Transverse relaxation time biosensing method based on differential diameter magnetic probe assembly strategy and application

Abstract

The invention discloses a transverse relaxation time biosensing method based on a differential magnetic probe assembly strategy and application, and belongs to the field of biochemical analysis. The invention provides a differential magnetic probe assembly signal sensing strategy based on DNA base complementary pairing, and provides a novel transverse relaxation time biosensing method based on the strategy. The difference of the particle sizes of the magnetic probes can reduce the steric hindrance of the magnetic particles with different particle sizes in the assembly process, and the efficient aggregation of the magnetic particles is realized, so that the signal sensing efficiency and the signal response are improved, the operation steps are reduced, and the practicability is improved. The constructed biosensor has the advantages of being wide in linear range, high in sensitivity, low in cost, easy to operate, wide in application range and the like, adapters and corresponding connection DNA can be replaced according to target objects, good applicability and universality are achieved, detection of different target objects such as macromolecules and micromolecules can be achieved, and the biosensor has good application prospects. Good application prospects are realized in the aspects of food safety, clinical diagnosis and the like.

Description

Technical Field

[0001] The present invention relates to a transverse relaxation time biosensing method and application based on a differential-diameter magnetic probe assembly strategy, belonging to the field of biochemical analysis. Background Art

[0002] With the improvement of living standards, biochemical analysis technology plays an increasingly prominent role in various fields of life science. Due to its advantages such as simplicity and rapidity, biosensors have attracted extensive attention in the field of biochemical analysis and have been widely applied in the rapid analysis of food safety hazard factors, clinical diagnosis of disease-related biomarkers, etc. A biosensor is a biochemical analysis method that converts information such as the concentration of a target substance into a readable signal through a biological recognition element (such as an antibody, aptamer, etc.) via a biochemical reaction and a signal conversion system. To achieve highly sensitive detection of the target substance, biosensors usually also introduce a signal amplification system, such as enzymatic signal amplification, click chemical reaction, etc. According to the different signal readout methods, biosensors can be divided into optical biosensors (such as fluorescence, colorimetric, chemiluminescent biosensors, etc.), electrochemical biosensors (such as amperometric, conductivity-type, etc.), magnetic biosensors (such as transverse relaxation, giant magnetoresistance sensors, etc.).

[0003] Among many biosensors, transverse relaxation time biosensors have attracted much attention due to their high signal-to-noise ratio, strong anti-interference ability, etc. The analysis principle of traditional transverse relaxation time biosensors is based on the recognition of a target substance by a magnetic probe modified with a recognition molecule, which changes the state of the magnetic probe from dispersion to aggregation. This state change will cause an increase in the inhomogeneity of the local magnetic field, accelerate the relaxation rate of the hydrogen protons of water molecules, and cause a decrease in its transverse relaxation time. Since biological, food and other samples basically do not contain magnetic signal interfering substances, the transverse relaxation time sensor has a high signal-to-noise ratio. However, traditional transverse relaxation time biosensors use superparamagnetic nanoparticles of the same size as magnetic probes, and the aggregation degree that can be caused during the signal conversion and sensing process is limited, resulting in low sensitivity and narrow linear range, and it is difficult to meet the requirements of sensitive, rapid and simple detection of target substances.

[0004] To solve this problem, the applicant previously proposed a transverse relaxation time immunosensor based on polydopamine nanoparticle signal conversion and click reaction signal amplification (Biosensors and Bioelectronics, 2022, 207, 114127) for the sensitive and stable detection of chlorpyrifos residues in fruits and vegetables. The sensor synthesized polydopamine nanoparticles with good stability and biocompatibility, modified the secondary antibody on its surface, and used it as a signal conversion element to regulate the Cu 2+ concentration, combined with the immune reaction, and converted the target substance concentration into Cu 2+Concentration. Subsequently, based on the redox reaction, the Cu + -mediated click chemical reaction is initiated, causing the aggregation of magnetic particles of the same size. This method has the following advantages: 1) The click chemical reaction is an effective strategy to change the state of magnetic probes, which can significantly improve the conversion efficiency of magnetic signals and the sensitivity of the method; 2) The polydopamine nanoparticle complex has a high ability to bind Cu 2+ ions, significantly changing the concentration of Cu 2+ ions, resulting in an effective change in magnetic signals and achieving the amplification of sensing signals; 3) The signal conversion system mediated by polydopamine nanoparticles can effectively reduce food matrix interference, reduce background signals, and improve sensitivity and stability. This method effectively improves the sensitivity and detection linear range of the method. However, in this method, a signal conversion system mediated by polydopamine and a signal amplification system mediated by click chemical reaction are introduced, increasing the complexity of the sensing system and the tediousness of operation, limiting its practical application. Therefore, developing a sensing system with high signal sensing efficiency, simplicity, rapidity, and a wide signal response range is an important way to improve the transverse relaxation time sensor. Summary of the Invention

[0005] To solve the above problems, based on the controllable synthesis characteristics of the particle size of magnetic probes, the present invention proposes a differential-diameter magnetic probe assembly signal sensing strategy, and based on this strategy, a sensitive, simple, and wide linear range transverse relaxation time biosensing method is invented. The difference in the particle size of magnetic probes can reduce the steric hindrance during the assembly of magnetic particles of different particle sizes, achieve the efficient aggregation of magnetic particles, thereby improving the signal sensing efficiency and signal response, while reducing the operation steps and improving the practicality.

[0006] The present invention provides a biosensor based on the assembly of differential-diameter magnetic probes, including:

[0007] (1) A magnetic bead probe complex with a DNA-1-aptamer complex immobilized on its surface, and the particle size of the magnetic bead probe is 180 - 300 nm;

[0008] (2) A magnetic particle probe complex with a DNA-2 immobilized on its surface, and the particle size of the magnetic particle probe is 50 - 150 nm;

[0009] The difference in the particle size between the magnetic bead probe and the magnetic particle probe is greater than 50 nm and does not exceed 150 nm.

[0010] In one embodiment, the biosensor is used to detect chloramphenicol, and the nucleotide sequence of the aptamer is as shown in SEQ ID NO.1; the nucleotide sequence of DNA-1 is as shown in SEQ ID NO.2; the nucleotide sequence of DNA-2 is as shown in SEQ ID NO.3.

[0011] In one embodiment, the biosensor is used to detect procalcitonin, wherein the nucleotide sequence of the aptamer is as shown in SEQ ID NO.4; the nucleotide sequence of DNA-1 is as shown in SEQ ID NO.5; the nucleotide sequence of DNA-2 is as shown in SEQ ID NO.6.

[0012] In one embodiment, the biosensor is used to detect chloramphenicol, wherein the nucleotide sequence of the aptamer is as shown in SEQ ID NO.7; the nucleotide sequence of DNA-1 is as shown in SEQ ID NO.8; the nucleotide sequence of DNA-2 is as shown in SEQ ID NO.9.

[0013] The present invention also provides a method for preparing the biosensor, which comprises the following steps:

[0014] S1. After mixing the aptamer that connects DNA-1 and the target, incubate at 90 - 95 °C and slowly cool to room temperature to form a DNA-1-aptamer complex.

[0015] S2. Couple the DNA-1-aptamer complex prepared in S1 on the surface of the magnetic bead probe, and couple DNA-2 on the surface of the magnetic particle probe to obtain a magnetic bead-DNA-1-aptamer complex and a magnetic particle-DNA-2 complex respectively.

[0016] In one embodiment, the particle size of the magnetic bead probe is 180 - 300 nm; the particle size of the magnetic particle probe is 50 - 150 nm.

[0017] In one embodiment, the difference in particle size between the magnetic bead probe and the magnetic particle probe is greater than 50 nm and not higher than 150 nm.

[0018] In one embodiment, the groups or molecules modified on the surfaces of the magnetic bead probe and the magnetic particle probe include amino group, carboxyl group or streptavidin.

[0019] In one embodiment, the aptamer is an aptamer corresponding to different target substances and can be replaced according to the target substance.

[0020] In one embodiment, the aptamer for detecting Listeria monocytogenes is single-stranded DNA, and any reported aptamer can be used in the present invention.

[0021] In one embodiment, DNA-1 and DNA-2 are single-stranded DNA with a length of 30 - 60 bases.

[0022] In one embodiment, the number of bases where DNA-1 binds to the aptamer is not less than 12.

[0023] In one embodiment, the 5'-end modification groups or molecules connecting DNA-1 and DNA-2 include, but are not limited to, amino group, carboxyl group, biotin, etc.

[0024] The present invention also provides a method for detecting a target using the biosensor, comprising the following steps:

[0025] S1. Mix the sample to be detected with the magnetic bead - linked DNA-1 - aptamer complex of the biosensor, and perform a biorecognition reaction;

[0026] S2. After magnetic separation, washing, and resuspension, add the magnetic particle - linked DNA-2 complex, and perform magnetic probe assembly based on the principle of base complementary pairing;

[0027] S3. Measure the transverse relaxation time of the reaction solution in step S2.

[0028] In one embodiment, the method comprises the following steps:

[0029] (1) Mix the sample to be detected with the magnetic bead - linked DNA-1 - aptamer complex for a biorecognition reaction;

[0030] (2) After subjecting the reactant in step (1) to magnetic separation and washing at a magnetic field strength above 0.1 T for 0.5 - 2 min, add the magnetic particle - linked DNA-2 probe, and initiate differential - diameter magnetic probe aggregation through DNA complementary pairing;

[0031] (3) Directly measure the transverse relaxation time of the system.

[0032] In one embodiment, the particle size difference between the magnetic bead probe and the magnetic particle probe is set to satisfy: when the particle size of the magnetic bead probe is 180 nm, the particle size of the magnetic particle probe is set to 100 nm.

[0033] In one embodiment, the detected target includes at least one of biomarkers, pathogenic bacteria, antibiotics, pesticide residues, veterinary drug residues, or biotoxins, and the detection matrix includes, but is not limited to, fruits and vegetables, animal tissues, serum, or dairy products.

[0034] In one embodiment, in step (2), the mass concentration ratio of the magnetic bead probe to the magnetic particle probe is 1:0.5 - 1:2, the assembly reaction temperature is 25 - 37 °C, and the reaction time is 5 - 10 min.

[0035] The present invention also provides the application of the biosensor or the method in the fields of food safety detection, clinical diagnosis, or environmental monitoring.

[0036] In one embodiment, the application includes the quantitative detection of Listeria monocytogenes, procalcitonin, or chloramphenicol in food.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] (1) Novel sensing mode and wide linear range: The magnetic probe differential diameter assembly of the present invention is in the mode of weakening the transverse relaxation time signal, which is not limited by the signal distortion caused by excessive aggregation of traditional magnetic probes. Even with excessive aggregation, stable signal reading can still be achieved, improving the response range of the transverse relaxation time signal and broadening the linear range of the method, up to 1×10 3 ~1×10 8 CFU / mL;

[0039] (2) High sensitivity: The differential diameter magnetic probe assembly strategy is developed for the first time, with high magnetic probe assembly efficiency. Through the low steric hindrance assembly of magnetic probes with different particle sizes, efficient sensing of the transverse relaxation time signal is achieved, avoiding the situation where traditional transverse relaxation time biosensors can only achieve signal sensing with loose aggregates in a similar sandwich structure when detecting bacteria or biological macromolecules, and at the same time avoiding the high steric hindrance phenomenon during the assembly of magnetic probes with the same particle size when detecting small molecules, which can effectively improve the detection sensitivity to 1×10 2 CFU / mL;

[0040] (3) Low cost and simple operation: The recognition elements used in the present invention are all DNA sequences, which are inexpensive and can greatly reduce the cost of using recognition elements such as antibodies. Moreover, the signal sensing is direct, the detection time is short, the scheme is simple, and the tester only needs to follow the brief step description to complete the detection, with good usability;

[0041] (4) Strong applicability and wide application range: The present invention uses aptamers as recognition elements and is combined with the connecting DNA mode. Finally, the assembly process is completed by the connecting DNA. It can be achieved by replacing the aptamer and the corresponding connecting DNA according to the target object, with good applicability and versatility, and can realize the detection of different target objects such as macromolecules and small molecules, and has good application prospects in food safety, clinical diagnosis, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the schematic diagram of the transverse relaxation time sensor detecting the target object in the present invention.

[0043] Figure 2 It is the Zeta potential and hydrated particle size result diagram of the magnetic probe and its assembly complex in the present invention.

[0044] Figure 3 It is the scanning electron microscope and elemental analysis diagram of the reaction product in the present invention.

[0045] Figure 4 is for MB 180 and MNP 100Transverse relaxation time result graphs of the magnetic probe complex before and after assembly.

[0046] Figure 5 Transverse relaxation time result graphs of magnetic probe complexes with different particle sizes before and after assembly.

[0047] Figure 6 Standard curve result graph for detecting Listeria monocytogenes by the method of the present invention.

[0048] Figure 7 Standard curve result graph for detecting Listeria monocytogenes by a traditional transverse relaxation time sensor.

[0049] Figure 8 Specificity result graph for detecting Listeria monocytogenes by the method of the present invention.

[0050] Figure 9 Result graph of using this transverse relaxation time biosensor to detect Listeria monocytogenes in fish meat and the comparison result with the plate counting method.

[0051] Figure 10 Result graph of using this transverse relaxation time biosensor to detect procalcitonin and the comparison result with the enzyme-linked immunosorbent assay method.

[0052] Figure 11 Result graph of using this transverse relaxation time biosensor to detect chloramphenicol in milk and the comparison result with the enzyme-linked immunosorbent assay method. Detailed implementation manners

[0053] Description of the sources of reagents and equipment:

[0054] Carboxylated superparamagnetic nanoparticles: 100 nm, purchased from Ocean Nanotech, LLC; 180 nm, purchased from Shanghai Aorun Micro-Nano New Materials Technology Co., Ltd.

[0055] All nucleic acid sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0056] Listeria monocytogenes (ATCC 19114) was purchased from Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.

[0057] Procalcitonin and magnetic separation racks were purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0058] Chloramphenicol, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysulfosuccinimide active ester (sulfo-NHS), and Tween-20 were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0059] Nuclear magnetic resonance imaging analyzer (MesoMR23 - 060V - 1): Suzhou Newmai Analytical Instruments Co., Ltd.

[0060] Example 1 Construction of differential diameter signal sensing strategy based on base complementary pairing in transverse relaxation time biosensor

[0061] (1) Preparation of magnetic probes

[0062] Select carboxyl - modified magnetic beads (COOH - MB 180 ) with a particle size of 180 nm and carboxyl - modified magnetic particles (COOH - MNP 100 ) with a particle size of 100 nm as magnetic probes for signal sensing. The magnetic beads and magnetic particles can use commercial products or be prepared by oneself, and prepare "MB 180 - linked DNA - 1 - aptamer" and "MNP 100 - linked DNA - 2" complexes respectively. The preparation process of the complexes is a conventional method well - known in the art, specifically as follows:

[0063] Aptamer: -5 ’ -ATC CAT GGG GCG GAG ATG AGG GGG AGG AGG GCG GGT ACC CGGTTGAT-3’(SEQ ID NO.1);

[0064] DNA - 1: -5 ’ -NH2-(CH2)6-TTT TTT ATCAA CCG GGT ACC CGC CCT-3 ’ (SEQ ID NO.2);

[0065] DNA - 2: -5 ’ -NH2-(CH2)6-TTT TTT AGG GCG GGT ACC CGG TTGAT-3 ’ (SEQ ID NO.3);

[0066] MB 180 Preparation of - linked DNA - 1 - aptamer complex: Wash 1 mg of COOH - MB 180 with MEST buffer (pH = 6.0 containing 0.05% Tween 20) 2 - 3 times, and resuspend with 0.5 mL of MEST (pH = 6.0) buffer for standby. Add the following to the above COOH - MB 180Add 100 μL of EDC and 50 μL of NHS, mix well and activate at room temperature for 15 - 25 min. After activation, perform magnetic separation, resuspend with 0.5 mL of PBS (pH = 7.4) buffer, add a certain amount of the hybrid product of amino - modified DNA - 1 and aptamer, and react the mixture by slow oscillation at 37 °C for 2 - 3 h; after the reaction, wash 3 - 5 times with PBST (pH = 7.4 containing 0.05% Tween 20) buffer, and finally resuspend with PBS buffer and store at 4 °C for later use. Among them, the preparation method of the amino - modified DNA - 1 - aptamer hybrid product is as follows: Mix the 5'-amino - modified DNA - 1 and the aptamer in a ratio of 1:1, and heat at 95 °C for 5 min, then slowly cool to room temperature.

[0067] MB 180 - Preparation of the MB - linked DNA - 1 complex: Only replace the amino - modified DNA - 1 - aptamer hybrid product with amino - modified DNA - 1, and the other steps are the same as the above protocol.

[0068] MNP 100 - Preparation of the MNP - linked DNA - 2 complex: Replace the amino - modified DNA - 1 - aptamer hybrid product with amino - modified DNA - 2, and replace 180 MB 100 with MNP, and the other steps are the same as the above protocol.

[0069] In this example, the Zeta potential and hydrodynamic diameter of the magnetic probe were measured by dynamic light scattering technology. The results are as Figure 2 shown. After coupling the linked DNA, the Zeta potentials of both 180 MB 100 and MNP magnetic probes decreased, indicating that the coupling of the linked DNA or the linked DNA - aptamer complex increased the dispersibility of the magnetic probe. In addition, the hydrodynamic diameter showed that the particle size of the magnetic probe increased to a certain extent after coupling. The above results prove that the linked DNA or the linked DNA - aptamer complex was successfully modified on the surface of the magnetic probe.

[0070] (2) Construction of the differential - diameter magnetic probe signal sensing strategy based on base complementary pairing

[0071] Mix a certain concentration of the 180 MB - linked DNA - 1 complex with 100-The DNA-2 complex was mixed evenly with a mass concentration ratio of 1:0.5 to 1:2, and reacted at room temperature for 5 to 10 minutes. After the reaction was completed, the liquid was transferred to a small glass bottle with a cover, and its transverse relaxation time was measured by a nuclear magnetic resonance imaging analyzer. The transverse relaxation time was measured using a CPMG pulse sequence with the following parameters: NMR frequency: 19 MHz; TW (ms): 5000; TE (ms): 1; PRG: 3; NECH: 12000; SW: 100.

[0072] The morphology and elemental composition of the reaction products were observed by scanning electron microscopy. Figure 3 As shown, MB 180 With MNP 100 The elements in the aggregates are tightly bound together and contain each other. The results of elemental analysis show that the elements contained in the aggregates include Fe, O, C, N, and P, which mainly come from the magnetic probe and its coupled DNA sequence.

[0073] The Zeta potential and hydrated particle size of the magnetic probe complex before and after assembly were measured by dynamic light scattering. Figure 2 As shown, after the formation of the complex, the Zeta potential of the magnetic probe complex decreased significantly, and the hydrated particle size increased significantly and was greater than 1 μm, proving that MB 180 With MNP 100 Micron-sized aggregates were formed.

[0074] By comparing the changes in the transverse relaxation time of the magnetic probe complex before and after assembly, the feasibility of the differential path signal sensing strategy based on base complementary pairing was verified. Figure 4 As shown, MB before assembly 180 - Connecting DNA-1 complex with MNP 100 The transverse relaxation time values ​​of the -connected DNA-2 complex were 354.6ms and 303.9ms, respectively. After the formation of aggregates, the transverse relaxation time value changed to 758.6ms, with a significant signal change, proving that the differential path signal sensing strategy is feasible.

[0075] In this embodiment, the particle size of the magnetic probe is optimized, and the magnetic probe signal sensor is prepared according to the above method, except that magnetic beads with particle sizes of 100, 180, 250, and 300 nm are selected respectively. Figure 5 As shown, the positive changes in the transverse relaxation time of the magnetic probe complex before and after assembly include MB 250 -MNP 100 、MB 180 -MNP 100 、MB 300 -MNP 180 、MB 250 -MNP180 , it is proved that under the above combination, the differential diameter signal sensing strategy is feasible, and the optimal combination is MB 180 -MNP 100 .

[0076] Example 2 Quantitative detection of microbial targets by transverse relaxation time biosensor (taking Listeria monocytogenes as an example)

[0077] Add 100 μL of the "MB 180 -Linked DNA-1-Listeria monocytogenes aptamer" complex prepared in Example 1 with a certain concentration into the gradient-diluted Listeria monocytogenes (ATCC 19114) bacterial solution, and react at 37 °C for 15 - 30 min. After the reaction is completed, magnetically separate to remove the supernatant, wash 2 - 3 times with PBST (PBS buffer containing 0.05% Tween-20) buffer, and then resuspend with 200 μL of PBS. Then, add 100 μL of MNP 100 -Linked DNA-2 complex, and slowly vortex and react at room temperature (20 - 25 °C) or 37 °C for 5 - 10 min. After the reaction is over, transfer all the reaction solution into a sample injection bottle and measure its transverse relaxation time. Taking the change value of the transverse relaxation time as the ordinate and the concentration of Listeria monocytogenes as the abscissa, the standard curve of this method is obtained.

[0078] In this example, the key parameter concentrations of the biochemical reactions involved were optimized. When the concentrations of MB 180 -Linked DNA-1-Listeria monocytogenes aptamer and MNP 100 -Linked DNA-2 complex are 2 μg / mL and 1 μg / mL respectively, and the reaction time of MB 180 -Linked DNA-1-Listeria monocytogenes aptamer with the Listeria monocytogenes (ATCC 19114) bacterial solution is 20 min, and the reaction time of MNP 100 -Linked DNA-2 complex is 8 min, which are the optimal conditions. The detection results of Listeria monocytogenes under the optimized optimal conditions are as Figure 6 shown. The linear range of the method of the present invention for detecting Listeria monocytogenes is 1×10 3 ~1×10 8 CFU / mL (Y = 55.98X – 133.97, R 2 = 0.99), and the detection sensitivity is 1×10 2 CFU / mL.

[0079] Comparative Example 1

[0080] Take the traditional transverse relaxation time immunosensor as a control. The traditional transverse relaxation time sensor uses "MNP 30"Instead of 'MB' in the sensor of Example 2" 180 " and 'MNP'" 100 ", and other reaction conditions are the same as those in Example 2."

[0081] Listeria monocytogenes was detected according to the optimized reaction method in Example 2, and the results are as Figure 7 shown. The linear range for detecting Listeria monocytogenes by the traditional transverse relaxation time sensor is 1×10 4 ~1×10 7 CFU / mL (Y = 13.44X – 14.17, R 2 = 0.98), and the detection sensitivity is 1×10 3 CFU / mL."

[0082] In comparison, the method of the present invention has higher sensitivity (an order of magnitude improvement) and a wider linear range (two orders of magnitude broadening), indicating that the invented differential diameter assembly strategy can effectively improve the analytical performance of the transverse relaxation time sensor."

[0083] Determination of Specificity and Recovery Rate of the Transverse Relaxation Time Biosensor in Example 3"

[0084] In this example, different foodborne pathogenic bacteria (Salmonella ATCC 14028, Staphylococcus aureus ATCC29213, Vibrio parahaemolyticus ATCC17802, Escherichia coli ATCC25922) were used as interfering substances to detect the specificity of this sensor for detecting Listeria monocytogenes. The concentrations of the above interfering bacterial species and Listeria monocytogenes were both 1×10 5 CFU / mL, and the determination was carried out under the optimal conditions of Example 2. The experimental results are as Figure 8 shown. Only Listeria monocytogenes can cause a significant change in the transverse relaxation time, and the influence of other analogs on the transverse relaxation time can be ignored."

[0085] In this example, the recovery rate was studied by the standard addition method. Different concentrations of Listeria monocytogenes (1×10 4 , 1×10 5 and 1×10 6 CFU / mL) were added to the homogenized blank fish meat samples, and then measured with this sensor. The results are shown in Table 1. The recovery rate of this sensor for detecting Listeria monocytogenes is between 81.23% - 93.22%, and the relative standard deviation is between 3.65% - 10.28%, indicating that this method has good accuracy."

[0086] Table 1 Recovery Rate of the Sensor for Detecting Listeria monocytogenes in Fish Meat Samples"

[0087] Labeled Concentration (CFU / mL) Detected Concentration (CFU / mL) Recovery Rate (%) Relative Standard Deviation (%) <![CDATA[1×10 4 > <![CDATA[(9.32±0.34)×10 3 > 93.22 3.65 <![CDATA[1×10 5 > <![CDATA[(8.85±0.91)×10 4 > 88.51 10.28 <![CDATA[1×10 6 > <![CDATA[(8.12±0.85)×10 5 > 81.23 10.10

[0088] Example 4 Transverse Relaxation Time Biosensor for Quantitative Detection of Listeria monocytogenes in Fish Samples

[0089] Homogenize the fish meat, take 5 g of fish meat sample into a centrifuge tube, add 9 mL of sterile normal saline, after centrifugation, aspirate the supernatant, detect it with this transverse relaxation time biosensor, and compare the methodology with the plate counting method. The experimental results are as Figure 9 shown. Samples No. 4, 7, and 8 were detected as positive for Listeria monocytogenes, and the others were negative. The plate counting method (referring to the method of GB4789.30-2016) was used to verify the number of Listeria monocytogenes in the samples. The results showed that the detection results of this transverse relaxation time biosensor were similar to those of the plate counting method, proving that this sensor has good accuracy.

[0090] Example 5 Quantitative Detection of Biomacromolecule Markers by Transverse Relaxation Time Biosensor (Taking Procalcitonin as an Example)

[0091] To test the versatility of this sensor, in this example, procalcitonin was taken as an example to determine the applicability of this transverse relaxation time biosensor for quantitative detection of biomacromolecule markers. The specific implementation method is the same as that of Examples 1-2, except that the "procalcitonin aptamer" and its corresponding DNA-1 and DNA-2 are used to replace the Listeria monocytogenes aptamer and the corresponding DNA in Example 2 for detecting procalcitonin in serum, and the methodology is compared with the enzyme-linked immunosorbent assay.

[0092] Aptamer: 5’-GCGGATGAAGACTGGTGTGTGGGGGAGGGGTGAGTTTTAGTGTTTTTGTTGGTTGGCGGCCCTAAATACGAGCAAC-3’ (SEQ ID NO.4);

[0093] DNA-1: 5’-NH2-(CH2)6-TTTTTTGTTGCTCGTATTTAGGGCC G-3’ (SEQ ID NO.5);

[0094] DNA-2: 5’-NH2-(CH2)6-TTTTTTCGGCCCTAAATACGAGCAAC-3’ (SEQ ID NO.6).

[0095] The results are as Figure 10 shown. Samples No. 2, 5, 8, and 10 were detected as positive for procalcitonin, and the others were negative. The detection results of the transverse relaxation time biosensor constructed in this application were similar to those of the enzyme-linked immunosorbent assay, proving that this sensor has good accuracy.

[0096] Example 6 Quantitative Detection of Small Molecule Hazardous Substance Residues by Transverse Relaxation Time Biosensor (Taking Chloramphenicol as an Example)

[0097] To test the versatility of this sensor, in this example, chloramphenicol was taken as an example to determine the applicability of this transverse relaxation time biosensor for quantitatively detecting small molecule hazardous substances in biology. The specific implementation manner was similar to that of Examples 1-2, except that the "chloramphenicol aptamer" and its corresponding DNA-1 and DNA-2 were used to replace the Listeria monocytogenes aptamer and the corresponding DNA sequence in Example 2 for detecting chloramphenicol in milk, and the enzyme-linked immunosorbent assay was used for methodological comparison.

[0098] Aptamer: 5’-ACT TCAGTGAGTTGTCCCACGGTCGGCGAGTCGGTGGTAG-3’ (SEQ ID NO.7);

[0099] DNA-1: 5’-NH2-(CH2)6-TTT TTTCTACCACCGACTCGC-3’ (SEQ ID NO.8);

[0100] DNA-2: 5’-NH2-(CH2)6-TTTTTTTCGGCGAGTCGGTGG TAG-3’ (SEQ ID NO.9).

[0101] The results were as Figure 11 shown. Samples No. 3 and No. 5 were detected as positive for chloramphenicol, and the others were negative. The detection results of this transverse relaxation time biosensor were similar to those of the enzyme-linked immunosorbent assay, proving that this sensor had good accuracy.

[0102] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A biosensor assembled based on a differential-diameter magnetic probe, characterized in that, Comprising: (1) A magnetic bead probe complex with a DNA-1-aptamer complex immobilized on its surface, wherein the magnetic bead probe has a particle size of 180 - 300 nm; (2) A magnetic particle probe complex with a DNA-2 immobilized on its surface, wherein the magnetic particle probe has a particle size of 50 - 150 nm; The difference in particle size between the magnetic bead probe and the magnetic particle probe is greater than 50 nm and not more than 150 nm.

2. The biosensor according to claim 1, wherein The aptamer is an aptamer for detecting *Listeria monocytogenes*, and its nucleotide sequence is as shown in SEQ ID NO.1; the nucleotide sequence of DNA-1 is as shown in SEQ ID NO.2; the nucleotide sequence of DNA-2 is as shown in SEQ ID NO.

3.

3. The biosensor according to claim 1, characterized in that, The aptamer is an aptamer for detecting procalcitonin, and its nucleotide sequence is as shown in SEQ ID NO.4; the nucleotide sequence of DNA-1 is as shown in SEQ ID NO.5; the nucleotide sequence of DNA-2 is as shown in SEQ ID NO.

6.

4. The biosensor according to claim 1, characterized in that, The aptamer is an aptamer for detecting chloramphenicol, and its nucleotide sequence is as shown in SEQ ID NO.7; the nucleotide sequence of DNA-1 is as shown in SEQ ID NO.8; the nucleotide sequence of DNA-2 is as shown in SEQ ID NO.

9.

5. A method for preparing the biosensor according to any one of claims 1 to 4, characterized in that, Including the following steps: S1. Mix the aptamer that links DNA-1 and the target substance, incubate at 90 - 95 °C, and slowly cool to room temperature to form a DNA-1-aptamer complex; S2. Couple the DNA-1-aptamer complex prepared in S1 to the surface of the magnetic bead probe, and couple DNA-2 to the surface of the magnetic particle probe to obtain a magnetic bead-DNA-1-aptamer complex and a magnetic particle-DNA-2 complex respectively; the magnetic bead probe has a particle size of 180 - 300 nm; the magnetic particle probe has a particle size of 50 - 150 nm.

6. The method according to claim 5, wherein The magnetic bead probe and the magnetic particle probe include at least one of the following characteristics: (a) The difference in particle size between the magnetic bead probe and the magnetic particle probe is greater than 50 nm and not higher than 150 nm; (b) The groups or molecules modified on the surfaces of the magnetic bead probe and the magnetic particle probe include amino groups, carboxyl groups or streptavidin.

7. A method for quantitatively detecting a target substance, characterized in that, When using the biosensor according to any one of claims 1 - 4 for detection, it includes the following steps: S1. Mix the sample of the target substance to be detected with the magnetic bead-DNA-1-aptamer complex of the biosensor for a biorecognition reaction; S2. After magnetic separation, washing and resuspension, add the magnetic particle-DNA-2 complex, and perform magnetic probe assembly based on the principle of base complementary pairing; S3. Measure the transverse relaxation time of the reaction solution in step S2.

8. The method according to claim 7, wherein Directly measure the transverse relaxation time of the reaction solution in step S2.

9. The method according to claim 7 or 8, characterized in that, The target substance to be detected includes at least one of biomarkers, pathogenic bacteria, antibiotics, pesticide residues, veterinary drug residues or biotoxins, and the detection matrix includes but is not limited to fruits and vegetables, animal tissues, serum or dairy products.

10. Use of the biosensor according to any one of claims 1 to 4 or the method according to any one of claims 5 to 9 in the fields of food safety detection, clinical diagnosis or environmental monitoring.

Images