Escherichia coli ph-specific aptamer, magnetically driven fluorescent micro-machines and uses thereof

Abstract

This invention discloses a pH-specific nucleic acid aptamer for *E. coli*, a magnetically driven fluorescent micromotor, and their applications, belonging to the field of pathogen detection technology. The nucleic acid aptamers include Apt-A suitable for acidic conditions, Apt-B suitable for neutral conditions, and Apt-C suitable for alkaline conditions. The magnetically driven fluorescent micromotor prepared using the above-mentioned pH-specific nucleic acid aptamers can be used for the detection and sterilization of *E. coli* in food, achieving accurate detection in complex food matrices without adjusting the sample pH, with a detection limit as low as 3.89 CFU / mL. Simultaneously, the micromotor integrates the photothermal effect of polydopamine, achieving over 99.9% bacterial inactivation within 60 seconds under near-infrared irradiation, realizing integrated "detection-sterilization". The magnetically driven fluorescent micromotor of this invention has advantages such as strong pH adaptability, high detection sensitivity, excellent sterilization efficiency, and good motion controllability, and has promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pathogen detection technology, specifically relating to pH-specific nucleic acid aptamers for Escherichia coli, magnetically driven fluorescent micromotors, and their applications. Background Technology

[0002] Foodborne illnesses have become a major threat to global public health. According to the World Health Organization, approximately 600 million people fall ill each year from consuming contaminated food, with pathogens (such as Escherichia coli O157:H7) being a primary cause. Therefore, developing integrated technologies for rapid, accurate, and in-situ detection and sterilization of pathogens is crucial for ensuring food safety and reducing public health risks.

[0003] Currently, the detection methods for foodborne pathogens are mainly divided into three categories: traditional biochemical culture methods, molecular biological detection techniques, and emerging biosensor technologies.

[0004] 1. Traditional biochemical culture method

[0005] Colony counting (CFU) and selective medium isolation and identification are considered the "gold standard" for pathogen detection. However, these methods have long detection cycles (usually 24-72 hours) and are cumbersome to operate, failing to meet the needs of rapid on-site testing.

[0006] 2. Molecular biological detection technology

[0007] Methods such as polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) have the advantages of high sensitivity and specificity. However, these methods rely on sophisticated instruments and professional operators, and require complex sample pretreatment (such as nucleic acid extraction), making them difficult to port and implement in real-time online monitoring.

[0008] 3. Biosensing technology

[0009] In recent years, nucleic acid aptamer-based biosensing technology has become a research hotspot in the field of pathogen detection due to its advantages such as high affinity, ease of chemical modification, and good stability. Aptamers are usually obtained through systematic evolutionary exponential enrichment of ligands (SELEX) screening, and specific aptamers against a variety of pathogens have been developed.

[0010] However, existing aptamers and their sensing systems still face the following technical bottlenecks in practical applications:

[0011] (1) Poor pH adaptability and insufficient detection stability

[0012] Traditional SELEX screening is usually performed under a single pH condition (such as pH 7.4). The resulting aptamers are prone to conformational changes in complex food matrices (such as the acidic environment of fruit juice or the weakly alkaline environment of kelp soup), which leads to a significant decrease in binding affinity and makes it difficult to guarantee the accuracy and reliability of the detection results.

[0013] (2) The detection function is limited and lacks integrated processing capability.

[0014] Existing detection platforms are mostly limited to a single detection function of "identification-signal output," and cannot achieve in-situ inactivation of contaminants. Positive samples still need to be transferred to sterilization equipment for processing after detection, which increases the risk of secondary contamination and processing time.

[0015] (3) Passive diffusion limitation, resulting in low detection efficiency.

[0016] Traditional nanoprobes rely on Brownian motion and random collisions with targets in liquid foods. In particular, they have low diffusion efficiency in high-viscosity food matrices such as milk, making it difficult to actively capture and enrich trace amounts of pathogens, thus limiting further improvements in detection sensitivity.

[0017] Therefore, how to obtain a pathogen detection system that can adapt to different pH food environments, has active movement capabilities, and integrates detection and sterilization functions has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0018] In view of the problems existing in the prior art, the purpose of this invention is to provide a pH-specific nucleic acid aptamer for Escherichia coli, a magnetically driven fluorescent micromotor, and their applications.

[0019] To achieve the above objectives, the present invention adopts the following technical solution:

[0020] A pH-specific nucleic acid aptamer for Escherichia coli, wherein the nucleic acid aptamer is any one of the following:

[0021] (a) The aptamer Apt-A, which is suitable for acidic pH conditions, has the nucleic acid sequence shown in SEQ ID NO:1;

[0022] (b) The aptamer Apt-B, which is suitable for neutral pH conditions, has the nucleic acid sequence shown in SEQ ID NO:2;

[0023] (c) The aptamer Apt-C, which is suitable for alkaline pH conditions, has the nucleic acid sequence shown in SEQ ID NO:3.

[0024] Based on the above scheme, the acidic pH condition is pH 3.0-4.0, the neutral pH condition is pH 6.0-7.5, and the alkaline pH condition is pH 7.5-8.5.

[0025] The above-mentioned uses of the Escherichia coli pH-specific nucleic acid aptamer are for preparing reagents for detecting Escherichia coli content or for sterilizing Escherichia coli.

[0026] A magnetically driven fluorescent micromotor, wherein the magnetically driven fluorescent micromotor is formed by loading a magnetic fluorescent molecular probe onto Spirulina through electrostatic adsorption;

[0027] The magnetic fluorescent molecular probe is formed by using magnetic beads coated with polydopamine as the core, covalently coupling a double-stranded DNA A1@A2, and then connecting A2 in the double-stranded DNA A1@A2 to streptavidin-modified carbon quantum dots via non-covalent bonds.

[0028] The nucleic acid sequence of A1 in the DNA double strand A1@A2 is any one of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, and its 5' end is modified with a carboxyl group; A2 in the DNA double strand A1@A2 is partially complementary to A1, and its 5' end is modified with biotin.

[0029] Based on the above scheme, when the sequence of A1 in the DNA double strand A1@A2 is as shown in SEQ ID NO:4, the sequence of A2 is as shown in SEQ ID NO:5;

[0030] When the sequence of A1 in the DNA double strand A1@A2 is as shown in SEQ ID NO:6, the sequence of A2 is as shown in SEQ ID NO:7;

[0031] When the sequence of A1 in the DNA double strand A1@A2 is as shown in SEQ ID NO:8, the sequence of A2 is as shown in SEQ ID NO:9.

[0032] Based on the above scheme, the preparation method of the magnetically driven fluorescent micromotor includes the following steps:

[0033] (1) React magnetic beads with dopamine hydrochloride in Tris-HCl buffer to form MBs@PDA;

[0034] (2) Mix the aptamer A1 modified with the carboxyl group at the 5' end and the complementary strand A2 modified with the biotin at the 5' end in an equal molar ratio and anneal to form A1@A2-DNA double strand;

[0035] (3) Carbon quantum dots were prepared by hydrothermal reaction using urea and citric acid as precursors and modified with streptavidin to obtain CQDs-SA;

[0036] (4) The A1@A2-DNA double strand from step (2) is immobilized on the surface of MBs@PDA from step (1) by EDC / NHS chemical method, and then CQDs-SA from step (3) is added. CQDs-SA is linked to A2 by biotin-streptavidin to obtain a magnetic fluorescent molecular probe.

[0037] (5) The cleaned and sterilized Spirulina and the magnetic fluorescent molecular probe prepared in step (4) are mixed and incubated in a buffer solution. The probe is loaded onto the surface of Spirulina by electrostatic adsorption to obtain a magnetically driven fluorescent micromotor.

[0038] The aforementioned magnetically driven fluorescent micromotor is used to detect or kill E. coli in food.

[0039] Based on the above scheme, the method for detecting the content of E. coli in food is as follows:

[0040] (1) Determine the pH value of the food matrix; select the appropriate magnetically driven fluorescent micromotor based on the determined pH value:

[0041] When the pH is 3.0-4.0, the magnetically driven fluorescent micromotor with sequence A1 as shown in SEQ ID NO:4 is selected;

[0042] When the pH is 6.0-7.5, the magnetically driven fluorescent micromotor with sequence A1 as shown in SEQ ID NO:6 is selected;

[0043] When the pH is 7.5-8.5, the magnetically driven fluorescent micromotor with sequence A1 as shown in SEQ ID NO:8 is selected;

[0044] (2) The selected magnetically driven fluorescent micromotor was incubated with the food matrix, the fluorescence intensity of the supernatant was detected, and the concentration of Escherichia coli was calculated by referring to the standard curve.

[0045] Based on the above approach, the method for eliminating E. coli in food comprises the following steps:

[0046] (1) Determine the pH value of the food matrix; select the appropriate magnetically driven fluorescent micromotor based on the determined pH value:

[0047] When the pH is 3.0-4.0, the magnetically driven fluorescent micromotor with sequence A1 as shown in SEQ ID NO:4 is selected;

[0048] When the pH is 6.0-7.5, the magnetically driven fluorescent micromotor with sequence A1 as shown in SEQ ID NO:6 is selected;

[0049] When the pH is 7.5-8.5, the magnetically driven fluorescent micromotor with sequence A1 as shown in SEQ ID NO:8 is selected;

[0050] (2) Mix the magnetically driven fluorescent micromotor with the food matrix at room temperature and irradiate with 808nm, 2W NIR for 30-60 seconds.

[0051] Based on the above scheme, the magnetically driven fluorescent micromotor is mixed with the food matrix under the drive of an external rotating magnetic field to enhance the movement capability of the micromotor and the target capture efficiency.

[0052] Advantages of the technical solution of this invention:

[0053] This invention, based on a rational design strategy using molecular dynamics simulations, successfully constructs pH-specific nucleic acid aptamers for *E. coli* covering acidic, neutral, and alkaline conditions. This solves the core problems of conformational instability and decreased binding affinity of traditional aptamers in food matrices with different pH values. The three pH-specific nucleic acid aptamers for *E. coli* cover a wide pH range, enabling accurate detection of *E. coli* content in real foods with different acid-base properties in most food matrices without pH adjustment.

[0054] The magnetically driven fluorescent micromotor constructed using the aforementioned pH-specific nucleic acid aptamer for *E. coli* integrates detection and sterilization functions. It can determine the *E. coli* content in the food matrix by detecting the fluorescence intensity of the supernatant, and can also achieve in-situ inactivation of *E. coli* under near-infrared irradiation, significantly improving the efficiency of the detection-sterilization process and effectively avoiding denaturation and nutrient loss in heat-sensitive food matrices caused by high-temperature sterilization. Furthermore, under the application of an external rotating magnetic field, the micromotor achieves helical directional motion, overcoming diffusion limitations in complex food matrices such as diluted milk, and improving target recognition efficiency. Therefore, the magnetically driven fluorescent micromotor of this invention has advantages such as strong pH adaptability, high detection sensitivity, excellent sterilization efficiency, and good motion controllability, and has significant application prospects in the detection and sterilization of *E. coli* in food. Attached Figure Description

[0055] Figure 1 DNA sequences and predicted secondary structures of aptamers Apt-A, Apt-B, and Apt-C;

[0056] Figure 2 Molecular docking of aptamer Apt-A with the target protein (a) and visualization analysis of key interactions (b);

[0057] Figure 3 Molecular docking of aptamer Apt-B with the target protein (a) and visualization analysis of key interactions (b);

[0058] Figure 4 Molecular docking of aptamer Apt-C with the target protein (a) and visualization analysis of key interactions (b);

[0059] Figure 5 Flow cytometry was used to verify the pH specificity of the three aptamers.

[0060] Figure 6 A schematic diagram (a) showing the four-coil setup on the microscope and a schematic diagram (b) showing the positions of the four coils.

[0061] Figure 7 Magnetic field distribution diagram simulated by a four-stage rotating magnetic field coil;

[0062] Figure 8 Image of the movement of the micromotor under a microscope when the four-stage coil drives the micromotor to move to the upper left.

[0063] Figure 9 Image of the movement of the micromotor under a microscope when the four-stage coil drives the micromotor to move to the lower right.

[0064] Figure 10 The results show the sensitivity detection of the Apt-A magnetically driven fluorescent micromotor.

[0065] Figure 11 This is a specific detection result for Apt-A magnetically driven fluorescent micromotors;

[0066] Figure 12 The Apt-A magnetically driven fluorescent micromotor provides in-situ sterilization performance;

[0067] Figure 13 To analyze the sterilization effect of Apt-A magnetically driven fluorescent micromotor treatment at 0 s (a) and 30 s (b) using PI staining flow cytometry;

[0068] Figure 14 Thermal images of Apt-A magnetically driven fluorescent micromotors of different masses after being irradiated with NIR light for different durations;

[0069] Figure 15 Line graphs showing temperature variations in the Apt-A magnetically driven fluorescent micromotor system under different experimental conditions;

[0070] Figure 16 The results of the biosafety assessment of the Apt-A magnetically driven fluorescent micromotor. Detailed Implementation

[0071] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and are not intended to limit the scope of the invention in any way.

[0072] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the experimental materials, reagents, and chemicals used in the following embodiments can be obtained through general channels.

[0073] Spirulina is a natural algae that can be used directly after being harvested from the sea without any additional processing. The spirulina used in the following examples was purchased from the Taobao store "Benchong Yiya Seafood Store".

[0074] The detection principle of the magnetically driven fluorescent micromotor for Escherichia coli described in this invention is as follows:

[0075] The magnetic fluorescent molecular probe is initially in a "fluorescence off" state. This is because the polydopamine (PDA) coating in the probe is extremely close to the carbon quantum dots (CQDs), and PDA, as a highly efficient fluorescence quencher, quenches the fluorescence of the CQDs through the fluorescence resonance energy transfer (FRET) mechanism.

[0076] When the micromotor comes into contact with the sample, the aptamer A1, immobilized on the probe surface, specifically recognizes and binds to the target protein on the surface of *E. coli*. Because the binding affinity of A1 to the target is significantly higher than its complementary pairing affinity with the complementary strand A2, a competitive displacement occurs: A1 detaches from A2 and binds to *E. coli*. The displaced A2-CQDs complex is released from the probe surface into the solution, escaping the short-range quenching effect of the PDA, and the fluorescence signal is recovered.

[0077] The fluorescence recovery intensity is positively correlated with the concentration of Escherichia coli in the solution. By detecting the fluorescence intensity of the supernatant, quantitative detection of Escherichia coli can be achieved.

[0078] The sterilization principle of the magnetically driven fluorescent micromotor for Escherichia coli described in this invention is as follows:

[0079] The polydopamine (PDA) coating on the surface of the micro-motor exhibits excellent photothermal conversion performance. When irradiated with 808 nm near-infrared (NIR) light, the PDA can efficiently absorb light energy and rapidly convert it into heat energy through non-radiative transitions, resulting in a sharp increase in local temperature.

[0080] Because the micromotor has specifically bound to the surface of *E. coli* via aptamer A1, the heat generated by the photothermal effect is highly concentrated around the bacteria, creating a localized high-temperature microenvironment. This high temperature acts on the bacteria, achieving sterilization primarily through the following mechanisms:

[0081] (1) It disrupts the integrity of the bacterial cell membrane, leading to increased membrane permeability and leakage of contents;

[0082] (2) It denatures and inactivates the proteins inside the bacteria, causing key enzymes and structural proteins to lose their function;

[0083] (3) Damages bacterial nucleic acid, inhibiting DNA replication and transcription.

[0084] The above-mentioned multiple effects work together to kill bacteria, achieving efficient and rapid in-situ sterilization.

[0085] The core advantage of this invention lies in integrating detection and sterilization functions onto the same micro-motor platform, forming an integrated workflow of "identification-detection-processing":

[0086] Step 1: Active Capture and Recognition

[0087] Driven by a rotating magnetic field, the micromotor actively moves within the sample, overcoming diffusion limitations and increasing the probability of collision with target bacteria. Aptamer A1 specifically recognizes and binds to E. coli.

[0088] Step 2: Signal Output and Detection

[0089] Event-triggered competitive substitution releases the A2-CQDs complex, restoring the fluorescence signal. Qualitative or quantitative analysis of E. coli is achieved by detecting fluorescence intensity.

[0090] Step 3: In-situ sterilization

[0091] After the test is completed, there is no need to transfer the sample; just apply near-infrared light directly. The PDA on the surface of the micro-motor generates localized high temperatures, which rapidly inactivate the E. coli attached to it.

[0092] The aforementioned collaborative working mechanism represents a technological leap from "passive detection" to "active searching - immediate reporting - in-situ treatment," significantly improving the efficiency and practicality of foodborne pathogen detection and control.

[0093] Example 1

[0094] The Escherichia coli pH-specific nucleic acid aptamers are Apt-A, which is suitable for acidic conditions (pH 3.0~4.0), Apt-B, which is suitable for neutral conditions (pH 6.0~7.5), and Apt-C, which is suitable for alkaline conditions (pH 7.5~8.5).

[0095] The nucleic acid sequence of aptamer Apt-A is shown in SEQ ID NO:1, the nucleic acid sequence of aptamer Apt-B is shown in SEQ ID NO:2, and the nucleic acid sequence of aptamer Apt-C is shown in SEQ ID NO:3.

[0096] Apt-A: 5'-AGTGCGTTTGTTGCGTTTTGGCAGTCCT-3' (SEQ ID NO: 1);

[0097] Apt-B: 5'-AATCACACTCTTGCATTTGGAAGACCT-3' (SEQ ID NO: 2);

[0098] Apt-C: 5'-AATCACCCTCTCACATCTGGAAGACAT-3' (SEQ ID NO: 3);

[0099] The secondary structures of the above aptamers Apt-A, Apt-B, and Apt-C were predicted using NUPACK as follows: Figure 1 As shown, the three aptamers exhibit significant differences in their base sequences. Apt-B is obtained through a mutation of the Apt-A base, and Apt-C is obtained through a mutation of the Apt-B base. Key mutations introduced through rational design were used to alter the pH-responsive properties of the aptamers. All three aptamers formed a secondary conformation dominated by a stem-loop structure, but the base pairing patterns in the loop and stem regions differed, directly affecting their binding mode to the target protein and their pH dependence.

[0100] The aptamers Apt-A, Apt-B, and Apt-C were molecularly docked with target proteins on the surface of *E. coli* using Discovery Studio 2021 software. A schematic diagram of the molecular docking is shown below. Figures 2-4 As shown, the top row displays the conformations of the three aptamers and target proteins, revealing different binding postures. The bottom row provides a visualization analysis, magnifying the binding interface and annotating the hydrogen bonds and key interaction sites between the aptamer and the protein. This validates the rational design strategy guided by molecular dynamics simulations, namely, modulating the pH responsiveness of the aptamer by modifying key residues.

[0101] Flow cytometry was used to verify the pH specificity of the three aptamers.

[0102] Three aptamers were labeled with FAM fluorescent dyes to ensure that the labeling did not affect binding activity. The pH of apple juice was approximately 3.5, milk approximately 6.5, and kelp soup approximately 8.0. *E. coli* was pre-cultured in media with pH values ​​of 3.5 (apple juice), 6.5 (milk), and 8.0 (kelp soup) to simulate the pH conditions of different food environments. Each FAM-labeled aptamer was incubated with the three pH-compatible bacteria in their respective binding buffers at 37°C for 30 minutes. After incubation, the bacteria were collected by centrifugation, washed three times with PBS of the same pH to remove unbound aptamers, resuspended, and analyzed using a BD FACS Aria III flow cytometer. Excitation wavelength was 488 nm, and emission wavelengths were 530 / 30 nm. At least 10,000 cells were recorded per sample, and binding affinity was quantified by mean fluorescence intensity (MFI).

[0103] The results are as follows Figure 5 As shown, Apt-A exhibited a strong fluorescence peak only in apple juice at pH 3.5, with weak signals in milk at pH 6.5 and kelp soup at pH 8, indicating a strict acidic pH preference. Apt-B produced significant fluorescence only in milk at pH 6.5, with extremely low signals in the other two matrices, validating its neutral pH specificity. Apt-C showed a strong fluorescence peak only in kelp soup at pH 8, with almost no response in acidic and neutral matrices, demonstrating its alkaline pH specificity. Therefore, each aptamer can efficiently recognize targets only in its optimized pH environment, and accurate detection of pathogens in complex food matrices can be achieved without pH adjustment of the samples.

[0104] Example 2

[0105] The Apt-A magnetically driven fluorescent micromotor was prepared by the following method:

[0106] (1) Preparation of magnetic beads-polydopamine (MBs@PDA):

[0107] 7.5 mg of dopamine hydrochloride was added to 30 mL of Tris-HCl buffer (pH 8.0) and shaken at room temperature for 3 h. 10 mL of the mixture was then added to 10 mg of magnetic beads and shaken for another 3 h. The mixture was magnetically separated using a magnetic rack and washed three times, then resuspended in 10 mL of ultrapure water. Dopamine self-polymerized at room temperature to form an MBs@PDA complex, which exhibits both magnetic responsiveness and photothermal conversion capabilities. The mass ratio of dopamine hydrochloride to magnetic beads was 1:4; the magnetic beads (MBs) were made of iron oxide (Fe3O4) with a particle size of 200 nm.

[0108] (2) Preparation of DNA double strands: E. coli-specific aptamer A1 (5' end modified with carboxyl group) and its complementary strand A2 (5' end modified with biotin) were mixed in equimolar ratio and annealed to form A1@A2-DNA double strands; the nucleic acid sequences of A1 and A2 are as follows:

[0109] A1 (carboxyl modification): 5'-COOH-AGTGCGTTTGTTGCGTTTGGCAGTCCT-3' (SEQ ID NO:4);

[0110] A2 (Biotin modification): 5'-Biotin-AGGACTGCCAAAGCAGCAAACGCA-3' (SEQ ID NO:5);

[0111] (3) Preparation of carbon quantum dots (CQDs):

[0112] 0.25 g of urea and 1.065 g of citric acid were dissolved in 20 mL of ultrapure water and transferred to a high-temperature, high-pressure reactor at 180 °C for 6 h. After the reaction, the high-temperature, high-pressure reactor was cooled to room temperature, and the resulting liquid was centrifuged at 14,000 rpm at 25 °C for 20 min to remove the uncarbonized portion. The supernatant was then dialyzed through a 1000 Da regenerated cellulose dialysis bag for 48 h, with the water changed every 4 h to remove small molecule impurities. The liquid inside the bag was retained to obtain CQDs with uniform particle size, which were then stored at 4 °C protected from light until use.

[0113] (4) Preparation of streptavidin-modified carbon quantum dots (CQDs-SA):

[0114] Add 5 mg of EDC powder and 5 mg of NHS powder to 300 μL of pure water and shake to dissolve completely to prepare a mixed EDC and NHS solution. Dilute the CQDs solution prepared in step (3) above by 4 times (1 mL CQDs solution + 3 mL water), add 400 μL of SA solution with a concentration of 1 mg / mL and 50 μL of EDC and NHS mixed solution, mix evenly and place in a constant temperature air bath shaker at 37°C for 3 h, and then place in a refrigerator at 4°C overnight. By activating CQDs-COOH, it reacts with SA to form an amide, so that SA is coated on the surface of CQDs to synthesize CQDs-SA, so as to connect with A2 5'-Biotin.

[0115] (5) Synthesis of magnetic fluorescent molecular probes:

[0116] Add 5 mg of EDC powder and 5 mg of NHS powder to 300 μL of purified water and shake to dissolve completely to prepare a mixed EDC and NHS solution; take 100 μL of 10 μM A1 / A2-DNA solution, dilute it to 2 μM with ultrapure water, add 40 μL of the mixed EDC and NHS solution, mix well and shake at room temperature for 30 min to activate the 5'-COOH of A1. Then, 800 μL of MBs@PDA with a concentration of 1 mg / mL was taken, magnetically separated, and the supernatant was discarded. The mixture was then thoroughly mixed with the activated A1 / A2-DNA solution and slowly shaken at room temperature for 1 h. The 5'-COOH of A1 in the A1 / A2-DNA double strand will undergo an amide reaction with the NH2 on the surface of MBs@PDA, loading the A1 / A2-DNA double strand onto the MBs@PDA surface, thus obtaining the A1 / A2-MBs@PDA complex. 100 μL of streptavidin-modified carbon quantum dot (CQDs-SA) solution (concentration approximately 14.8 mg / mL) was mixed with 250 μg (the optimal amount determined in preliminary experiments) of the synthesized A1 / A2-MBs@PDA complex and reacted at room temperature for 1 h. This allowed the SA in CQDs-SA to link with the 5'-Biotin of A2 in the A1 / A2-MBs@PDA complex, yielding a magnetic fluorescent molecular probe. This probe was magnetically separated, washed three times with ultrapure water, and resuspended. Because PDA has a fluorescence quenching effect on CQDs, this intact magnetic fluorescent molecular probe does not show fluorescence.

[0117] (6) Pretreatment of Spirulina template and functionalization of probe loading: Spirulina (SP) was washed, filtered through a 0.22 μm filter membrane for sterilization, and then dispersed in 1×PBS buffer at pH 6-7 (spirulina concentration 1 mg / mL) to ensure the integrity of its helical structure. Potentiometric measurements showed that under pH 6-7 conditions, Spirulina exhibited a negative potential, while the magnetic fluorescent molecular probe showed a positive potential; the two could bind together through electrostatic adsorption. The prepared magnetic fluorescent probe was mixed with the Spirulina suspension at a 1:1 volume ratio and incubated at room temperature for 24 hours. Through electrostatic interaction, the probe was uniformly adsorbed onto the surface of Spirulina, thus obtaining the Apt-A magnetically driven fluorescent micromotor.

[0118] Example 3

[0119] The Apt-B magnetically driven fluorescent micromotor was prepared by the following method:

[0120] Except for the following steps, the remaining steps are the same as in Example 2:

[0121] (2) Preparation of DNA double strands: E. coli-specific aptamer A1 (5' end modified with carboxyl group) and its complementary strand A2 (5' end modified with biotin) were mixed in equimolar ratio and annealed to form A1@A2-DNA double strands; the nucleic acid sequences of A1 and A2 are as follows:

[0122] A1 (carboxyl modification): 5'-COOH-AATCACACTCTTGCATTTGGAAGACCT-3' (SEQ ID NO:6);

[0123] A2 (Biotin modification): 5'-Biotin-AGGTCTTCCAAATGCAA-3' (SEQ ID NO:7).

[0124] Example 4

[0125] Apt-C magnetically driven fluorescent micromotors are fabricated by the following method:

[0126] Except for the following steps, the remaining steps are the same as in Example 2:

[0127] (2) Preparation of DNA double strands: E. coli-specific aptamer A1 (5' end modified with carboxyl group) and its complementary strand A2 (5' end modified with biotin) were mixed in equimolar ratio and annealed to form A1@A2-DNA double strands; the nucleic acid sequences of A1 and A2 are as follows:

[0128] A1 (carboxyl modification): 5'-COOH-AATCACCCTCTCACATCTGGAAGACAT-3' (SEQ ID NO:8);

[0129] A2 (Biotin modification): 5'-Biotin-ATGTCTTCCAGATGTG-3' (SEQ ID NO:9).

[0130] Example 5

[0131] A method for detecting Escherichia coli in food, comprising the following steps:

[0132] (1) Measure the pH value of the food matrix to be tested, and select a suitable magnetically driven fluorescent micromotor based on the measured pH results:

[0133] When the pH of the food matrix to be tested is 3.0-4.0, the Apt-A magnetically driven fluorescent micromotor is selected for detection.

[0134] When the pH of the food matrix to be tested is 6.0-7.5, the Apt-B magnetically driven fluorescent micromotor is selected for detection.

[0135] When the pH of the food matrix to be tested is 7.5-8.5, the Apt-C magnetically driven fluorescent micromotor is selected for detection.

[0136] When the pH value of the food matrix to be tested is not within the operating pH range of any of the above-mentioned magnetically driven fluorescent micromotors, the pH value of the food matrix to be tested is adjusted to the nearest pH range, and a suitable magnetically driven fluorescent micromotor is selected based on the adjusted pH value.

[0137] (2) The magnetically driven fluorescent micromotor and the food matrix to be tested are gently shaken and incubated at room temperature. Escherichia coli specifically binds to Apt-A1 in the magnetically driven fluorescent micromotor and competitively releases A2-CQDs, resulting in fluorescence recovery. In order to increase the binding efficiency of the magnetically driven fluorescent micromotor to Escherichia coli in the food matrix to be tested, the movement speed of the magnetically driven fluorescent micromotor can be increased under the drive of an external magnetic field. After the reaction is completed, the fluorescence intensity of the supernatant is detected, and the concentration of Escherichia coli in the food matrix to be tested is calculated by referring to the standard curve.

[0138] The standard curve is plotted using the following method:

[0139] A magnetically driven fluorescent micromotor was incubated with suspensions of E. coli of different concentrations at room temperature with gentle shaking, and the fluorescence intensity of the supernatant was measured. A standard curve was plotted with the concentration of E. coli (CFU / mL) on the x-axis and the fluorescence intensity on the y-axis.

[0140] Example 6

[0141] A method for killing E. coli in food, comprising the following steps:

[0142] (1) Measure the pH value of the food matrix to be tested, and select a suitable magnetically driven fluorescent micromotor based on the measured pH results:

[0143] When the pH of the food matrix to be tested is 3.0-4.0, the Apt-A magnetically driven fluorescent micromotor is selected for detection.

[0144] When the pH of the food matrix to be tested is 6.0-7.5, the Apt-B magnetically driven fluorescent micromotor is selected for detection.

[0145] When the pH of the food matrix to be tested is 7.5-8.5, the Apt-C magnetically driven fluorescent micromotor is selected for detection.

[0146] When the pH value of the food matrix to be tested is not within the operating pH range of any of the above-mentioned magnetically driven fluorescent micromotors, the pH value of the food matrix to be tested is adjusted to the nearest pH range, and a suitable magnetically driven fluorescent micromotor is selected based on the adjusted pH value.

[0147] (2) Mix the magnetically driven fluorescent micromotor with the food matrix to be tested at room temperature and irradiate with 808nm, 2W NIR for 30-60 seconds. In order to increase the sterilization efficiency and sterilization coverage of Escherichia coli in the food matrix to be tested by the magnetically driven fluorescent micromotor, this process can be carried out under the drive of an external magnetic field to increase the movement speed of the magnetically driven fluorescent micromotor.

[0148] Characterization of magnetic drive motion characteristics of micro motors

[0149] Taking the Apt-A magnetically driven fluorescent micromotor as an example, the magnetic drive motion characteristics of the micromotor were characterized. The specific operation is as follows:

[0150] like Figure 6 and Figure 7 As shown, a customized quadrupole electromagnetic coil system was placed under an optical microscope. The positional relationship of the four coils is illustrated in the figure, enabling magnetic drive control of a micromotor. This system can generate a uniform rotating magnetic field in a horizontal plane, driving a micromotor loaded with magnetic particles to propel itself in a helical manner.

[0151] The structure and performance of the coils were characterized using finite element simulation: two pairs of coils were orthogonally arranged around the sample cavity. Coils 1 and 2 were driven by a sinusoidal current source with an amplitude of 1 ampere and a frequency adjustable within the range of 0 to 100 Hz, with a phase of 0°. Coils 3 and 4 were driven by a current source of the same specification, with a phase offset of 90°. This orthogonal excitation method can generate a magnetic field vector that rotates smoothly in the XY plane at the center of the coils.

[0152] By adjusting the frequency of the input current, the rotation frequency of the magnetic field can be precisely controlled, thereby achieving linear regulation of the micromotor's movement speed. The movement process of the micromotor is recorded in real time using an optical microscope equipped with a digital camera.

[0153] By controlling the magnitude and direction of the magnetic field, the micromotor can be moved to the upper left and lower right. The movement of the micromotor can be recorded using a microscope equipped with a camera. Figure 8 and Figure 9 As shown.

[0154] Sensitivity and specificity of micro motor detection

[0155] Sensitivity detection: Apt-A magnetically driven fluorescent micromotors were incubated with suspensions of different concentrations of *E. coli* (pH 3.5) at room temperature with gentle shaking. *E. coli* specifically binds to Apt-A1 in the Apt-A magnetically driven fluorescent micromotor, competitively releasing A2-CQDs, leading to fluorescence recovery. The fluorescence intensity of the supernatant was measured using a Hitachi F-2700 fluorescence spectrophotometer, and a calibration curve was plotted to determine the detection limit. A standard curve was plotted with the concentration of *E. coli* (CFU / mL) on the x-axis and fluorescence intensity on the y-axis, as shown below. Figure 10 As shown, y = 1502.1lg(x) - 245.79 (R 2 =0.9578) The mean blank fluorescence intensity μ0 = 494 a.u.; the standard deviation of blank fluorescence intensity σ0 = 49 a.u. Therefore, the LOD signal is 641 a.u. and the detection limit is 3.89 CFU / mL.

[0156] Specificity testing: The Apt-A magnetically driven fluorescent micromotor was incubated with *Escherichia coli* (target), *Staphylococcus aureus*, *Salmonella typhimurium*, and *Klebsiella pneumoniae* (non-target) under the same conditions, and the fluorescence response intensity was measured to verify specificity. The specificity testing results of the Apt-A magnetically driven fluorescent micromotor are as follows: Figure 11 As shown, the Apt-A magnetically driven fluorescent micromotor has excellent specificity, producing a significant fluorescent response only to the target bacteria, while the fluorescence signal for non-target strains such as Staphylococcus aureus, Salmonella typhimurium, and Klebsiella pneumoniae is low and negligible.

[0157] Micro-motor in-situ sterilization performance evaluation

[0158] Apt-A magnetically driven fluorescent micromotor was mixed with E. coli suspension (10) 6 A mixture of CFU / mL (pH 3.5) was prepared and irradiated with an 808nm, 2W NIR light for 0-60 seconds. Traditional pasteurization at 72℃ was used as the control group. Bacterial viability was calculated using the plate count method. The results are as follows: Figure 12 As shown, the bactericidal effect of E. coli suspension treated with Apt-A magnetically driven fluorescent micromotor increased with prolonged NIR irradiation time. After 30 seconds of NIR treatment, the number of bacterial colonies on the plate was significantly reduced, and the bacterial survival rate was reduced by more than 99.9% within 60 seconds. In contrast, the bacterial suspension treated with pasteurization at 72℃ only showed a significant bactericidal effect after 60 seconds of treatment. Therefore, compared to pasteurization at 72℃, Apt-A magnetically driven fluorescent micromotor treatment achieves better bactericidal effects in a shorter time.

[0159] The sterilization effect of Apt-A magnetically driven fluorescent micromotor treatment for 0 s and 30 s was analyzed by PI staining and flow cytometry. The results are as follows: Figure 13 As shown, when the treatment time is 0s, the bacterial community is concentrated in the Q4 region. After 30s of treatment, the killed pathogens gradually move to the Q1 region after being stained with PI dye, indicating that the 30s NIR treatment killed some of the pathogens. The above experiments all demonstrate that the Apt-A magnetically driven fluorescent micromotor has a good bactericidal effect under NIR irradiation.

[0160] The photothermal conversion efficiency of MBs@PDA was verified by monitoring the solution temperature change during irradiation using a Thermo Scientific Evolution 201 UV-visible spectrophotometer, and the results are as follows: Figure 14 and Figure 15 As shown.

[0161] Figure 14 The images show thermal images of Apt-A magnetically driven fluorescent micromotors of different masses (0 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg) after irradiation with NIR light for different times (0 s, 10 s, 20 s, 30 s, 40 s, 50 s, 60 s). The results show that the brightness of the thermal images gradually increases with the increase of the amount of fluorescent motor added and the extension of the NIR irradiation time, indicating that the sample amount of the fluorescent motor and the NIR irradiation time are positively correlated with its photothermal effect. Figure 15 The figure shows a line graph illustrating the temperature change of the Apt-A magnetically driven fluorescent micromotor system under different experimental conditions. It can be seen that with the extension of NIR irradiation time, the system temperature changes in two stages: a stable increase and a tendency to plateau. Furthermore, the final temperature after the system reaches plateau is positively correlated with the amount of fluorescent motor added. These results fully demonstrate that the modification with a polydopamine (PDA) coating can significantly enhance the photothermal effect of the fluorescent motor.

[0162] Application validation in actual food matrices

[0163] Three types of food samples with different pH values ​​were selected: acidic (apple juice), neutral (milk), and alkaline (kelp soup). No pH adjustment was required; known concentrations of *E. coli* were directly added. Apt-A, Apt-B, and Apt-C magnetically driven fluorescent micromotors were used to detect the food samples at the three pH values, and the recovery rates were calculated. The volume ratio of the micromotor to the sample was 1:2, and the final concentration of the micromotor after mixing was approximately 0.6 mg / mL. The results are shown in Table 1.

[0164] Table 1. Recovery rates of Escherichia coli in food samples at different pH values ​​using three magnetically driven fluorescent micromotors.

[0165]

[0166] Table 1 shows that the three magnetically driven fluorescent micromotors exhibit a pH-dependent recognition pattern: each aptamer demonstrates high accuracy only in pH environments that match its pH. Apt-A achieved a recovery rate of 93.8±4.5% in apple juice, Apt-B 98.5±5.6% in milk, and Apt-C 94.9±1.1% in kelp soup. Conversely, when the aptamers were used with matrices that did not match the pH, the recovery rate decreased significantly; for example, Apt-A's recovery rate was only 55.2±6.1% in milk and 68.7±5.3% in kelp soup. This significant difference fully validates the pH specificity of the three magnetically driven fluorescent micromotors, enabling reliable detection of pathogens in complex, unmodified food samples.

[0167] Micro-electrode biosafety assessment

[0168] Taking the Apt-A magnetically driven fluorescent micromotor as an example, the biosafety of the micromotor was evaluated as follows:

[0169] Apt-A magnetically driven fluorescent micromotor, magnetic fluorescent molecular probe, SP, and ultrapure water were incubated with mammalian Caco-2 cells for 0, 24, 48, and 72 hours, respectively. Cell viability was then assessed using the CCK-8 assay to simulate a scenario where residual micromotors might remain after actual use. Results are as follows: Figure 16 As shown, even at the high concentration tested (3.8 mg / mL), cell viability remained above 85%, with no statistically significant difference compared to the control group without the micromotor. This result clearly demonstrates that the components of the micromotor and its degradation products have extremely low cytotoxicity.

[0170] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A pH-specific nucleic acid aptamer for Escherichia coli, characterized in that, The nucleic acid aptamer is any one of the following: (a) The aptamer Apt-A, which is suitable for acidic pH conditions, has the nucleic acid sequence shown in SEQ ID NO:1; (b) The aptamer Apt-B, which is suitable for neutral pH conditions, has the nucleic acid sequence shown in SEQ ID NO:2; (c) The aptamer Apt-C, which is suitable for alkaline pH conditions, has the nucleic acid sequence shown in SEQ ID NO:3; The acidic pH conditions are pH 3.0-4.0, the neutral pH conditions are pH 6.0-7.5, and the alkaline pH conditions are pH 7.5-8.

5.

2. The use of the Escherichia coli pH-specific nucleic acid aptamer according to claim 1, characterized in that, Used to prepare reagents for detecting Escherichia coli content or for sterilizing Escherichia coli.

3. A magnetically driven fluorescent micromotor, characterized in that, The magnetically driven fluorescent micromotor is formed by loading a magnetic fluorescent molecular probe onto Spirulina through electrostatic adsorption. The magnetic fluorescent molecular probe is formed by using magnetic beads coated with polydopamine as the core, covalently coupling a double-stranded DNA A1@A2, and then connecting A2 in the double-stranded DNA A1@A2 to streptavidin-modified carbon quantum dots via non-covalent bonds. The nucleic acid sequence of A1 in the DNA double strand A1@A2 is any one of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, and its 5' end is modified with a carboxyl group; A2 in the DNA double strand A1@A2 is partially complementary to A1, and its 5' end is modified with biotin. When the sequence of A1 in the DNA double strand A1@A2 is as shown in SEQ ID NO:4, the sequence of A2 is as shown in SEQ ID NO:5; When the sequence of A1 in the DNA double strand A1@A2 is as shown in SEQ ID NO:6, the sequence of A2 is as shown in SEQ ID NO:7; When the sequence of A1 in the DNA double strand A1@A2 is as shown in SEQ ID NO:8, the sequence of A2 is as shown in SEQ ID NO:

9.

4. The magnetically driven fluorescent micromotor according to claim 3, characterized in that, The fabrication method of the magnetically driven fluorescent micromotor includes the following steps: (1) React magnetic beads with dopamine hydrochloride in Tris-HCl buffer to form MBs@PDA; (2) Mix the aptamer A1 modified with the carboxyl group at the 5' end and the complementary strand A2 modified with the biotin at the 5' end in an equal molar ratio and anneal to form A1@A2-DNA double strand; (3) Carbon quantum dots were prepared by hydrothermal reaction using urea and citric acid as precursors and modified with streptavidin to obtain CQDs-SA; (4) The A1@A2-DNA double strand from step (2) is immobilized on the surface of MBs@PDA from step (1) by EDC / NHS chemical method, and then CQDs-SA from step (3) is added. CQDs-SA is linked to A2 by biotin-streptavidin to obtain a magnetic fluorescent molecular probe. (5) The cleaned and sterilized Spirulina and the magnetic fluorescent molecular probe prepared in step (4) are mixed and incubated in a buffer solution. The probe is loaded onto the surface of Spirulina by electrostatic adsorption to obtain a magnetically driven fluorescent micromotor.

5. The use of the magnetically driven fluorescent micromotor according to claim 3 or 4 for purposes other than disease diagnosis and treatment, characterized in that, Used to detect or kill E. coli in food.

6. The use of the magnetically driven fluorescent micromotor according to claim 5 for purposes other than disease diagnosis and treatment, characterized in that, The method for detecting the content of E. coli in food is as follows: (1) Determine the pH value of the food matrix; select the appropriate magnetically driven fluorescent micromotor based on the determined pH value: When the pH is 3.0-4.0, the magnetically driven fluorescent micromotor with sequence A1 as shown in SEQ ID NO:4 is selected; When the pH is 6.0-7.5, the magnetically driven fluorescent micromotor with sequence A1 as shown in SEQ ID NO:6 is selected; When the pH is 7.5-8.5, the magnetically driven fluorescent micromotor with sequence A1 as shown in SEQ ID NO:8 is selected; (2) The selected magnetically driven fluorescent micromotor was incubated with the food matrix, the fluorescence intensity of the supernatant was detected, and the concentration of Escherichia coli was calculated by referring to the standard curve.

7. The use of the magnetically driven fluorescent micromotor according to claim 5 for purposes other than disease diagnosis and treatment, characterized in that, The steps for killing E. coli in food are as follows: (1) Determine the pH value of the food matrix; select the appropriate magnetically driven fluorescent micromotor based on the determined pH value: When the pH is 3.0-4.0, the magnetically driven fluorescent micromotor with sequence A1 as shown in SEQ ID NO:4 is selected; When the pH is 6.0-7.5, the magnetically driven fluorescent micromotor with sequence A1 as shown in SEQ ID NO:6 is selected; When the pH is 7.5-8.5, the magnetically driven fluorescent micromotor with sequence A1 as shown in SEQ ID NO:8 is selected; (2) Mix the magnetically driven fluorescent micromotor with the food matrix at room temperature and irradiate with 808nm, 2W NIR for 30-60 seconds.

8. The use of the magnetically driven fluorescent micromotor according to claim 6 or 7 for purposes other than disease diagnosis and treatment, characterized in that, The magnetically driven fluorescent micromotor is mixed with a food matrix under the drive of an external rotating magnetic field to enhance the micromotor's mobility and target capture efficiency.

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