crRNA / activator complexes, and the high initial background fluorescence makes it difficult to distinguish signal changes from additional activation. Therefore, the most sensitive state of the CRISPR / Cas12a system is the transition state, where even a small amount of activator can easily activate the Cas12a enzyme ( Figure 3 A(2)) to induce strong cleavage activity. Figure 3 It was observed in A(3) that under the same concentration of activator, the side branch cleavage activity of Cas12a enzymes corresponding to different crRNAs was different, which was attributed to the fact that the cleavage efficiency of the CRISPR / Cas12a system is driven by the properties of multiple components, so the structure and ratio of the components have a significant impact on the performance of the CRISPR / Cas12a system.

[0101] To determine the transition state, we first evaluated the effect of the activation complex (activator / crRNA) on the Cas12a enzyme. For a fixed concentration of Cas12a enzyme, the concentration of the activation complex was gradually increased to above 4 nM (maintaining the ratio of activator to crRNA at 1:1), and the collateral cleavage activity of Cas12a increased significantly ( Figure 3By plotting the first-order derivative of the activation curve, the molecular switch is divided into three states (i.e., closed state, transition state, and open state, such as Figure 3 ). The closed state and the open state were formed with low concentration (<3nM) and high concentration (>8nM) of the activation complex, respectively. The transition state was prepared with a medium concentration of the activation complex, and a high response was maintained to the change in the concentration of the activation complex, with the apex being 5.32nM. In addition, when the ratio of activator to crRNA was reduced in all three states, only the trans-cleavage activity of the Cas12a enzyme in the transition state changed significantly ( Figure 3 C).

[0102] To establish the transition state, we further adjusted the amount of activator while keeping the amount of crRNA constant ( Figure 3 The optimal transition state is defined as the vertex on the first-order derivative activation graph ( Figure 3 D), this defined transition state shows a further increase in responsiveness, with the Cas12a enzyme having the highest cleavage rate, while the closed and open states of the CRISPR / Cas12a system fail to produce any distinguishable signals ( Figure 3 E), the ratio-regulated Cas12a enzyme not only exhibited significant side branch cleavage activity when incubated with on-target fungal toxins, but also maintained low background activity when incubated with off-target fungal toxins. In the transition state CRISPR / Cas12a system, the specific concentration ratio of the enzymes in the three states was calculated to be: 5nM (inactivated state Cas12a): 100nM (intermediate state Cas12b): 195nM (activated state Cas12c). The transition state CRISPR / Cas12a system achieved faster activation kinetics for all six fungal toxins ( Figure 4 ), compared with molecular switches prepared in other states, the transition state CRISPR / Cas12a system can quickly activate the Cas12a enzyme, and low concentration targets can be recognized within 40 minutes of incubation at room temperature.

[0103] Fluid testing of pressure-driven microfluidic chips:

[0104] Fluid control on microfluidic chips is an important guarantee for high-throughput mycotoxin detection. A red dye solution with 25 μL of mineral oil on top was used for fluid testing. Inspired by the natural fractal tree system, the flow channel design ensures that the pressure applied at the inlet is evenly distributed in the reaction chamber to guide the liquid diversion. This distribution pattern is not affected by pressure changes during injection and ensures 100% distribution efficiency. Figure 5 The composition and the entire operation process of the pressure-driven microfluidic chip. Figure 5(A) shows the chip's structural layers: the bottom layer is a signal enhancement layer distributed in eight hollow holes with PC; the middle layer is a reaction layer with eight reaction chambers passing through the top and bottom and an exhaust channel extending from the bottom; the top layer is a flow channel layer with a fractal branching microchannel network that can evenly distribute the sample solution; Figure 5 (B) The initial and final states of the fluid test in the chip (sample solution with added red dye): the chip can drive the uniform distribution of the sample under variable external pressure; Figure 5 (C) is the whole process of sample flow in the chip: the liquid enters the flow channel of the chip under the action of external pressure; the fluid compresses the air in the reaction chamber and the flow channel, and the air is slowly discharged through the exhaust channel, thereby adjusting the pressure of each reaction chamber to be consistent; after the fluid completely enters the reaction chamber, mineral oil immediately enters to seal the flow channel and the reaction chamber; the CRISPR / Cas12a system in the reaction chamber starts working and releases fluorescence if the target fungal toxin is present.

[0105] from Figure 5 As can be seen in the figure, the red liquid is evenly distributed in the eight reaction chambers on the left and right, and is isolated by the subsequent mineral oil to avoid cross contamination. The exhaust channel at the bottom of the chip uses the capillary effect to form a stop valve to exhaust the air in the reaction chamber, but not the liquid. To ensure the accuracy of the results, the two reaction chambers on the right are used as negative and positive controls, and the other six reaction chambers are used to detect six different fungal toxins. In addition, chips with different fractal dimensions and branching levels can be further expanded to higher throughput detection schemes.

[0106] FTMB operation steps:

[0107] The fractal microchannels of the microfluidic chip in FTMB achieve pressure consistency in each reaction chamber. Therefore, the injection operation only requires the sample (200 μL) to be slowly injected into the chip through a syringe from the inlet until the liquid is dispersed into each reaction chamber. After the sample distribution is completed, 20 μL of mineral oil is immediately injected into the chip to form a thin film on the reaction solution for sealing to prevent contamination and solvent evaporation. Then, a layer of blue plastic film is covered on the surface of the chip to seal all the holes. The incubation is continued at room temperature for 40 minutes, and the fluorescence of each reaction chamber on the chip under irradiation of a 380nm portable laser (300mW) is collected using a smartphone in a dark environment.

[0108] Optimization of key parameters in FTMB.

[0109] As a substrate for the CRISPR / Cas12a system, quantum dots are usually required to support the entire reaction process. Figure 6It can be seen that the fluorescence intensity usually increases within a certain range with the increase of quantum dot concentration until it reaches the maximum value. By comprehensively considering the fluorescence intensity and reagent dosage, 300nM quantum dots were selected. On this basis, the concentrations of crRNA and trigger switch were optimized, and the concentration of Cas12a enzyme was measured at 110nM, such as Figure 7 As shown, the fluorescence intensity increases with the increase of crRNA concentration. Considering the different binding abilities of crRNA and activator to Cas12a enzyme, the optimal crRNA concentrations of six fungal toxins are 150nM (ZEN), 175nM (FB1, DON) and 200nM (AFB1, OTA, T-2). Similarly, the optimal dose of the trigger switch was determined by studying the changes in fluorescence intensity under different concentrations of trigger switches. The binding efficiency of the trigger switch with the target fungal toxin in the transition state is affected by its sequence. The concentration of the trigger switch was selected as 300nM (FB1), 350nM (OTA, ZEN, DON) and 400nM (AFB1, T-2) ( Figure 8 ).

[0110] Quantitative standard curves and detection limits of various mycotoxins:

[0111] Under the optimal experimental conditions, the FTMB standard curves for the detection of six mycotoxins were constructed. Fig. 9 As shown in A, the fluorescence images corresponding to different mycotoxin concentrations were analyzed, and the obtained data were used to fit the standard curve. The brightness of the sensing site in the microfluidic chip increased with the increase of the mycotoxin concentration. The signal transduction mechanism of FTMB does not require competition from the target analogs to achieve quantification by reducing the number of recognition molecules modified with signal markers. Therefore, the fluorescence intensity is positively correlated with the concentration of the target mycotoxin, which is beneficial to improve the detection sensitivity. The standard regression curves of AFB1, OTA, ZEN, FB1, T-2 and DON were obtained by the Michaelis-Menten equation ( Fig. 9 A). The correlation coefficient R ranged from 0.949 to 0.989. 2 The values ​​showed excellent correlation coefficients within the measurement range. According to the standard curve, the detection limits (LODs) of AFB1, OTA, ZEN, FB1, T-2, and DON were 2.3, 3.9, 2.6, 1.4, 1.7, and 1.5 fg mL -1 .

[0112] Specificity of FTMB:

[0113] To further evaluate the specificity of FTMB for single and multiplex detection, the fluorescence responses to individual AFB1, OTA, ZEN, FB1, T-2, DON and their mixtures were measured ( Fig. 9B). For a single test, the target fungal toxin corresponding to each detection unit can trigger the CRISPR / Cas12a system in the reaction chamber to restore the fluorescence of the quantum dots. Fig. 9 (B) In the lower left corner, it can be seen that the detection unit has no response to non-target mycotoxins, and the signal value is similar to the negative control. Fig. 9 (B) Fluorescence images of the eight chambers on the chip and the corresponding normalized fluorescence values ​​show that for multiplex detection, FMTB also provides accurate results when testing different combinations of multiple mycotoxins (mixtures of 2, 3, 4, and 5 mycotoxins). However, in some competitive immunoassays that require the preparation of haptens, the autofluorescence of some mycotoxins (such as AFB1) can interfere with the interpretation of the results. FTMB is not affected in this regard because it emits fluorescence by triggering a cascade reaction. These results show that the non-competitive detection method of the FTMB system can accurately and specifically detect single or multiple target toxins.

[0114] Detection of mycotoxins in real samples:

[0115] In order to accurately evaluate the efficacy of mycotoxin detection in FTMB, standard solutions of AFB1, OTA, ZEN, FB1, T-2 and DON were added to the corn, peanut and wheat samples prepared in advance above for recovery experiments. As shown in Tables 2-7, the recoveries of AFB1, OTA, ZEN, FB1, T-2 and DON were 86.60%-116.40%, 92.08%-108.26%, 91.00%-102.35%, 91.20%-103.60%, 90.80%-114.85% and 94.62%-115.96%, respectively. In addition, the RSD of the test results was between 0.58%-5.67%, indicating that the FTMB system has high accuracy in the detection of mycotoxins in real samples. More importantly, all the results were highly consistent with the HPLC method (88.76%-109.99%), indicating the reliability of this strategy for mycotoxin monitoring in real samples.

[0116] Table 2 Recovery results of AFB1 detected using FTMB and consistency between FTMB and HPLC

[0117]

[0118] Table 3 Recovery results of OTA detected using FTMB and consistency between FTMB and HPLC

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[0120]

[0121] Table 4 Recovery results of ZEN detected using FTMB and consistency between FTMB and HPLC

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[0123] Table 5 Recovery results of FB1 detected using FTMB and consistency between FTMB and HPLC

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[0125]

[0126] Table 6 Recovery results of T-2 detected using FTMB and consistency between FTMB and HPLC

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[0128] Table 7 Recovery results of DON detected using FTMB and consistency between FTMB and HPLC

[0129]

[0130] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be regarded as exemplary and non-limiting from any point of view, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention, and any reference numerals in the claims should not be regarded as limiting the claims involved.

Claims

1. A detection method based on a microfluidic chip, characterized in that: The microfluidic chip comprises: A sample injection port is located in the upper layer; at least one reaction chamber is located in the middle layer, the reaction chamber is connected to the sample injection port through a flow channel, and a detection reagent is pre-installed in the reaction chamber, and the detection reagent responds to the content of the object to be detected based on fluorescence; a light-transmitting lower layer, and a signal amplification layer is also provided between the lower layer and the reaction chamber; the lower layer has a hydrophobic polydimethylsiloxane layer; An exhaust channel is also provided at the bottom of the reaction chamber, the diameter of the exhaust channel is smaller than the diameter of the reaction chamber, when the injection pressure of the sample solution is less than a threshold value, the sample solution cannot be discharged from the exhaust channel due to the capillary force, and when the injection pressure of the sample solution is greater than the threshold value, the sample solution can be discharged; The number of the reaction chambers is multiple, forming a reaction chamber array; The detection method comprises the following steps: Step a, adding the sample solution from the sample adding port, and entering the reaction chamber through the flow channel, and reacting with the detection reagent to generate fluorescence; Step b, after the fluorescence is amplified by the signal amplification layer, the fluorescence is collected from the lower layer, and the concentration of the target substance in the sample solution is measured according to the light intensity; Step c: after the detection is completed, the sample addition port is pressurized to discharge the reaction system.

2. The detection method according to claim 1, characterized in that: The outlet end of the exhaust passage is located at the upper layer.

3. The detection method according to claim 1, characterized in that: The signal amplification layer is composed of carboxyl-modified polystyrene microspheres, which are prepared into a suspension, dripped into the surface of the bottom layer and left to stand to form a film.

4. The detection method according to claim 3, characterized in that: The conditions for static film formation are as follows: temperature is 30-60° C. and time is 10-15 hours.

5. The detection method according to claim 3, characterized in that: The detection reagent is a microsphere obtained after freeze-drying.

6. The detection method according to claim 1, characterized in that: In the step a, after the sample solution is added, mineral oil is continuously added to seal the reaction chamber.

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