A small molecule detection system based on MIP-CRISPR / Cas12a cascade amplification and application

Abstract

The application discloses a small molecule detection system based on MIP-CRISPR / Cas12a cascade amplification and application, and the detection system comprises the following: a molecularly imprinted polymer prepared by taking the small molecule as a template; single-stranded DNA which can be combined with the molecularly imprinted polymer and can be competitively replaced by the small molecule to be released from the molecularly imprinted polymer; a CRISPR / Cas12a system, wherein the CRISPR / Cas12a reaction system comprises a Cas12a protein, crRNA and a fluorescent reporter probe. The application first organically integrates the competitive replacement mechanism of the molecularly imprinted polymer and the trans cleavage activity of the CRISPR / Cas12a, constructs a complete cascade amplification detection chain, and ingeniously converts the small molecule chemical signal which is difficult to directly detect into nucleic acid signal which is easy to amplify, thereby opening up a new path for the ultra-sensitive detection of small molecule organic compounds.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a small molecule detection system based on MIP-CRISPR / Cas12a cascade amplification and its application. Background Technology

[0002] Small molecule compounds generally refer to organic compounds with a molecular weight of less than 1500 Da, encompassing amino acids, hormones, sugars, lipids, vitamins, drug residues, and environmental pollutants. Among these, endogenous small molecule metabolites, as key intermediates or end products of the metabolic network of living organisms, exhibit concentration changes highly correlated with disease occurrence and physiological regulation, and have significant application value in clinical diagnosis, food safety, and environmental monitoring. Existing detection technologies are mainly divided into two categories: large-scale instrumental analysis and biosensing. The former involves expensive equipment, cumbersome sample preparation, and difficulty in rapid on-site detection; the latter relies on antibodies and nucleic acid aptamers, which suffer from high cost, poor stability, easy inactivation, or easy enzymatic degradation, making it difficult to meet the demand for stable and rapid detection of trace small molecules in complex matrices.

[0003] Molecularly imprinted polymers (MIPs) are artificial biomimetic recognition materials prepared using molecular imprinting technology. Often referred to as "plastic antibodies," they polymerize using a target molecule as a template, then elute the template to form a specific cavity that highly matches the spatial structure, size, and functional groups of the target molecule, achieving precise "key-and-lock" recognition. MIPs possess advantages such as simple preparation, low cost, resistance to high and low temperatures, acid and alkali resistance, resistance to enzymatic degradation, and resistance to matrix interference, making them widely used for the recognition and enrichment of small molecules in complex systems. However, traditional MIPs are inert polymer materials, and their signal transduction relies on physical interfaces such as electrochemistry and SPR. They lack liquid-phase biochemical signal amplification capabilities, making it difficult to couple with amplification systems such as nucleic acids and enzymes, and their detection sensitivity is insufficient to reach clinical trace detection levels.

[0004] CRISPR / Cas12a is a next-generation, highly sensitive nucleic acid detection tool. Guided by crRNA, it can be specifically activated by ssDNA, generating highly efficient non-specific trans-cleavage activity and achieving exponential signal amplification triggered by a single molecule. It has outstanding advantages such as isothermal reaction, high sensitivity, and strong specificity. However, Cas12a can only recognize and respond to nucleic acid molecules and cannot directly bind to or detect small molecules. Existing small molecule detection strategies heavily rely on nucleic acid aptamers as signal transduction mediators, and still suffer from drawbacks such as difficulty in screening, insufficient stability, and susceptibility to matrix interference. There is a lack of a stable and universal mechanism to efficiently convert small molecule recognition into Cas12a activation signals.

[0005] In summary, the field of small molecule detection still faces three major technical bottlenecks: insufficient stability of biometric elements, weak MIP signal amplification capability, and the difficulty of CRISPR systems in directly detecting non-nucleic acid targets. To address this, this invention provides a small molecule detection method based on MIP-CRISPR / Cas12a cascade amplification, meeting the demand for high-sensitivity, high-specificity, and rapid detection of trace small molecules in various scenarios. Summary of the Invention

[0006] The first objective of this invention is to provide a small molecule detection system based on MIP-CRISPR / Cas12a cascade amplification, and the second objective is to provide applications of this small molecule detection system.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a small molecule detection system based on molecularly imprinted polymer-CRISPR / Cas12a cascade amplification, wherein the small molecule refers to an organic compound with a molecular weight less than 1500 Da, and the detection system comprises: (1) A molecularly imprinted polymer prepared using the small molecule as a template; (2) Single-stranded DNA that can bind to the molecularly imprinted polymer and can be competitively replaced by the small molecule and thus released from the molecularly imprinted polymer; (3) CRISPR / Cas12a system, wherein the CRISPR / Cas12a reaction system comprises Cas12a protein, crRNA, and fluorescent reporter probe; in: The crRNA can assemble with the Cas12a protein to form a Cas12a-crRNA complex and contains a guide sequence that can complementarily pair with the single-stranded DNA; the single-stranded DNA released from the molecularly imprinted polymer can bind complementaryly to the crRNA to activate the trans-cleavage activity of the Cas12a protein. The fluorescent reporter probe is a single-stranded DNA with fluorescent and quenching groups modified at both ends, respectively. The Cas12a protein, whose trans-cleavage activity is activated, can non-specifically cleave the single-stranded DNA of the fluorescent reporter probe, thereby relieving the quenching effect and outputting a fluorescent detection signal.

[0008] In one specific embodiment, the functional monomers used to prepare the molecularly imprinted polymer are o-phenylenediamine and 4-vinylpyridine. The functional monomers may be one or more selected from o-phenylenediamine, methacrylic acid, 4-vinylpyridine, acrylamide, trifluoromethacrylic acid, and N-isopropylacrylamide.

[0009] In one specific embodiment, the crosslinking agent used to prepare the molecularly imprinted polymer is divinylbenzene or trimethylolpropane trimethacrylate. The crosslinking agent may be one or more of divinylbenzene, trimethylolpropane trimethacrylate, N,N'-methylenebisacrylamide, and polyethylene glycol dimethacrylate.

[0010] In one specific embodiment, the sequence of the single-stranded DNA is shown in SEQ ID NO.1, and the sequence of the crRNA is shown in SEQ ID NO.2.

[0011] In one specific embodiment, the fluorescent reporter probe has a fluorophore of FAM and a quencher of BHQ1, which are linked by a TTATT sequence.

[0012] In one specific embodiment, the small molecule detection system further includes a buffer solution for binding the single-stranded DNA to the molecularly imprinted polymer.

[0013] The application of any of the above small molecule detection systems in the preparation of detection reagents for determining the content of phenylalanine or cortisol in samples.

[0014] In one specific embodiment, a linear relationship between the concentration of phenylalanine or cortisol and the fluorescence growth rate is first established, and then the concentration of phenylalanine or cortisol in the sample is calculated based on the linear relationship and the fluorescence growth rate of the sample.

[0015] In one specific embodiment, the sample is a biological sample.

[0016] In one specific embodiment, the biological sample is urine or sweat.

[0017] The detection principle and challenges of this invention: The core idea of ​​the detection system provided by this invention is to couple the specific recognition capability of MIP with the signal amplification function of the CRISPR / Cas12a system. The process of using this detection system for the detection of target small molecules can be understood as a cascade event chain triggered by the target small molecule.

[0018] In the initial state of the system, the MIP is pre-loaded with single-stranded DNA, which is anchored to the specific cavity constructed by the MIP for the target small molecule through affinity. When the sample to be tested contains the target small molecule, the target small molecule has a stronger binding affinity to the MIP cavity, competitively displacing the single-stranded DNA from the cavity and releasing it into the sample solution. Simultaneously, the pre-assembled Cas12a protein and crRNA binary complex in the reaction system are in a ready state. The released single-stranded DNA contains a sequence perfectly complementary to the crRNA guide sequence; upon binding, the two induce conformational rearrangement of the Cas12a protein, thereby unlocking its highly efficient trans-cleavage activity. At this point, the activated Cas12a non-specifically cleaves the fluorescent reporter probes (which contain ssDNA, and the activated Cas12a continuously and indiscriminately cleaves all free ssDNA probes) modified at both ends with fluorophores and quenchers, respectively, causing the fluorophore and quencher to separate, thus eliminating the fluorescence resonance energy transfer effect and emitting a continuous and stable fluorescence signal.

[0019] The sensitivity of this system comes from the amplification effect: the target molecule displaces single-stranded DNA, realizing the transformation of a chemical event into a nucleic acid event; the released single-stranded DNA acts as an activation sequence and binds to the Cas12a / crRNA complex, inducing Cas12a to acquire trans-cleavage activity; and a single activated Cas12a can enzymatically cleave several reporter probes, resulting in several times the signal gain.

[0020] The accurate quantification of target small molecules in this detection system relies heavily on a stable and reliable linear correlation between the concentration of the target small molecule and the terminal fluorescence signal of its induced cascade reaction. This linear correlation is a prerequisite for the quantitative analysis of small molecules in samples. Because this detection system involves multiple continuous and coupled reaction processes, including target small molecule recognition, DNA release, Cas12a activation, and fluorescence signal amplification, each reaction step affects the final fluorescence signal output. Therefore, the response relationship between the target small molecule concentration and the terminal fluorescence signal or fluorescence growth rate is difficult to predict using theoretical models.

[0021] Beneficial effects of this invention: This invention is the first to organically integrate the competitive substitution mechanism of molecularly imprinted polymers (MIPs) with the trans-cleavage activity of CRISPR / Cas12a, constructing a complete cascade amplification detection chain: the target small molecule releases the single-stranded DNA anchored on the MIP into solution through competitive substitution. The released DNA acts as the activating strand, complementary to crRNA, thereby activating the trans-cleavage activity of Cas12a. The activated Cas12a further non-specifically cleaves the fluorescent reporter probe and outputs a quantitatively detectable fluorescent signal. This three-stage cascade design, which propagates stepwise from chemical events to nucleic acid events and then to enzymatic signal amplification, ingeniously transforms the difficult-to-detect small molecule chemical signals into easily amplified nucleic acid signals, opening up a completely new pathway for the ultrasensitive detection of small molecule organic compounds.

[0022] The preparation of molecularly imprinted polymers (MIPs) is highly designable; for any target small molecule, a corresponding MIP can be prepared by selecting appropriate functional monomers and cross-linking agents. Therefore, this invention is not limited to the detection of a specific small molecule, but rather is a universal detection platform. By simply changing the template molecule of the MIP and adjusting the sequence of the anchored single-stranded DNA accordingly, this system can be extended to the detection of various small molecule organic compounds, covering multiple target categories such as environmental hormones, pesticide residues, veterinary drug residues, biotoxins, and prohibited additives, demonstrating extremely broad application coverage and high technological reusability. Attached Figure Description

[0023] Figure 1 Characterization of Phe-MIP nanoparticles; where: A is a scanning electron microscope (SEM) image of Phe-MIP nanoparticles (scale bar: 300 nm); B is the UV-Vis absorbance of Phe-MIP and Phe-NIP before template extraction; C is a comparison of UV-Vis absorbance of Phe-MIP before and after template extraction; D is a comparison of the peak values ​​of energy dispersive X-ray spectroscopy (EDX) of carbon in Phe-MIP before and after template extraction.

[0024] Figure 2 Figure 1 shows the binding and adsorption properties of Phe-MIP nanoparticles; where: A is the UV-vis spectrum of Phe-MIP after incubation with 500 μM-20 mM phenylalanine in the high concentration range; B is the linear fitting calibration curve of the characteristic peak absorbance in the high concentration range of A (SD, n=3); C is the UV-vis spectrum of Phe-MIP after incubation with 5 μM–500 μM phenylalanine in the low concentration range; D is the adsorption kinetics curve of Phe-MIP (the trend of adsorption amount Q with incubation time).

[0025] Figure 3This document outlines the construction and optimization of a MIP nucleic acid loading workflow. Specifically: A) optimization of nucleic acid incubation concentration (SD, n=3); B) optimization of nucleic acid incubation time (SD, n=3); and C) optimization of the basic loading and elution buffer system (SD, n=3). Different combinations of loading and elution buffers were compared (PBS, conventional phosphate buffer; PBST, 1% Tween-20 PBS; PBSM, 10mM MgSO4). 2+ Differences in probe binding amount and specific binding amount between PBS-MIP and NIP; D represents the reverse dynamic occupancy process construction (SD, n=3), PBS wash5': after the original incubation and washing process, PBS blank competition for 5 min, 0.2×DNA: 0.2×DNA reverse loading for 5 min, 0.5×DNA: 0.5×DNA reverse loading for 5 min.

[0026] Figure 4 To construct and optimize the conditions for competitive release of MIP-DNA and CRISPR / Cas12a cascade amplification system; where A is the optimization of MIP-DNA dosage; B is the optimization of target competitive release temperature and time; C is the optimization of Cas12a concentration; and D is the optimization of the assembly ratio of Cas12a and crRNA (SD, n=3).

[0027] Figure 5 This study evaluates the specificity and selectivity of Phe-MIP and MIP-CRISPR / Cas12a detection systems in artificial urine matrices. A represents a comparison of the equilibrium adsorption capacity (Qe) of Phe-MIP for the target molecule and structurally similar amino acids; B represents the fluorescence response of the MIP-CRISPR / Cas12a detection system to the target molecule and structurally similar amino acids. Phe: phenylalanine; Trp: tryptophan; His: histidine; Ala: alanine; Tyr: tyrosine; AA: a mixed amino acid sample of Phe, Trp, and Tyr (SD, n=3).

[0028] Figure 6 To evaluate the detection performance of the detection system in PBS buffer and artificial urine matrix; where A is the fluorescence kinetic curve of Phe samples with different concentrations in PBS buffer system; B is the linear fitting result of Phe concentration and fluorescence growth rate (dF / dT) in PBS buffer system; C is the fluorescence kinetic curve of Phe samples with different concentrations in artificial urine matrix; D is the linear fitting result of Phe concentration and dF / dT in artificial urine matrix (SD, n=3).

[0029] Figure 7The images show the SEM characterization results of Cort-MIP; where A is the SEM image of Cort-MIP nanoparticles (scale bar: 1 μm); and B is the high-magnification SEM image of Cort-MIP nanoparticles (scale bar: 200 nm).

[0030] Figure 8 The adsorption and recognition performance of Cort-MIP for cortisol was evaluated. Among them, A is the adsorption kinetic curve of Cort-MIP for cortisol; B is the fitting result of the pseudo-second-order kinetic model of adsorption kinetic data; C is the adsorption isotherm after Cort-MIP reacts with different concentrations of cortisol; D is the fitting result of Langmuir model of adsorption isotherm data (SD, n=3).

[0031] Figure 9 This study evaluates the feasibility and performance of the MIP-Cas12a detection system. A represents the competitive release behavior of the MIP-DNA system and the feasibility verification of the Cas12a response. B represents the effects of different buffer systems on probe immobilization and release, and the construction of thermodynamic thresholds: Tris-Mg2: 10 mM Tris + 2 mM MgCl2 (pH 7.5), Tris-Mg5: 10 mM Tris + 5 mM MgCl2 (pH 7.5), McIlvaine: citrate-phosphate buffer (pH 6.0), Na-PBS: phosphate buffer (pH 7.0, NaCl-free). C represents the fluorescence kinetic response curve of the MIP-Cas12a system for cortisol detection. D represents the linear fitting results of cortisol concentration versus dF / dT (SD, n=3). Detailed Implementation

[0032] The substantive content of the present invention will be described in detail below with reference to specific embodiments. However, those skilled in the art should know that the scope of protection of the present invention should not be limited to these specific embodiments.

[0033] Example 1: Small molecule detection system based on molecularly imprinted polymer-CRISPR / Cas12a cascade amplification This embodiment provides a small molecule detection system based on molecularly imprinted polymer-CRISPR / Cas12a cascade amplification, wherein the small molecule refers to an organic compound with a molecular weight less than 1500 Da, and the detection system comprises: (1) A molecularly imprinted polymer prepared using the small molecule as a template; (2) Single-stranded DNA that can bind to the molecularly imprinted polymer and can be competitively replaced by the small molecule and thus released from the molecularly imprinted polymer; (3) CRISPR / Cas12a system, wherein the CRISPR / Cas12a reaction system comprises Cas12a protein, crRNA, and fluorescent reporter probe; in: The crRNA can assemble with the Cas12a protein to form a Cas12a-crRNA complex and contains a guide sequence that can complementarily pair with the single-stranded DNA; the single-stranded DNA released from the molecularly imprinted polymer can bind complementaryly to the crRNA to activate the trans-cleavage activity of the Cas12a protein. The fluorescent reporter probe is a single-stranded DNA with fluorescent and quenching groups modified at both ends, respectively. The Cas12a protein, whose trans-cleavage activity is activated, can non-specifically cleave the single-stranded DNA of the fluorescent reporter probe, thereby relieving the quenching effect and outputting a fluorescent detection signal.

[0034] The functional monomers used to prepare the molecularly imprinted polymer are o-phenylenediamine and 4-vinylpyridine. Other functional monomers may be selected from one or more of o-phenylenediamine, methacrylic acid, 4-vinylpyridine, acrylamide, trifluoromethacrylic acid, and N-isopropylacrylamide.

[0035] The crosslinking agent used to prepare the molecularly imprinted polymer is divinylbenzene or trimethylolpropane trimethacrylate. Alternatively, the crosslinking agent may be one or more of divinylbenzene, trimethylolpropane trimethacrylate, N,N'-methylenebisacrylamide, and polyethylene glycol dimethacrylate.

[0036] The sequence of the single-stranded DNA is shown in SEQ ID NO.1, and the sequence of the crRNA is shown in SEQ ID NO.2. The fluorescent reporter probe has a FAM fluorescent group and a BHQ1 quenching group, which are linked by the TTATT sequence.

[0037] The small molecule detection system also includes a buffer solution for binding the single-stranded DNA to the molecularly imprinted polymer.

[0038] Example 2: Application Case - Detection of Phenylalanine in Urine Matrix I. Experimental Materials 1. Target small molecules and their similar compounds Phenylalanine (Phe), tyrosine (Tyr), histidine (His), alanine (Ala), and tryptophan (Trp) were all purchased from Sigma-Aldrich.

[0039] 2. MIP synthesis reagents Functional monomer: o-phenylenediamine (OPD), purchased from Aladdin.

[0040] Crosslinking agent: divinylbenzene (DVB), 80% purity, purchased from Aladdin.

[0041] Initiator: Azobisisobutyronitrile (AIBN), 99% pure, purchased from Aladdin.

[0042] Solvents and elution reagents: methanol, acetonitrile, and acetic acid, all HPLC grade, purchased from Merck; ultrapure water was prepared using a Milli-Q water purifier.

[0043] 3. Nucleic acid and CRISPR / Cas12a system Cas12a protein: LbCas12a, 100 μM, purchased from New England Biolabs (NEB).

[0044] Single-stranded DNA (ssDNA, SEQ ID NO.1): sequence 5'-CAATAGCTTATCAGACTGTCT-3', synthesized by Beijing BGI.

[0045] Guide RNA (crRNA, SEQ ID NO.2): sequence 5'-UAAUUUCUACUAAGUGUAGAUAGACAGUCUGAUAAGCUAUUG-3'.

[0046] Single-chain fluorescent quenching probe (FQ probe): sequence and modification are 5'-FAM-TTATT-BHQ1-3'.

[0047] 4. Buffer system and matrix Nucleic acid loading buffer: 10 mM Tris, 2 mM MgCl2, pH 7.5.

[0048] CRISPR reaction buffer (1×NEB r2.1): 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 100 μg / ml BSA, pH 7.9.

[0049] Detection substrate: Artificial urine, purchased from Shanghai Yuanye Biotechnology, catalog number R23032.

[0050] II. Experimental Methods and Results 1. Characterization of Phe-MIP synthesis and recognition performance 82.6 mg Phe and 216.3 mg OPD functional monomer were added to 10 mL of a methanol / water mixture (4:1, v / v) at a molar ratio of 1:4. The mixture was magnetically stirred at 300 rpm for 10 minutes in the dark until completely dissolved, yielding a homogeneous and transparent prepolymer solution. This solution was then transferred to an anaerobic chamber. Under anaerobic and light-protected conditions, 72 μL of DVB crosslinking agent pre-cooled to 25°C and 41.0 mg AIBN initiator were added, and the mixture was stirred for 5 minutes to ensure uniform dispersion (the molar ratio of template molecule: functional monomer: crosslinking agent: initiator in the system was 1:4:1:0.5). After rigorous anaerobic purging of the reaction system in the anaerobic chamber, the thick-walled vacuum reaction vessel cap with a polytetrafluoroethylene (PTFE) gasket was quickly tightened to achieve an airtight seal. The sealed reaction vessel was removed from the anaerobic chamber and immersed in a 60°C constant-temperature water bath for thermally initiated bulk polymerization at 200 rpm for 18 hours. After the reaction was completed and cooled to room temperature, 5 mL of eluent methanol / acetic acid (3:7, v / v) was added to the system. The mixture was continuously stirred and eluted at room temperature for 16 hours to disrupt the template binding sites. The suspension was centrifuged at 7000 rpm for 2 minutes, and the supernatant was discarded. The resulting precipitate was resuspended in 10 mL of deionized water and 10 mL of methanol, and washed three times by centrifugation. Finally, the purified precipitate was dried overnight in a vacuum drying oven at 30°C until it became powder, thus obtaining Phe-MIP nanoparticles. A synthesis control was also set up; the steps were the same except that the template molecule Phe was not added, resulting in the synthesis of the non-imprinted polymer (Phe-NIP).

[0051] To verify the successful synthesis of Phe-MIP and the formation of its cavity structure, its morphology and composition were characterized. Figure 1 As shown in Figure A, SEM results confirmed that the synthesized Phe-MIP was uniform in size (approximately 50 nm), possessing a uniform morphology and a significantly rough porous structure. Its large specific surface area provided a physical basis for subsequent efficient target molecule adsorption. Figure 1 As shown in Figure B, the UV-visPhe characteristic absorption peaks of Phe-MIP and Phe-NIP before elution were compared, revealing significant differences. This confirms that the template molecule and functional monomer underwent effective and specific assembly and interaction during MIP synthesis. Figure 1 As shown in Figure C, the absorbance changes before and after template elution were compared using UV-Vis. The results show that after eluent treatment, the characteristic absorbance of the Phe template molecule in the polymer significantly decreased, objectively proving that the Phe template molecule within the polymer backbone had been effectively stripped, successfully releasing the specific imprinted cavity. Figure 1As shown in Figure D, EDX analysis revealed a significant attenuation in the signal intensity of carbon (C) in the template-eluted polymer. This data further confirms, at the elemental composition level, that the carbon-rich Phe template molecules have been eluted, and the imprinted cavity has been successfully prepared.

[0052] To verify the target-binding ability of the prepared Phe-MIP, gradient adsorption experiments and kinetic evaluations were conducted. The results showed that Phe-MIP exhibited excellent adsorption response over a very wide concentration range. Figure 2 As shown in Figures A and B, the absorbance exhibits a good linear relationship within the high concentration range of 500 μM to 20 mMPhe; Figure 2 As shown in Figure C, even at extremely low concentrations (covering the physiological concentration range of urine) ranging from 5 μM to 500 μMPhe, this material still maintains sensitive adsorption and binding capabilities. Figure 2 The adsorption kinetics curve shown in Figure D further demonstrates that Phe-MIP has an extremely fast mass transfer rate, reaching thermodynamic equilibrium of adsorption capacity within a very short time (5 minutes), which provides direct data support for achieving point-of-care testing (POCT).

[0053] 2. Nucleic acid loading process and kinetic threshold construction This embodiment systematically validated and optimized the reaction conditions and elution process for loading ssDNA probes onto the surface of MIPs. The specific loading procedure was as follows: 1 mg / mL MIP nanoparticles were dispersed in a loading solution containing specific metal ions, the ssDNA probe was added, and the mixture was incubated at 37°C and 200 rpm for a specific time; subsequently, centrifugation, washing, and dynamic occupancy treatment were performed. The experimental results of the optimized conditions are as follows: Probe concentration optimization, such as Figure 3 Figure A shows the effect of incubation with different concentrations of ssDNA from 0.5 μM to 10 μM on the detection signal. The results show that the signal-to-noise ratio (S / N) of the detection system reaches its peak when the probe concentration is 1 μM. Too low a concentration leads to insufficient effective loading within the blot cavity; too high a concentration exacerbates the non-specific adsorption of the probe on the polymer surface, resulting in a significant increase in background fluorescence. Therefore, 1 μM is preferred as the standard loading concentration.

[0054] Figure 3 Incubation time was optimized in the study, and the effects of different loading times from 0.5 hours to 4 hours on the effective probe load and release rate were investigated. The results showed that the system response value was highest at 30 minutes of incubation; however, the signal progressively decreased with increasing incubation time. This data objectively confirms the existence of a reverse dynamic equilibrium process between binding and leakage at the solid-liquid interface, meaning that prolonged incubation leads to secondary detachment of the already bound probe. Therefore, the optimal incubation kinetic window was cut off at 30 minutes.

[0055] To establish the optimal initial nucleic acid loading and elution buffer, such as Figure 3 As shown in Figure C, the experiment investigated cross-combinations of different buffers used as loading and elution media. The buffers investigated included: standard PBS buffer, PBST buffer (i.e., PBS with 1% Tween-20 added), and PBSM buffer (i.e., PBS with 10 mM divalent magnesium ions added). The results showed that when standard PBS buffer was used as both the loading and elution system (PBS group), the specific binding capacity of the Phe-MIP to the probe reached its maximum. This indicates that in the current Phe target system, the pure PBS environment provides the most suitable initial electrostatic and hydrogen bonding interaction network; while the additional introduction of nonionic surfactants (PBST group) or higher concentrations of divalent metal ions (PBSM group) failed to further enhance the specific confined binding of the probe, and may even have interfered with the micro-matching conformation formed based on template imprinting. Therefore, a fixed procedure based on pure PBS incubation and washing with pure PBS was established.

[0056] like Figure 3As shown in Figure D, this embodiment constructs and optimizes the elution process after MIP probe loading, breaking the inherent thermodynamic constraint between high background and low positive signal at the solid-liquid interface. Conventional PBS washing group: The probes were washed three times with conventional PBS buffer after incubation. Results showed that with increasing washing cycles, both the positive signal and background leakage decreased synchronously, leading to a deterioration in the overall signal-to-noise ratio and failing to fundamentally solve the high background problem caused by non-specific adsorption of probes at surface defect sites. To further remove surface background, a 5-minute PBS blank incubation (simulating the main source of background without target detection) was introduced after PBS washing. Although this step eluted a large number of weakly bound background probes and improved the signal-to-noise ratio, prolonged background elution caused a significant desorption and loss of effective probes within the blot cavity, resulting in a substantial attenuation of the overall positive signal, which could not meet the sensitivity requirements of the downstream CRISPR system. Construction of a reverse dynamic occupancy system: To address the aforementioned contradiction, this method creatively introduces low-concentration (0.2× and 0.5× of the target loading concentration) homologous probe solutions for 5 minutes of reverse incubation after 5 minutes of competition with a PBS blank, followed immediately by centrifugation for phase separation. Experiments show that the 0.5× concentration of homologous probes achieved the best results. The rapid introduction of low-concentration probes enabled reverse occupancy at the microscopic interface, with free probes preferentially and rapidly occupying the weakly binding competitive sites on the MIP surface, thereby stabilizing the background leakage at a controllable low level. Simultaneously, this operation constructed an appropriate concentration buffer, curbing further loss of deep effective probes. Under this optimized microscopic energy state, the subsequent target molecule Phe can more easily and efficiently trigger the steric hindrance competition mechanism, releasing a large amount of probes bound to the MIP surface. Ultimately, this resulted in a significant enhancement of the positive signal and stable suppression of the background, achieving an optimal signal-to-noise ratio.

[0057] 3. Competition release and cascading amplification To establish a small molecule detection system based on MIP competitive release and CRISPR / Cas12a cascade amplification, this embodiment systematically optimized the MIP-DNA dosage, target competitive release conditions, and CRISPR / Cas12a reaction system. Figure 4 As shown in Figure A, with the increase of MIP-DNA dosage, the amount of ssDNA released induced by the target gradually increases, and the fluorescence response of the system continuously enhances. When the MIP-DNA dosage reaches 1.6 mg, the detection system achieves the highest signal-to-noise ratio; with further increases in dosage, the background signal of the system increases synchronously, leading to a decrease in the overall signal-to-noise ratio. This indicates that appropriately increasing the amount of MIP-DNA is beneficial to improving the amount of nucleic acid released by the target, but excessive dosage will increase non-specific background release. Therefore, 1.6 mg was determined to be the optimal MIP-DNA dosage.

[0058] like Figure 4 As shown in Figure B, the reaction temperature and reaction time during the competitive release process were optimized. At 25℃, the overall fluorescence response of the system was low, indicating limited competitive exchange efficiency between the target molecule and the imprinted cavity. However, at 37℃, the release efficiency significantly improved, achieving the highest fluorescence response and maximum signal-to-noise ratio at 5 min. Further extending the reaction time resulted in a gradual decrease in the detection signal, indicating a dynamic equilibrium between the released probe and the polymer interface. Excessive incubation time is detrimental to maintaining the effective concentration of the released probe. These results demonstrate that an optimal competitive release kinetic window exists in this system. Therefore, incubation at 37℃ for 5 min was determined as the optimal competitive release condition, and this 5-min incubation time is consistent with the previous adsorption kinetic results, indicating that the competitive release is based on a highly efficient Phe-MIP mass transfer rate.

[0059] like Figure 4 As shown in Figure C, the concentration of Cas12a in the CRISPR / Cas12a system was optimized. The results showed that the fluorescence response of the system continuously increased with increasing Cas12a concentration. When the Cas12a concentration reached 80 nM, the system achieved the highest detection signal and the optimal signal-to-noise ratio; further increases in Cas12a concentration did not significantly improve the signal. This indicates that 80 nM Cas12a is sufficient to respond adequately to the released ssDNA activation signal and achieve efficient trans-cleavage; therefore, 80 nM was determined to be the optimal working concentration of Cas12a.

[0060] like Figure 4 As shown in Figure D, the assembly ratio of Cas12a and crRNA was optimized. The results showed that different assembly ratios significantly affected the formation efficiency of the Cas12a / crRNA complex. The system achieved the highest fluorescence response when the molar ratio of Cas12a to crRNA was 1:2, significantly better than the 1:1.5 group and the negative control group (NTC). This indicates that an appropriate excess of crRNA promotes the full assembly of Cas12a and crRNA, improves the formation efficiency of the active complex, and thus enhances the subsequent trans-cleavage ability. Therefore, the optimal assembly ratio of Cas12a to crRNA was determined to be 1:2.

[0061] Based on the optimization results above, it was finally determined that 1.6 mg of MIP-DNA was used for competitive target release, and the reaction was carried out at 37℃ for 5 min. A CRISPR / Cas12a detection system was constructed using 80 nM Cas12a and a Cas12a molar ratio of 1:2. Under these conditions, the system achieved the highest signal-to-noise ratio and optimal detection performance, providing a stable and reliable experimental basis for subsequent highly sensitive detection of small molecules.

[0062] 4. Specificity and selectivity To verify the specific recognition ability of the constructed Phe-MIP for the target molecule Phe and the selectivity of the MIP-CRISPR / Cas12a detection system, several structurally similar amino acids were selected as interfering agents in an artificial urine matrix for evaluation. The selected interfering agents included Trp, His, Ala, and Tyr. Trp and Tyr both contain aromatic ring structures and have high structural similarity to Phe; His contains an imidazole side chain; and Ala is a typical aliphatic amino acid. Furthermore, a mixed amino acid sample (AA) composed of Phe, Trp, and Tyr was set up to simulate the coexistence interference environment in complex biological samples.

[0063] like Figure 5 As shown in Figure A, the recognition specificity of Phe-MIP for different amino acids was evaluated by LC-MS / MS determination. The results showed that Phe-MIP exhibited the highest adsorption capacity for the target molecule Phe, significantly higher than structural analogs such as Trp, His, Ala, and Tyr. Although Trp and Tyr contain aromatic side chains, their adsorption capacities were still significantly lower than Phe, indicating that the recognition cavity formed during imprinting polymerization depends not only on molecular size matching but also on the combined influence of functional group spatial distribution and interaction modes. These results demonstrate that Phe-MIP possesses excellent molecular recognition ability and high selective adsorption characteristics.

[0064] like Figure 5 As shown in Figure B, the above amino acid samples were further detected using the MIP-CRISPR / Cas12a system under the same conditions. The results showed that the Phe group produced the strongest fluorescence response, while the Trp, His, Ala, and Tyr groups all produced only low-level background signals. Figure 5 The adsorption selectivity trend reflected in group A remained consistent. This indicates that only the target molecule Phe can effectively compete for the blotted site and trigger ssDNA release, thereby activating the Cas12a trans-cleavage activity and generating a significant fluorescence signal. For group AA, the fluorescence response remained at a high level, indicating that even under conditions where multiple structural analogs coexist, Phe can still be preferentially recognized and effectively trigger the detection system.

[0065] The above results demonstrate that the Phe-MIP constructed in this invention not only possesses excellent selective adsorption capacity but also effectively transfers this molecular recognition specificity to the CRISPR / Cas12a signal amplification process, achieving highly selective detection of target molecules. Even in complex biological matrices and environments with multiple analogues, the system maintains good anti-interference capabilities and detection accuracy.

[0066] 5. Detection performance evaluation To evaluate the quantitative detection performance of the MIP-CRISPR / Cas12a detection system for Phe, concentration gradient detection experiments were conducted in PBS buffer and artificial urine matrix. Following the established standard detection procedure, different concentrations of Phe samples were incubated with MIP-DNA to complete the competitive release reaction. The supernatant was collected and added to the CRISPR / Cas12a detection system for fluorescence detection. By recording fluorescence kinetic curves and calculating dF / dT as a quantitative analysis index, a quantitative relationship between Phe concentration and detection signal was established.

[0067] like Figure 6 As shown in Figure A, in the PBS buffer system, as the Phe concentration gradually increased from 0.1 μM to 500 μM, the fluorescence response of the detection system gradually increased, and the kinetic curves corresponding to different concentrations of samples could be clearly distinguished. With the increase of the target molecule concentration, the amount of released ssDNA increased, thereby activating more Cas12a / crRNA complexes and improving trans-cleavage efficiency, which was manifested as a continuous increase in the fluorescence growth rate.

[0068] like Figure 6 As shown in Figure B, linear regression analysis was performed using dF / dT as the quantitative analysis index. The results showed that within the investigated concentration range, Phe concentration and dF / dT exhibited a good linear relationship, with a linear correlation coefficient (R) of over 0.99, indicating that the detection system of this invention has excellent quantitative analysis capabilities.

[0069] To further verify the applicability of the system in complex biological matrices, detection experiments with the same concentration gradient were conducted in an artificial urine matrix. For example... Figure 6 As shown in Figure C, different concentrations of Phe samples also produced distinct fluorescence kinetic responses, with the overall trend consistent with the PBS system. The results indicate that background components in artificial urine do not significantly affect the competitive release process of MIP and the subsequent CRISPR / Cas12a signal amplification process, demonstrating good matrix tolerance of the system.

[0070] like Figure 6 As shown in Figure D, after linear fitting of the detection results in artificial urine, the Phe concentration and dF / dT maintained a good linear relationship, with R exceeding 0.99. Further, the limit of detection (LOD) was calculated based on LOD = 3σ / S, where σ is the standard deviation of the negative control and S is the slope of the calibration curve. The calculated LOD for Phe using the detection system of this invention is 0.0741 μM. This LOD is significantly lower than the clinically relevant concentration range of phenylalanine in urine, meeting the requirements for trace detection.

[0071] The above results show that the MIP-CRISPR / Cas12a cascade detection system constructed in this invention exhibits good linearity, high detection sensitivity, and excellent matrix adaptability in both PBS buffer and artificial urine matrix, enabling rapid quantitative detection of phenylalanine.

[0072] 6. Methodological Validation To further evaluate the methodological performance of the MIP-CRISPR / Cas12a detection system of this invention, its precision and accuracy were systematically investigated in an artificial urine matrix. The results are shown in Tables 1-3.

[0073] As shown in Table 1, under the same batch experimental conditions, Phe samples at concentration levels of 1, 10, 100, and 1000 μM were measured six times repeatedly, with the endpoint fluorescence signal (Fluorescence) and dF / dT used as evaluation indicators. The results showed that the RSD of Fluorescence was 1.41%–3.48%, and the RSD of dF / dT was 2.17%–8.89%, both less than 10%. This indicates that the detection system of this invention has good intra-batch precision and detection stability.

[0074] Table 1. Intra-batch precision evaluation of the MIP-CRISPR / Cas12a detection system (n=6) To further evaluate the stability of the detection system across different experimental batches, Phe samples at three concentration levels (10, 100, and 500 μM) were independently and repeatedly tested in an artificial urine matrix. The results are shown in Table 2. The measured concentrations of each sample showed good consistency with the theoretical concentrations, with inter-batch RSDs ranging from 8.37% to 12.80%, all below 15%. These results demonstrate that the detection system of this invention maintains stable detection performance under different batch experimental conditions, exhibiting good inter-batch precision.

[0075] Table 2. Inter-batch precision evaluation of the MIP-CRISPR / Cas12a detection system (n=9) To evaluate the detection accuracy of the detection system of this invention, three concentration levels of samples were constructed in an artificial urine matrix to simulate healthy individuals (30 μM), patients with mild phenylketonuria (120 μM), and patients with classic phenylketonuria (600 μM). The results are shown in Table 3. The detection recoveries for each concentration level ranged from 92.80% to 98.26%, with corresponding RSDs of 7.90% to 10.30%, all below 15%. These results indicate that the detection system of this invention possesses good detection accuracy and matrix adaptability.

[0076] Table 3. Accuracy evaluation of Phe detection in artificial urine (n=6) The concentration range examined in this embodiment covers the typical concentration range of phenylalanine in the urine of healthy individuals, patients with mild phenylketonuria, and patients with classic phenylketonuria. This demonstrates that the MIP-CRISPR / Cas12a detection system constructed in this invention can meet the detection needs of phenylalanine levels under different disease states and has good application potential.

[0077] Example 3: Application Case - Detection of Cortisol in Sweat Matrix I. Experimental Materials 1. Target small molecule Cortisol (Cort) was purchased from Sigma-Aldrich.

[0078] 2. MIP synthesis reagents Functional monomer: 4-vinylpyridine (4-VP), purity ≥96%, purchased from Shanghai Yuanye Biotechnology.

[0079] Crosslinking agent 1: DVB, 80% purity, purchased from Aladdin.

[0080] Crosslinking agent 2: Trimethylolpropane trimethacrylate (TRIM), purity ≥98%, purchased from Shanghai Yuanye Biotechnology.

[0081] Initiator: AIBN, 99% purity, purchased from Aladdin.

[0082] Solvents and elution reagents: methanol, acetonitrile, and acetic acid, all HPLC grade, purchased from Merck; ultrapure water was prepared using a Milli-Q water purifier.

[0083] 3. Nucleic acid and CRISPR / Cas12a system Cas12a protein: LbCas12a, 100 μM, purchased from New England Biolabs (NEB).

[0084] Single-stranded DNA (ssDNA, SEQ ID NO.1): sequence 5'-CAATAGCTTATCAGACTGTCT-3', synthesized by Beijing BGI.

[0085] Guide RNA (crRNA, SEQ ID NO.2): sequence 5'-UAAUUUCUACUAAGUGUAGAUAGACAGUCUGAUAAGCUAUUG-3'.

[0086] Single-chain fluorescent quenching probe (FQ probe): sequence and modification are 5'-FAM-TTATT-BHQ1-3'.

[0087] 4. Buffer system and matrix Nucleic acid loading buffer: 10 mM Tris, 2 mM MgCl2, pH 7.5.

[0088] CRISPR reaction buffer (1×NEB r2.1): 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 100 μg / ml BSA, pH 7.9.

[0089] Test matrix: artificial sweat, purchased from Shanghai Yuanye Biotechnology, product number R26245.

[0090] II. Experimental Methods and Results 1. Synthesis and Characterization of Cort-MIP Weigh 34 mg of cortisol and add it to 15 mL of acetonitrile. After ultrasonic dissolution, add 40 μL of 4-VP and 34 μL of TRIM sequentially, and pre-assemble at room temperature in the dark for 30 min. Then, magnetically stir for 10 min until the system is completely dissolved to obtain a homogeneous and transparent prepolymer solution. Transfer the prepolymer solution to an anaerobic chamber, and add 600 μL of DVB and 40 mg of AIBN under anaerobic and dark conditions. Continue stirring for 5 min to ensure uniform dispersion. Then, perform anaerobic purging of the reaction system and quickly tighten the thick-walled reaction flask with a PTFE gasket to achieve an airtight seal. Remove the reaction flask from the anaerobic chamber and place it in a 60°C constant temperature water bath for polymerization at 200 rpm for 18 h. After the reaction was completed and cooled to room temperature, the template was eluted twice with methanol / acetic acid (8:2, v / v) eluent for 4 hours each time; then eluted twice more with methanol / acetic acid (9:1, v / v) eluent for 4 hours each time; finally, pure methanol was used for continuous elution until cortisol was undetectable in the eluent. The polymer was collected and vacuum dried to obtain the cortisol molecularly imprinted polymer (Cort-MIP).

[0091] After polymerization and elution with template molecules, the resulting Cort-MIP was characterized by SEM. Figure 7 As shown in Figure A, the obtained Cort-MIP exhibits a distinct particle aggregation structure, with a three-dimensional network structure formed by the accumulation of numerous microspheres, indicating that the precipitation polymerization method successfully obtained well-dispersed polymer particles. Figure 7As shown in Figure B, at higher magnification, the Cort-MIP surface is relatively rough, with abundant pores and depressions. This structure facilitates the formation of imprinted recognition sites after template molecule elution and increases the mass transfer efficiency of target molecules, thereby improving the polymer's binding capacity and recognition efficiency for cortisol. SEM results indicate that the prepared Cort-MIP possesses typical molecularly imprinted polymer morphology characteristics, providing a good structural basis for the subsequent rapid recognition and adsorption of cortisol molecules.

[0092] 2. Evaluation of Cort-MIP Adsorption and Recognition Performance To evaluate the recognition and binding ability of the prepared Cort-MIP to cortisol, adsorption experiments were conducted by adding Cort-MIP to solutions of cortisol at different concentrations. The change in cortisol concentration in the solution before and after adsorption was measured by ultraviolet spectrophotometry, and Qe was calculated. Further analysis was performed on adsorption kinetics, adsorption isotherms, and adsorption model fitting.

[0093] like Figure 8 As shown in Figure A, the adsorption capacity of Cort-MIP increases rapidly upon contact with cortisol, achieving rapid binding in the early stages of the reaction and reaching adsorption equilibrium in approximately 5 minutes. The results indicate that the constructed imprinted sites have good accessibility, allowing the target molecule to rapidly diffuse to the recognition site and complete the binding process, meeting the recognition time requirements of rapid detection systems. Figure 8 As shown in Figure B, a good linear relationship was obtained after fitting the adsorption kinetic experimental data using a pseudo-second-order kinetic model. The results indicate that the adsorption process of cortisol by Cort-MIP is mainly controlled by the specific interaction between the imprinted recognition site and the target molecule, rather than a simple physical adsorption process, suggesting that specific binding sites capable of effectively recognizing cortisol are formed in the polymer. Figure 8 As shown in Figure C, the adsorption capacity of Cort-MIP gradually increased with the increase of cortisol concentration from 0.1 nM to 10 μM. When the cortisol concentration further increased, the growth trend of adsorption capacity gradually slowed down, indicating that the imprinted recognition sites on the polymer surface gradually reached saturation. Figure 8 As shown in Figure D, the Langmuir model was used to fit the adsorption isotherm data, yielding a good fit. This indicates that the distribution of recognition sites on the Cort-MIP surface is relatively uniform, and the adsorption process conforms to the monolayer adsorption model. The maximum adsorption capacity (Qmax) calculated according to the Langmuir model is approximately 13.6 mg / g, indicating that the constructed Cort-MIP has high cortisol binding and enrichment capabilities.

[0094] The results show that the constructed Cort-MIP can effectively identify and enrich cortisol within a wide concentration range, covering the physiological concentration of cortisol in sweat from 0.1 nM to 10 μM.

[0095] 3. Feasibility and performance evaluation of the MIP-Cas12a detection system A Cort-MIP-DNA complex recognition system was constructed, in which nucleic acid signal probes were immobilized on the surface of Cort-MIPs through physical adsorption or interfacial interaction, forming a stable MIP-DNA complex structure. Based on this, cortisol standard solutions of different concentrations (0.1 nM–1 μM) were added to the reaction system and incubated at 37°C to induce specific competitive binding of target molecules to the imprinted sites within the MIPs, thereby releasing surface-bound DNA. After the reaction, 10 μL of the supernatant was added to the Cas12a detection system (containing Cas12a protein, crRNA, and FQ fluorescent reporter probe), and fluorescence signal changes were monitored in real time at 37°C. Cas12a was activated upon recognizing the target DNA and non-specifically cleaved the FQ probe, generating an amplified fluorescence signal. The fluorescence-time curve was recorded, and dF / dT was calculated to achieve quantitative detection of the target molecule.

[0096] like Figure 9 As shown in Figure A, without the addition of cortisol, the nucleic acid probe stably binds to the surface of the Cort-MIP, and the DNA content in the supernatant is low, indicating that the MIP has a good immobilization ability for the probe. When cortisol is added, the target molecule enters the MIP recognition site and competes with the template molecule for binding, thereby disrupting the original molecularly imprinted binding site and releasing the DNA bound to the surface. Experimental results show that the DNA concentration in the supernatant increases significantly, while the residual DNA on the MIP surface decreases. Furthermore, when the released DNA is introduced into the Cas12a system, a significant enhancement of the fluorescence signal is observed, indicating that the released DNA successfully activates the trans-cleavage activity of Cas12a, achieving effective transduction from small molecule recognition signal to nucleic acid amplification signal. This result demonstrates that this system possesses a complete and feasible pathway for small molecule recognition to trigger competitive release and subsequently trigger enzyme cleavage amplification.

[0097] like Figure 9As shown in Figure B, this embodiment compares the effects of five different buffer systems on probe immobilization and release to evaluate the relationship between interfacial binding strength and signal output. Experimental results show that when McIlvaine buffer is used, the overall fluorescence release is significantly suppressed. This indicates that the nucleic acid probe undergoes excessively strong non-specific adsorption on the Cort-MIP surface, making it difficult for target molecules to effectively compete for replacement, thus reducing release efficiency. Conversely, when sodium-free phosphate buffer or conventional PBS buffer is used, the lack of divalent metal ions (such as Mg) that can stabilize interfacial binding reduces the binding stability of the nucleic acid probe on the MIP surface, making it prone to spontaneous detachment. This results in a high background signal even under target-free conditions, thus reducing the overall signal-to-noise ratio. Introducing Tris buffer containing divalent magnesium ions significantly improves the interfacial binding state of the system. Among the tested parameters, the probe immobilization and release reached a dynamic equilibrium at a MgCl concentration of 2 mM, exhibiting the highest signal-to-noise ratio. However, when the MgCl concentration was further increased to 5 mM, the DNA release process was inhibited due to the excessively strong coordination bridging effect of metal ions on the DNA-MIP interface, resulting in a decrease in the positive signal. Therefore, this system determined 2 mM MgCl as the optimal reaction condition, thereby establishing a suitable thermodynamic binding threshold at the solid-liquid interface and achieving a balance between stable probe immobilization and target-induced release.

[0098] like Figure 9 As shown in Figure C, under different concentrations of cortisol, the fluorescence signal of the Cas12a system gradually increased over time, and its growth rate increased with increasing concentration. This result indicates that with increasing cortisol concentration, the amount of DNA released by the MIP increases synchronously, leading to an increased Cas12a trans-cleavage rate, ultimately manifested as enhanced fluorescence signal. This dynamic response demonstrates that the system exhibits good time- and concentration-dependent signal output capabilities.

[0099] like Figure 9 As shown in Figure D, using dF / dT as a quantitative indicator, a good linear relationship (R≈0.98) was observed between cortisol concentration and dF / dT in the range of 1 nM–1 μM. This result demonstrates that the MIP-Cas12a system constructed in this invention can achieve stable quantitative detection through kinetic parameters, rather than relying solely on the endpoint fluorescence value, thereby improving the repeatability and anti-interference ability of the detection.

[0100] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. A small molecule detection system based on molecularly imprinted polymer-CRISPR / Cas12a cascade amplification, characterized in that, The small molecules refer to organic compounds with a molecular weight of less than 1500 Da, and the detection system comprises: (1) A molecularly imprinted polymer prepared using the small molecule as a template; (2) Single-stranded DNA that can bind to the molecularly imprinted polymer and can be competitively replaced by the small molecule and thus released from the molecularly imprinted polymer; (3) CRISPR / Cas12a system, wherein the CRISPR / Cas12a reaction system comprises Cas12a protein, crRNA, and fluorescent reporter probe; in: The crRNA can assemble with the Cas12a protein to form a Cas12a-crRNA complex and contains a guide sequence that can complementarily pair with the single-stranded DNA; the single-stranded DNA released from the molecularly imprinted polymer can bind complementaryly to the crRNA to activate the trans-cleavage activity of the Cas12a protein. The fluorescent reporter probe is a single-stranded DNA with fluorescent and quenching groups modified at both ends, respectively. The Cas12a protein, whose trans-cleavage activity is activated, can non-specifically cleave the single-stranded DNA of the fluorescent reporter probe, thereby relieving the quenching effect and outputting a fluorescent detection signal.

2. The small molecule detection system according to claim 1, characterized in that: The functional monomers used to prepare the molecularly imprinted polymer are selected from one or more of o-phenylenediamine, methacrylic acid, 4-vinylpyridine, acrylamide, trifluoromethacrylic acid, and N-isopropylacrylamide.

3. The small molecule detection system according to claim 2, characterized in that: The crosslinking agent used to prepare the molecularly imprinted polymer is selected from one or more of divinylbenzene, trimethylolpropane trimethacrylate, N,N'-methylenebisacrylamide, and polyethylene glycol dimethacrylate.

4. The small molecule detection system according to claim 1, characterized in that: The sequence of the single-stranded DNA is shown in SEQ ID NO.1, and the sequence of the crRNA is shown in SEQ ID NO.

2.

5. The small molecule detection system according to claim 1, characterized in that: The fluorescent reporter probe has a fluorophore of FAM and a quencher of BHQ1, which are linked by a TTATT sequence.

6. The small molecule detection system according to claim 1, characterized in that, It also includes a buffer solution for binding the single-stranded DNA to the molecularly imprinted polymer.

7. The use of the small molecule detection system according to any one of claims 1 to 6 in the preparation of a detection reagent for determining the content of phenylalanine or cortisol in a sample.

8. The application according to claim 7, characterized in that: First, establish a linear relationship between the concentration of phenylalanine or cortisol and the fluorescence growth rate. Then, calculate the concentration of phenylalanine or cortisol in the sample based on this linear relationship and the fluorescence growth rate of the sample.

9. The application according to claim 7 or 8, characterized in that: The sample is a biological sample.

10. The application according to claim 9, characterized in that: The biological sample is urine or sweat.

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