A magnetic sensor device based on DNA binding force and its application for bacteria detection

Abstract

This invention discloses a magnetic sensor device based on DNA binding force and its application in bacterial detection. This invention relates to the field of magnetic sensing technology, specifically to a magnetic sensor device based on DNA binding force and its application in bacterial detection. The device for biological sample analysis of this invention includes a magnetic sensor, a DNA walking strand (WS), a nucleic acid aptamer specific to the biological sample to be detected, and an exonuclease III. The magnetic sensor is an array containing N types of double-stranded DNA molecules named TP-AP-FP immobilized on a substrate and a magnetic solid modified with streptavidin. When used for bacterial detection, this invention eliminates the need for bacterial labeling and bacterial nucleic acid extraction, allowing direct interaction with the bacterial surface. It is simple to operate, saves time, has high accuracy, can construct magnetic sensors for detecting different targets, and is versatile with a wide detection range.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of magnetic sensing, in particular to a magnetic sensor device based on DNA binding force and application thereof in bacterial detection. BACKGROUND

[0002] Outbreaks of infectious diseases caused by different pathogenic bacteria are becoming a serious threat to human health. Each year, more than 300 million cases of serious and even fatal diseases caused by bacterial infections, with more than 2 million deaths. If no action is taken, the number of deaths caused by bacterial infections can increase to 100 million people per year by 2050, with a cumulative cost of 100 trillion dollars to the global economy. Therefore, accurate detection of multiple pathogenic bacteria is helpful for the use of appropriate antibiotics for treatment, which is crucial for improving patient survival rate.

[0003] Bacterial blood culture is currently the gold standard for detecting pathogenic bacteria in clinical practice. This detection method is greatly limited due to the need for a large amount of blood samples, cumbersome procedures (e.g., bacterial culture and microbial phenotype identification), time-consuming processes (up to several days), and often depends on the professional knowledge of the operator. Genotypic methods based on pathogen nucleic acid analysis, such as polymerase chain reaction (PCR), can shorten the analysis time to a few hours after blood culture. However, the error of PCR amplification or replication can cause errors in microbial quantification, and it is very cumbersome in the process of handling samples, making it difficult to perform high-throughput analysis of clinical samples. Biosensors based on immunological methods are also commonly used to detect bacteria. This method is usually based on antigen-antibody reaction, and the target molecule is identified by immune reaction, which has good specificity and fast detection speed. However, in actual sample detection, it takes a long time to obtain the corresponding antibody, and the specificity is easily affected by the interference from the antibody, which affects the detection sensitivity. Therefore, there is an urgent need to develop a sensitive, multi-channel, high-throughput pathogenic bacteria detection method in clinical diagnosis. SUMMARY

[0004] The technical problem to be solved by the present application is how to perform sensitive, multi-channel, high-throughput pathogenic bacteria detection.

[0005] In order to solve the above problems, the present application provides a device for biological sample analysis.

[0006] The device for biological sample analysis provided by the present application comprises a magnetic sensor, a DNA walking chain WS, a nucleic acid aptamer specific to the biological sample to be detected, and an exonuclease III.

[0007] The magnetic sensor is an array containing N types of double-stranded DNA molecules named TP-AP-FP fixed on a substrate and a magnetic solid modified with streptavidin. The double-stranded DNA molecules of TP-AP-FP are composed of single-stranded DNA molecules named template probe TP, auxiliary probe AP and binding force probe FP, respectively. The 5' end of the template probe TP is modified with a biotin molecule, and the 3' end has m1 deoxythymidine nucleotides. The first m2 nucleotides from the 5' end are complementary to the auxiliary probe AP.

[0008] The auxiliary probe AP pairs complementaryly with the template probe TP for the first m2 nucleotides starting from the 5' end, and pairs complementaryly with the DNA traveling strand WS for the first m3 nucleotides starting from the 3' end, forming a blunt 3' end with the DNA traveling strand WS; the DNA traveling strand WS pairs complementaryly with a nucleic acid aptamer specific to the biological sample to be tested;

[0009] The binding force probe FP has a group modified at its 5' end for immobilization on the substrate, and has m1 deoxythymidine nucleotides at its 3' end; the binding force probe FP is immobilized on the substrate; the binding force probe FP is complementary to the template probe TP, and the complementary pairing region of FP and TP overlaps with the complementary pairing region of AP and TP; the number of bases in the overlapping region is 3 or more.

[0010] N is a natural number greater than 1;

[0011] m1 is a natural number greater than or equal to 4, such as 10;

[0012] The m2 is a natural number greater than or equal to 6, such as 12;

[0013] The m3 is a natural number greater than or equal to 12, such as 21.

[0014] In the above-described device, the magnetic sensor further contains N types of double-stranded DNA molecules named TP-FP immobilized on the substrate. The TP-FP double-stranded DNA molecules are formed by linking the template probe TP and the binding force probe FP single-stranded DNA molecules.

[0015] In the above-mentioned device, the biological sample to be tested may be Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhimurium and / or Shigella, or the sample to be tested may be a sample containing Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhimurium and / or Shigella.

[0016] In the above-mentioned device, the nucleic acid aptamer specific to the biological sample to be tested may be a nucleic acid aptamer specific to Staphylococcus aureus, a nucleic acid aptamer specific to Bacillus subtilis, a nucleic acid aptamer specific to Escherichia coli, a nucleic acid aptamer specific to Shigella, or a nucleic acid aptamer specific to Salmonella typhimurium.

[0017] In the above-mentioned device, the magnetic sensor can be a magnetic sensor for detecting Staphylococcus aureus, the nucleotide sequence of the template probe TP is sequence 1, the nucleotide sequence of the auxiliary probe AP is sequence 2, and the nucleotide sequence of the binding force probe FP is sequence 3.

[0018] In the above-mentioned device, the magnetic sensor can be a magnetic sensor for detecting Bacillus subtilis, the nucleotide sequence of the template probe TP is sequence 6, the nucleotide sequence of the auxiliary probe AP is sequence 7, and the nucleotide sequence of the binding force probe FP is sequence 8.

[0019] In the above device, the magnetic sensor can be a magnetic sensor for detecting Escherichia coli, the nucleotide sequence of the template probe TP is sequence 11, the nucleotide sequence of the auxiliary probe AP is sequence 12, and the nucleotide sequence of the binding force probe FP is sequence 13.

[0020] In the above-mentioned device, the magnetic sensor can be a magnetic sensor for detecting Shigella, the nucleotide sequence of the template probe TP is sequence 16, the nucleotide sequence of the auxiliary probe AP is sequence 17, and the nucleotide sequence of the binding force probe FP is sequence 18.

[0021] In the above-mentioned device, the magnetic sensor can be a magnetic sensor for detecting Salmonella typhimurium, the nucleotide sequence of the template probe TP is sequence 21, the nucleotide sequence of the auxiliary probe AP is sequence 22, and the nucleotide sequence of the binding force probe FP is sequence 23.

[0022] In this invention, N can specifically be 2, 3, 4, or 5. The number of 3 or more can specifically be 3-10.

[0023] The aforementioned apparatus for analyzing biological samples also includes a device for measuring the binding force of different DNA double strands.

[0024] In this invention, the nucleotide sequence of the DNA walking chain WS can be sequence 4, and the corresponding nucleotide sequence of the nucleic acid aptamer specific to the biological sample to be tested is sequence 5.

[0025] In this invention, the nucleotide sequence of the DNA walking chain WS can be sequence 9, and the corresponding nucleotide sequence of the nucleic acid aptamer specific to the biological sample to be tested is sequence 10.

[0026] In this invention, the nucleotide sequence of the DNA walking chain WS can be sequence 14, and the corresponding nucleotide sequence of the nucleic acid aptamer specific to the biological sample to be tested is sequence 15.

[0027] In this invention, the nucleotide sequence of the DNA walking chain WS can be sequence 19, and the corresponding nucleotide sequence of the nucleic acid aptamer specific to the biological sample to be tested is sequence 20.

[0028] In this invention, the nucleotide sequence of the DNA walking chain WS can be sequence 24, and the corresponding nucleotide sequence of the nucleic acid aptamer specific to the biological sample to be tested is sequence 25.

[0029] This invention also provides a method for fabricating the magnetic sensor described above, comprising the following steps:

[0030] 1) A TP-AP double-stranded solution is prepared by mixing one or more sets of template probe solution TP and auxiliary probe solution AP and annealing at 95°C for 5 to 10 minutes.

[0031] 2) Take the TP-AP double-stranded solution from step 1) and mix it with one or more binding force probe solutions FP. Incubate at 37°C for 60-120 minutes to hybridize and form TP-AP-FP double-stranded probes. Fix them on a solid substrate and seal them to obtain a liquid containing TP-AP-FP double-stranded probes fixed on the substrate. Add magnetic beads modified with streptavidin to the liquid and magnetize them to prepare an array containing one or more sets of DNA double-stranded probes.

[0032] 3) A TP-FP double-chain solution is prepared by mixing one or more sets of template probe solution TP and binding force probe solution FP and annealing at 95℃ for 5 to 10 minutes.

[0033] 4) Take the TP-FP double-stranded probe solution from step 3), fix it on a solid substrate and seal it, and then magnetize it after coupling magnetic beads on the TP-FP double-stranded probe to prepare a nanoarray containing one or more sets of DNA double-stranded probes.

[0034] The present invention also provides a method for detecting microbial samples, comprising the following steps:

[0035] 1) Add mixed solution A and the sample to be tested to the magnetic sensor described above and incubate. The mixed sample A contains WS-Aptamer double-stranded probe solution and exonuclease III. The WS-Aptamer double-stranded probe solution is prepared by mixing one or more DNA walking strand solutions WS and complementary nucleic acid aptamer solutions Aptamer and annealing at 95°C for 5 to 10 minutes.

[0036] 2) After incubation, external mechanical force is applied to the magnetic sensor. The composition of the sample to be tested is determined by the decrease in magnetic signal and the binding force of the DNA double-stranded probe.

[0037] Different DNA binding forces were applied to the magnetic sensor, and the changes in magnetic signals before and after the application of the force were recorded. The changes in magnetic signals were positively correlated with the bacterial concentration. Each concentration of bacterial solution corresponds to a change in magnetic signal. A standard curve was constructed based on the logarithm of the change in magnetic signal and the bacterial concentration. Different magnitudes of force caused a decrease in magnetic signal corresponding to different types of bacteria. The type of bacteria in the sample can be determined based on the magnitude of the binding force corresponding to the change in magnetic signal.

[0038] In the above method, the content of TP-AP in the TP-AP double-chain solution is 0.4–6 μM;

[0039] Preferably, the final concentration of the TP-AP double-stranded solution is 3 μM.

[0040] In the above method, the TP-AP-FP double-stranded probe solution formed by hybridization is 0.4–6 μM;

[0041] Preferably, the final concentration of the TP-AP-FP double-stranded probe is 3 μM.

[0042] In the above method, the amount of magnetic beads added is 1-9 mg / mL;

[0043] Preferably, the final concentration of the magnetic beads is 2.5 mg / mL.

[0044] In the above method, magnetization involves placing a magnet with a magnetic field strength of 0.8 to 1 T at a distance of 1 to 5 cm from the solid substrate and leaving it to stand for 1 to 10 minutes.

[0045] Furthermore, the relationship between the centrifugal force and the centrifuge speed is as follows:

[0046] F=mω 2 r (1)

[0047] In equation (1), m is the mass of the magnetic bead, r is the radius of rotation, and ω is the rotational speed.

[0048] The present invention also provides the application of the apparatus or method for detecting microbial samples described above in the detection of proteins, nucleic acids, small molecules, metal ions, bacteria and / or cells.

[0049] The present invention also provides the application of the apparatus or method for detecting microbial samples described above in the analysis of whole blood samples.

[0050] This invention also provides double-stranded DNA molecules of TP-AP-FP and / or TP-FP as described above. The following probe combinations can be used for the identification of different bacteria:

[0051] 1) The TP1, AP1, FP1, WS1 and Aptamer1, which recognizes Staphylococcus aureus, form a set of probes (the nucleotide sequences of each are shown in Table 1) for detecting Staphylococcus aureus in the sample to be tested;

[0052] 2) The TP2, AP2, FP2, WS2 and Aptamer2, which recognizes Bacillus subtilis, form a set of probes (the nucleotide sequences of each are shown in Table 1) for detecting Bacillus subtilis in the sample to be tested;

[0053] 3) The TP3, AP3, FP3, WS3 and Aptamer3, which recognizes Escherichia coli, form a group of probes (the nucleotide sequences of each are shown in Table 1) used to detect Escherichia coli in the sample to be tested;

[0054] 4) The TP4, AP4, FP4, WS4 and Aptamer4, which recognizes Shigella, form a set of probes (the nucleotide sequences of each are shown in Table 1) for detecting Shigella in the sample to be tested;

[0055] 5) The TP5, AP5, FP5, WS5 and Aptamer5, which recognizes Salmonella typhimurium, form a group of probes (the nucleotide sequences of each are shown in Table 1) used to detect Salmonella typhimurium in the sample to be tested.

[0056] Table 1 Nucleotide sequences involved in this invention

[0057]

[0058]

[0059]

[0060] Note: Biotin represents biotin, and NH2C6 represents the amino group used in this invention to immobilize DNA molecules on an aldehyde substrate.

[0061] Compared with the prior art, the present invention has the following advantages:

[0062] 1) The magnetic sensor constructed in this invention uses magnetic signals as reporting signals, which are not affected by solution color and ambient light source, and the signal is stable and has good repeatability;

[0063] 2) This invention has a very strong multiplexing capability. Compared with the multiplexing capability of optical sensors which is limited by the number of spectral colors, it can regulate the binding force of DNA by controlling the sequence and length of the complementary part of the DNA double strand, thereby enabling the simultaneous detection of multiple targets and greatly improving the multiplexing capability.

[0064] 3) This invention utilizes the catalytic action of exonuclease III to enable the DNA walking strand to automatically travel on the substrate surface, hybridize with the helper probe AP, and trigger a large number of anchor-mediated strand displacement reactions, enabling the sensor to respond to a single target. Through a series of cyclic cascade reactions, the sensitivity of the sensor is improved, achieving bacterial detection at the single-cell level.

[0065] 4) When this invention is used for bacterial detection, there is no need to label the bacteria or extract bacterial nucleic acid. It can directly interact with the bacterial surface, making the operation simple, time-saving, and highly accurate.

[0066] 5) By changing different nucleic acid aptamer sequences and corresponding DNA probes, this invention can achieve the detection of other targets. The nucleic acid aptamers obtained through in vitro screening technology bind to the targets with high specificity. Based on the nucleic acid aptamer sequences, corresponding DNA walking strands, template probes, auxiliary probes, and binding force probes can be designed to construct magnetic sensors for detecting different targets. It has versatility and a wide detection range.

[0067] 6) This invention has excellent anti-interference ability and high detection accuracy. It can simultaneously detect multiple clinically relevant bacterial species in simulated blood samples without the need for a washing step, and has great potential to guide decision-making in various clinical situations. Attached Figure Description

[0068] Figure 1 This is a schematic diagram of the process for detecting bacteria in an embodiment of the present invention.

[0069] Figure 2 This is a schematic diagram illustrating the design of DNA double strands with different binding forces in an embodiment of the present invention.

[0070] Figure 3 In this embodiment of the invention, force-induced remanent magnetization spectroscopy is used to measure the binding force of different DNA double strands.

[0071] Figure 4 This is a polyacrylamide gel electrophoresis image used to verify the formation of DNA double-stranded probes in an embodiment of the present invention.

[0072] Figure 5 This is the result of detecting Staphylococcus aureus using a magnetic sensor based on DNA binding force in an embodiment of the present invention.

[0073] Figure 6This is a graph showing the relationship between experimental parameters (DNA double-strand concentration, exonuclease III concentration, incubation time, and magnetic bead solution concentration) and magnetic signal changes in embodiments of the present invention.

[0074] Figure 7 The results of detecting Staphylococcus aureus and Bacillus subtilis using a magnetic sensor based on DNA binding force in this embodiment of the invention are shown.

[0075] Figure 8 The results of detecting Staphylococcus aureus, Bacillus subtilis, and Shigella using a magnetic sensor based on DNA binding force in this embodiment of the invention are shown.

[0076] Figure 9 In this embodiment of the invention, a magnetic sensor based on DNA binding force is used to identify bacteria in whole blood samples.

[0077] Figure 10 This is a statistical analysis of the detection of bacteria in whole blood samples using a magnetic sensor based on DNA binding force in an embodiment of the present invention. Detailed Implementation

[0078] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0079] Unless otherwise specified, the experimental methods used in the following examples 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 materials and reagents used in the following examples are commercially available.

[0080] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0081] The magnetic bead M280 used in the following examples was purchased from Invitrogen, part number 11205D.

[0082] The Tris-HCl buffer in the following examples consists of 20 mM Tris-HCl, 100 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and the remainder is sterile water; the pH of the Tris-HCl buffer is 7.4.

[0083] The DNA hybridization buffer used in the following examples is 20 mM Tris-HCl, 1 mM EDTA, and 1 M NaCl; the pH of the Tris-HCl buffer is 8.0.

[0084] The Staphylococcus aureus used in the following examples is ATCC 6538, purchased from the China General Microbiological Culture Collection Center.

[0085] The Bacillus subtilis strains used in the following examples are designated CMCC(B)63501, Escherichia coli CMCC44103, Shigella CMCC 51572, and Salmonella typhimurium CMCC 50115. All were purchased from the China Medical Bacteriological Culture Collection Center. The China Medical Bacteriological Culture Collection Center has a dedicated website at http: / / www.cmccb.org.cn, where the public can directly order bacterial strains online.

[0086] The sterile, anticoagulated rabbit whole blood used in the following examples was purchased from Guangzhou Ruite Biotechnology Co., Ltd., with a product specification of 100 mL.

[0087] Example 1: Construction of a device for a magnetic sensor based on DNA binding force

[0088] 1. The specific steps for constructing a magnetic sensor based on DNA binding force are as follows:

[0089] 1) Clean the glass substrate twice with acetone, three times with ultrapure water, and for 10 minutes with 1 mol / L NaOH solution. Clean it twice with ultrapure water and twice with anhydrous ethanol. Place it in a drying oven at 250℃ for 3 hours to remove surface impurities and bacteria.

[0090] 2) Place the clean glass substrate in a plasma cleaner for 60 seconds to generate surface hydroxyl groups, thus obtaining a hydroxylated glass substrate;

[0091] 3) The hydroxylated glass substrate was added to an anhydrous ethanol solution containing 1% APTES (3-aminopropyltriethoxysilane), soaked at room temperature for 3 hours, washed 3 times with anhydrous ethanol solution, and dried with nitrogen gas to obtain an aminated glass substrate.

[0092] 4) Place the above-mentioned aminated glass substrate in Tris-HCl buffer containing 1% glutaraldehyde, incubate at 37°C for 2 hours, and wash 3 times with Tris-HCl buffer to obtain aldehyde-modified glass substrate.

[0093] 5) Anneal the template probe TP (with biotin labeled at the 5' end) and the auxiliary probe AP at 95°C for 5–10 minutes to form TP-AP double strands. Table 1 shows five template probes TP and five auxiliary probes AP; from Table 1, five TP-AP double strands are obtained, namely TP1-AP1 double strand, TP2-AP2 double strand, TP3-AP3 double strand, TP4-AP4 double strand, and TP5-AP5 double strand.

[0094] 6) Mix the TP-AP double strands formed in step 5) and the binding force probe FP (with an amino group NH2C6 at the 5' end, which can react with the aldehyde-modified glass substrate in step 4) in equal molar amounts, and incubate at 37°C for 1–2 h. Hybridization will then form a TP-AP-FP double strand at a concentration of 3 μM. Table 1 shows five binding force probes FP. From Table 1, five TP-AP-FP double strands were obtained: TP1-AP1-FP1 double strand, TP2-AP2-FP2 double strand, TP3-AP3-FP3 double strand, TP4-AP4-FP4 double strand, and TP5-AP5-FP5 double strand.

[0095] 7) Take 20 μL of one or more sets of TP-AP-FP double strands (3 μM) with a molar ratio of 1:1 and add them to the glass substrate sample cell in step 4) which has been aldehyde-modified. Reduce with sodium borohydride and incubate overnight at 4°C to form a DNA nanoarray.

[0096] 8) After incubation, seal the sample cell with Tris-HCl buffer containing 1% bovine serum albumin (BSA) and 0.05% Tween 20 to reduce non-specific adsorption;

[0097] 9) After blocking, Tris-HCl buffer containing streptavidin M280 magnetic beads was added to the sample cell and incubated at 37°C for 1 h to couple the magnetic beads to the template probe TP via biotin-streptavidin interaction and magnetize them with a magnet with a magnetic field strength of 0.8T.

[0098] 10) Mix equimolar amounts of the DNA traveling strand (WS) (nucleotide sequences shown in Table 1) and the nucleic acid aptamer sequence (nucleotide sequences shown in Table 1), and anneal at 95°C for 5–10 minutes to form a 3 μM WS-Aptamer double strand. Table 1 shows five DNA traveling strands (WS) and five nucleic acid aptamers (Aptamer). From Table 1, five WS-Aptamer double strands were obtained, namely WS1-Aptamer1, WS2-Aptamer2, WS3-Aptamer3, WS4-Aptamer4, and WS5-Aptamer5.

[0099] 11) Add exonuclease III to the WS-Aptamer double-stranded solution in step 10) to make the concentration of exonuclease III 2 U / μL, forming mixed solution A.

[0100] 2. Measure the binding force of different DNA double strands.

[0101] The binding force of different DNA double strands was measured by force-induced remanent magnetization spectroscopy.

[0102] The steps for measuring the TP-AP-FP double-chain bonding force in this embodiment are as follows:

[0103] 1) The template probe TP and the auxiliary probe AP were annealed at 95℃ for 5-10 minutes to form TP-AP double strands, and then mixed with the binding force probe FP in equimolar amounts. The mixture was incubated at 37℃ for 1-2 hours to form TP-AP-FP double strands at a concentration of 3 μM through hybridization.

[0104] 2) The formed TP-AP-FP double chain is fixed on an aldehyde-modified glass substrate according to step 7) in Example 1, sealed, connected to magnetic beads, and magnetized;

[0105] 3) Centrifuge the glass substrate obtained in step 2) at 1000 rpm / min for 5 minutes to remove physical adsorption. Place the glass substrate on the detection stage of an ultra-low field optical atomic magnetometer and measure the magnetic signal at this time;

[0106] 4) Gradually increase the centrifugal force, centrifuge for 5 minutes each time, and record the magnetic signal after centrifugation, corresponding one-to-one with the centrifugal force;

[0107] 5) According to the principle of measuring the bonding force, Brownian motion will occur when the TP-AP-FP double chain dissociates, the direction of the magnetic dipole of the magnetic bead will change, and the magnetic signal will decrease. Therefore, when the magnetic signal decreases significantly, the corresponding centrifugal force is the bonding force of the TP-AP-FP double chain.

[0108] The magnitude of the centrifugal force acting on the magnetic bead can be calculated using the following formula:

[0109] F=mω 2 r (1)

[0110] In equation (1), m is the mass of the magnetic bead, r is the radius of rotation, and ω is the centrifugal speed.

[0111] The steps for measuring the TP-FP double-chain bonding force in this embodiment are as follows:

[0112] 1) The template probe TP and the binding force probe FP were annealed at 95℃ for 5-10 minutes to form a TP-FP double strand with a concentration of 3μM;

[0113] 2) The formed TP-FP double chain is fixed on an aldehyde-modified glass substrate according to step 7) in Example 1, sealed, connected to magnetic beads, and magnetized.

[0114] 3) Centrifuge the glass substrate obtained in step 2) at 1000 rpm / min for 5 minutes to remove physical adsorption. Place the glass substrate on the detection stage of an ultra-low field optical atomic magnetometer and measure the magnetic signal at this time;

[0115] 4) Gradually increase the centrifugal force, centrifuge for 5 minutes each time, and record the magnetic signal after centrifugation, corresponding one-to-one with the centrifugal force;

[0116] 5) When the magnetic signal drops significantly, the corresponding centrifugal force is the binding force of the TP-FP double strand.

[0117] The results are as follows Figure 2 As shown, in the TP1-AP1-FP1 double strand, TP2-AP2-FP2 double strand, TP3-AP3-FP3 double strand, TP4-AP4-FP4 double strand, and TP5-AP5-FP5 double strand, the complementary pairing portions of TP and FP in the five double strands have the same base sequence and number, and therefore have the same binding force F0. According to Figure 3 Based on the force-induced remanent magnetization spectrum, F0 = 26 pN. Furthermore, the binding force of the TP1-FP1 double strand is F1 = 38 pN, the binding force of the TP2-FP2 double strand is F2 = 51 pN, the binding force of the TP3-FP3 double strand is F3 = 67 pN, the binding force of the TP4-FP4 double strand is F4 = 102 pN, and the binding force of the TP5-FP5 double strand is F5 = 110 pN.

[0118] Example 2: Feasibility verification of using a magnetic sensor device based on DNA binding force to detect Staphylococcus aureus

[0119] Based on the Aptamer1 sequence, which identifies Staphylococcus aureus, the following sequences were designed: DNA walking strand WS1 sequence, template probe TP1 sequence, helper probe AP1 sequence, and binding force probe FP1 sequence (nucleotide sequences of each sequence are shown in Table 1). The binding force of the TP1-AP1-FP1 double strand was measured to be F0 = 26 pN and the binding force of the TP1-FP1 double strand was F1 = 38 pN using force-induced remanent magnetization spectroscopy. The results are as follows: Figure 2 China A and Figure 3 As shown in A and B.

[0120] The feasibility of using a magnetic sensor based on DNA binding force to detect bacteria by targeting Staphylococcus aureus was verified, and the steps are as follows:

[0121] 1) A 3 μM TP1-AP1-FP1 double-stranded probe was immobilized on an aldehyde-modified glass substrate, sealed, coupled with magnetic beads, and magnetized.

[0122] 2) Mix WS1-Aptamer1 (final concentration 3 μM) double strands and exonuclease III (final concentration 2 U / μL) thoroughly to form mixed solution A;

[0123] 3) Add Tris-HCl buffer containing Staphylococcus aureus to mixed solution A. The concentration of Staphylococcus aureus is 10.8 CFU / mL;

[0124] 4) Add Tris-HCl buffer solution that does not contain Staphylococcus aureus to mixed solution A as a blank control experiment;

[0125] 5) Add the mixed solutions obtained in steps 3) and 4) to the magnetic sensor constructed in step 1) and incubate at 37°C for 80 minutes.

[0126] 6) Apply F0 (26pN) to the sensor to remove the unreacted TP1-AP1-FP1 double strand, and record the magnetic signal at this time as B0;

[0127] 7) Apply F1 (38pN) to the sensor and record the magnetic signal at this time as B1.

[0128] First, the formation of DNA double strands was verified by 15% polyacrylamide gel electrophoresis. For example... Figure 4 As shown, band M is a DNA marker, representing bands of DNA sequences with different base pairs. Band 1 represents the Aptamer1 sequence of Staphylococcus aureus, band 2 represents the traveling strand WS1, and band 3 represents the WS1-Aptamer1 double strand formed by annealing the Aptamer1 and the traveling strand WS1. Band 2 contains two bands because a portion of the traveling strand WS1 itself can form secondary structures. Band 3 shows a significant lag compared to bands 1 and 2, and the secondary structure band formed by WS1 itself is significantly lighter, indicating that the WS1-Aptamer1 double strand was formed through annealing. In addition, band 4 is the helper probe AP1, band 5 is the template probe TP1, band 6 is the binding force probe FP1, and band 7 is the TP1-AP1-FP1 double-stranded probe formed through annealing. Compared with bands 4, 5, and 6, band 7 showed a significant lag and a larger molecular weight, thus verifying the formation of the TP1-AP1-FP1 double-stranded probe.

[0129] Next, the feasibility of using a magnetic sensor based on DNA binding force to detect Staphylococcus aureus will be verified. For example... Figure 5As shown in Figure A, in a blank sample without Staphylococcus aureus, a significant decrease in magnetic signal occurred when F0 (26 pN) was applied to the sensor, corresponding to the dissociation of the TP1-AP1-FP1 double helix. In contrast, when the sample contained Staphylococcus aureus, the nucleic acid aptamer specifically recognized Staphylococcus aureus, allowing the DNA walking strand WS1 to be released from the WS1-Aptamer1 double helix. The released WS1 hybridizes with the helper probe AP1 via a foothold-mediated strand displacement reaction, inducing AP1 to dissociate from the TP1-AP1-FP1 double helix. Subsequently, the newly formed WS1-AP1 double helix is ​​catalyzed by exonuclease III, gradually hydrolyzing the blunt-ended 3' end, while the hydrolysis of other DNA strands is blocked by the terminal oligonucleotide T sequence. The re-released WS1 can walk on the substrate surface, continuing to hybridize with AP1 via a foothold-mediated strand displacement reaction and triggering hydrolysis. This results in the conversion of numerous TP1-AP1-FP1 double strands on the sensor surface into TP1-FP1 double strands. Therefore, when F0 (26 pN) is applied to the sensor, the magnetic signal shows almost no decrease. However, when F1 (38 pN) is applied, the magnetic signal decreases significantly. This rapid decrease in magnetic signal corresponds to the typical dissociation process of the TP1-FP1 double strands. This result demonstrates the successful fabrication of the sensor and its ability to be used for bacterial detection.

[0130] Example 3: Investigating the signal amplification mechanism of a magnetic sensor device based on DNA binding force

[0131] To verify the mechanism of biorecognition signal amplification, the change (ΔB) in magnetic signal before and after application of F1 (38pN) was measured in the presence of Staphylococcus aureus, WS1-Aptamer1 double-stranded DNA, or exonuclease III in mixed solution A. Exonuclease III typically amplifies the signal by hydrolyzing a portion of the DNA sequence to enable the DNA walking strand to move across the substrate surface.

[0132] The results are as follows Figure 5 As shown in Figure C, the change in magnetic signal in the blank group is negligible. Compared with the blank group, when Staphylococcus aureus and WS1-Aptamer1 double strands are present simultaneously in mixed solution A, a basement-mediated strand displacement reaction occurs, causing AP1 to dissociate from the TP1-AP1-FP1 double strand, resulting in a slight increase in magnetic signal change, reaching 2.6 ± 0.5 pT. In the experiment where Staphylococcus aureus, WS1-Aptamer1 double strands, and exonuclease III are present simultaneously in mixed solution A, the magnetic signal change is the largest, reaching 16.3 ± 0.9 pT, with sensitivity much higher than the control group in mixed solution A without WS1-Aptamer1 double strands or exonuclease III.

[0133] The results showed that the WS1-Aptamer1 double strand is crucial for recognizing target bacteria and initiating DNA strand displacement reactions, while exonuclease III-induced signal amplification significantly improved the sensitivity.

[0134] Example 4: Improving sensor performance by optimizing experimental parameters

[0135] To achieve optimal performance of the DNA binding-based magnetic sensor for bacterial detection, four experimental parameters were optimized: DNA double-strand concentration, exonuclease III concentration, incubation time, and magnetic bead concentration.

[0136] 1) DNA double-strand concentration

[0137] Double strands of TP1-AP1-FP1 at concentrations of 0.4, 0.8, 1.5, 3.0, 4.5, and 6.0 μM were immobilized on an aldehyde-modified glass substrate, sealed, and then coupled with magnetic beads. The substrate was incubated with magnetic beads at a concentration of 2.5 mg / mL to couple the substrate with the magnetic beads.

[0138] Mixed solution A containing equimolar amounts of WS1-Aptamer1 double strands and exonuclease III (5 U / μL) and 10 8 After thoroughly mixing CFU / mL of Staphylococcus aureus, 20 μL was added to the constructed magnetic sensor and incubated at 37°C for 60 minutes. F0 was applied to the sensor to remove unreacted TP1-AP1-FP1 double strands. F1 was applied to the sensor, and the change in magnetic signal (ΔB) before and after application was recorded.

[0139] The results are as follows Figure 6 As shown in Figure A, the magnetic signal gradually increases with increasing DNA double-strand concentration from 0.4 μM to 3.0 μM. This is because more DNA double strands are immobilized on the sensor surface, leading to greater dissociation of the TP1-FP1 double strands by external force. When the DNA double-strand concentration exceeds 3.0 μM, the magnetic signal decreases slightly, possibly due to increased steric hindrance at higher concentrations. Therefore, a DNA double-strand concentration of 3.0 μM is considered the optimal experimental parameter.

[0140] 2) Concentration of exonuclease III

[0141] 3 μM of TP1-AP1-FP1 double strands were immobilized on an aldehyde-modified glass substrate, sealed, and then connected to magnetic beads. The substrate was incubated with magnetic beads at a concentration of 2.5 mg / mL to couple the substrate with magnetic beads.

[0142] Mixed solution A containing 3 μM WS1-Aptamer1 double strands and 0, 1, 2, 3, 4, 5 U / μL exonuclease III and 10 4After thoroughly mixing CFU / mL of Staphylococcus aureus, 20 μL was added to the constructed magnetic sensor and incubated at 37°C for 60 minutes. F0 was applied to the sensor to remove unreacted TP1-AP1-FP1 double strands. F1 was applied to the sensor, and the change in magnetic signal (ΔB) before and after application was recorded.

[0143] The results are as follows Figure 6 As shown in Figure B, the magnetic signal gradually increased with increasing exonuclease III concentration from 0 U / μL to 2 U / μL, then plateaued between 2 U / μL and 5 U / μL. Therefore, an exonuclease III concentration of 2 U / μL was selected as the optimal experimental parameter.

[0144] 3) Magnetic bead incubation time

[0145] 3 μM of TP1-AP1-FP1 double strands were immobilized on an aldehyde-modified glass substrate, blocked, and then coupled with magnetic beads. The substrate was incubated with magnetic beads at a concentration of 2.5 mg / mL to allow for coupling. A mixed solution A containing 3 μM of WS1-Aptamer1 double strands, 2 U / μL exonuclease III, and 10... 8 After thoroughly mixing CFU / mL of Staphylococcus aureus, 20 μL was added to the constructed magnetic sensor and incubated at 37°C for 10, 20, 40, 60, 80, 100, and 120 minutes. F0 was applied to the sensor to remove unreacted TP1-AP1-FP1 double strands. F1 was applied to the sensor, and the change in magnetic signal (ΔB) before and after application was recorded.

[0146] The results are as follows Figure 6 As shown in Figure C, the magnetic signal changes the most when the incubation time is 80 minutes. Therefore, an incubation time of 80 minutes is taken as the optimal experimental parameter.

[0147] 4) Magnetic bead concentration

[0148] 3 μM of TP1-AP1-FP1 double strands were immobilized on an aldehyde-modified glass substrate, sealed, and then coupled with magnetic beads. The substrate was incubated with magnetic beads at concentrations of 1.0, 2.5, 5.0, 7.5, and 9.0 mg / mL to allow for coupling. A mixed solution A containing 3 μM of WS1-Aptamer1 double strands and 2 U / μL exonuclease III was prepared and 10... 8 After thoroughly mixing CFU / mL of Staphylococcus aureus, 20 μL was added to the constructed magnetic sensor and incubated at 37°C for 80 minutes. F0 was applied to the sensor to remove unreacted TP1-AP1-FP1 double strands. F1 was applied to the sensor, and the change in magnetic signal (ΔB) before and after application was recorded.

[0149] The results are as follows Figure 6As shown in Figure D, the magnetic signal showed the most significant change when the magnetic bead concentration was 2.5 mg / mL. Therefore, a magnetic bead concentration of 2.5 mg / mL was taken as the optimal experimental parameter.

[0150] Example 5: Sensitivity and dynamic response range of a DNA binding-based magnetic sensor device for bacterial detection.

[0151] Under the optimal experimental parameters in Example 4, the sensitivity and dynamic response range of the sensor for bacterial detection were investigated.

[0152] The steps are as follows: 3 μM of TP1-AP1-FP1 double strands were immobilized on an aldehyde-modified glass substrate, sealed, connected to magnetic beads, and incubated with magnetic beads at a concentration of 2.5 mg / mL to couple the substrate with magnetic beads.

[0153] Mixed solution A containing 3 μM WS1-Aptamer1 double strands and 2 U / μL exonuclease III, and solution B containing 10 μM WS1-Aptamer1 double strands and 10 μL exonuclease III respectively. 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 and 10 8 After thoroughly mixing CFU / mL of Staphylococcus aureus, 20 μL was added to the constructed magnetic sensor and incubated at 37°C for 80 minutes. F0 was applied to the sensor to remove unreacted TP1-AP1-FP1 double strands. F1 was applied to the sensor, and the change in magnetic signal (ΔB) before and after application was recorded.

[0154] By analyzing the relationship between the change in magnetic signal ΔB and the concentration of Staphylococcus aureus, a standard curve for the detection of Staphylococcus aureus using a magnetic sensor based on DNA binding force was obtained. The results are as follows: Figure 5 As shown in Figure B, with the increase of Staphylococcus aureus concentration, the change in magnetic signal ΔB also gradually increases, reaching a certain level at 10... 0 ~10 8 The sensor exhibits good linearity at CFU / mL and a wide dynamic response range. Furthermore, it features low background noise and built-in enzyme-induced cascade signal amplification, achieving a detection limit of 1 CFU / mL.

[0155] Example 6: Investigating the anti-interference performance of a magnetic sensor device based on DNA binding force

[0156] In this embodiment, Staphylococcus aureus is used as the target bacteria, and other non-target bacteria or mixtures are used to explore the anti-interference performance of the sensor.

[0157] The steps are as follows: 3 μM of TP1-AP1-FP1 double strands were immobilized on an aldehyde-modified glass substrate, sealed, and then coupled with magnetic beads. The substrate was incubated with magnetic beads at a concentration of 2.5 mg / mL to couple the fibers. A mixed solution A containing 3 μM of WS1-Aptamer1 double strands, 2 U / μL of exonuclease III, and different samples was thoroughly mixed. 20 μL of this solution was added to the constructed magnetic sensor and incubated at 37°C for 80 minutes. F0 was applied to the sensor to remove unreacted TP1-AP1-FP1 double strands. F1 was applied to the sensor, and the change in magnetic signal (ΔB) before and after application was recorded.

[0158] The sample groups are as follows:

[0159] 1) Tris-HCl buffer that contains no bacteria;

[0160] 2) Contains 10 8 CFU / mL E. coli Tris-HCl buffer;

[0161] 3) Contains 10 8 Tris-HCl buffer for CFU / mL Salmonella Typhimurium;

[0162] 4) Contains 10 8 CFU / mL Bacillus subtilis Tris-HCl buffer;

[0163] 5) Contains 10 8 CFU / mL Shigella Tris-HCl buffer;

[0164] 6) Contains 10 5 CFU / mL Staphylococcus aureus Tris-HCl buffer;

[0165] 7) Contains 10 8 A Tris-HCl buffer solution containing CFU / mL of Escherichia coli, Salmonella typhimurium, Bacillus subtilis, and Shigella is called Mixed Solution 1.

[0166] 8) Contains 10 8 CFU / mL Escherichia coli, Salmonella typhimurium, Bacillus subtilis, Shigella, 10 5 The Tris-HCl buffer solution containing CFU / mL Staphylococcus aureus is called Mixed Solution 2;

[0167] The results are as follows Figure 5 As shown in D, when the concentration of Staphylococcus aureus is 10... 5 CFU / mL, while the concentration of other non-target bacteria was 10. 8At CFU / mL, the platform's response to Staphylococcus aureus was significantly higher than other control groups before and after the application of F1 (38 pN), indicating its excellent specificity. 10 5 CFU / mL Staphylococcus aureus and 10 8 The magnetic signal change of the mixed solution 2, prepared by mixing other non-target bacteria at CFU / mL, was similar to that of Staphylococcus aureus alone, indicating that this method has good application potential for the detection of bacteria in complex samples.

[0168] Example 7: Simultaneous detection of Staphylococcus aureus and Bacillus subtilis using a magnetic sensor device based on DNA binding force.

[0169] Detecting the multiplexing capability of bacteria is a crucial aspect of bacterial identification. In this embodiment, using Staphylococcus aureus and Bacillus subtilis as examples, the ability of this sensor device to simultaneously detect two bacteria was investigated. The set of DNA probes for Staphylococcus aureus consisted of Aptamer1, WS1, TP1, AP1, and FP1, while the set of DNA probes for Bacillus subtilis consisted of Aptamer2, WS2, TP2, AP2, and FP2. The detection steps are as follows:

[0170] 1) The binding forces of the TP1-AP1-FP1 double strand, TP1-FP1 double strand, TP2-AP2-FP2 double strand, and TP2-FP2 double strand were measured by force-induced remanent magnetization spectroscopy. Among them, the complementary pairing parts of TP and FP in the TP1-AP1-FP1 double strand and TP2-AP2-FP2 double strand have the same base sequence and number, so the binding force of the TP1-AP1-FP1 double strand is F1 = 38 pN, and the binding force of the TP2-FP2 double strand is F2 = 51 pN.

[0171] 2) 3 μM of TP1-AP1-FP1 double strands and TP2-AP2-FP2 double strands were fixed on an aldehyde-modified glass substrate at a molar ratio of 1:1. After sealing, magnetic beads were attached and incubated with magnetic beads at a concentration of 2.5 mg / mL to couple the substrate with magnetic beads.

[0172] 3) Add 3 μM of WS1-Aptamer1 double strands and WS2-Aptamer2 double strands to an exonuclease III solution (final concentration 2 U / μL) at a molar ratio of 1:1 and mix well to form mixed solution A;

[0173] 4) Add bacteria to the mixed solution A to mix with the test sample, the bacterial concentration is 10. 5 CFU / mL;

[0174] 5) Add the solution obtained in step 4) to the sensor constructed in step 2) and incubate at 37°C for 80 minutes;

[0175] 6) Apply F0 (26pN) to the sensor in step 5) to remove the unreacted TP1-AP1-FP1 double strand and TP2-AP2-FP2 double strand, and record the magnetic signal at this time;

[0176] 7) Apply centrifugal force to the sensor sequentially from smallest to largest, and record the magnetic signals;

[0177] The results are as follows Figure 7 As shown, the magnetic signal changed significantly at 38 pN and 51 pN, corresponding to the dissociation of the TP1-FP1 and TP2-FP2 double strands, with proportions of 42.1 ± 10.4% and 42.8 ± 8.5%, respectively. Based on the binding forces of the TP1-FP1 and TP2-FP2 double strands corresponding to Staphylococcus aureus and Bacillus subtilis, respectively, at 38 pN and 51 pN, the bacterial species in the sample can be clearly identified as Staphylococcus aureus and Bacillus subtilis in a 1:1 ratio, consistent with the pre-designed pattern.

[0178] Example 8: Simultaneous detection of Staphylococcus aureus, Bacillus subtilis, and Shigella using a magnetic sensor device based on DNA binding force.

[0179] In this embodiment, Staphylococcus aureus, Bacillus subtilis, and Shigella are used as examples to further apply the sensor device to detect more types of bacteria in a mixed solution. Specifically, the DNA probes for Staphylococcus aureus are Aptamer1, WS1, TP1, AP1, and FP1; the DNA probes for Bacillus subtilis are Aptamer2, WS2, TP2, AP2, and FP2; and the DNA probes for Shigella are Aptamer4, WS4, TP4, AP4, and FP4. The detection steps are as follows:

[0180] 1) The binding forces of the TP1-AP1-FP1 double strand, TP1-FP1 double strand, TP2-AP2-FP2 double strand, TP2-FP2 double strand, TP4-AP4-FP4 double strand, and TP4-FP4 double strand were measured by force-induced remanent magnetization spectroscopy. Among them, the complementary pairing parts of TP and FP in the TP1-AP1-FP1 double strand, TP2-AP2-FP2 double strand, and TP4-AP4-FP4 double strand have the same base sequence and number, so the binding forces are all F0 = 26 pN. The binding force of the TP1-FP1 double strand is F1 = 38 pN, the binding force of the TP2-FP2 double strand is F2 = 51 pN, and the binding force of the TP4-FP4 double strand is F4 = 102 pN.

[0181] 2) 3 μM of TP1-AP1-FP1 double strands, TP2-AP2-FP2 double strands, and TP4-AP4-FP4 double strands were fixed on an aldehyde-modified glass substrate at a molar ratio of 1:1:1. After sealing, magnetic beads were attached and incubated with magnetic beads at a concentration of 2.5 mg / mL to couple the substrate with magnetic beads.

[0182] 3) Add 3 μM of WS1-Aptamer1 double strand, WS2-Aptamer2 double strand, and WS4-Aptamer4 double strand to an exonuclease III solution (final concentration 2 U / μL) at a molar ratio of 1:1:1 and mix well to form mixed solution A;

[0183] 4) Add bacteria to the mixed solution A to mix with the test sample, the bacterial concentration is 10. 5 CFU / mL;

[0184] 5) Add the solution obtained in step 4) to the sensor constructed in step 2) and incubate at 37°C for 80 minutes;

[0185] 6) Apply F0 (26pN) to the sensor in step 5) to remove unreacted TP1-AP1-FP1 double strands, TP2-AP2-FP2 double strands, and TP4-AP4-FP4 double strands, and record the magnetic signal at this time;

[0186] 7) Apply centrifugal force to the sensor sequentially from smallest to largest, and record the magnetic signals;

[0187] The results are as follows Figure 8 As shown, the magnetic signal exhibited three significant decreases at 38 pN, 51 pN, and 102 pN, corresponding to the dissociation of the TP1-FP1, TP2-FP2, and TP4-FP4 double strands, respectively, with dissociation rates of 28.1 ± 4.8%, 29.2 ± 6.1%, and 24.5 ± 4.0%. This indicates that the bacterial species in the mixed test sample were Staphylococcus aureus, Bacillus subtilis, and Shigella, in a ratio of approximately 1:1:1. The sample analysis results were consistent with the pre-defined bacterial species. Therefore, this demonstrates that the DNA binding-based magnetic sensor device can achieve multiplex detection and phenotypic analysis of bacteria, and can identify various combinations of different bacterial types.

[0188] Example 9: A magnetic sensor device based on DNA binding force for the analysis of whole blood samples.

[0189] Detecting bacteria directly from untreated biological matrices (such as blood and urine) is crucial to reduce biases introduced during pre-enrichment, extraction, and purification steps. Since magnetic sensors utilize magnetic signals as reporting signals, they are unaffected by solution color and ambient light. Therefore, in this example, blank samples and whole blood samples containing bacteria were prepared and added to the sensor. Different types of bacteria were then detected using DNA binding forces.

[0190] The sample groups are as follows:

[0191] 1) Sterilized rabbit whole blood containing no bacteria;

[0192] 2) Contains a concentration of 10 5 Sterilized rabbit whole blood containing CFU / mL Staphylococcus aureus;

[0193] 3) Contains a concentration of 10 5 Sterilized rabbit whole blood containing CFU / mL Shigella;

[0194] 4) Contains a concentration of 10 5 Sterilized rabbit whole blood containing CFU / mL Staphylococcus aureus and Escherichia coli (in a 1:1 ratio);

[0195] 5) Contains a concentration of 10 5 Sterilized rabbit whole blood containing CFU / mL Bacillus subtilis and Escherichia coli (in a 1:1 ratio);

[0196] 6) Contains a concentration of 10 5 Sterilized rabbit whole blood containing CFU / mL Staphylococcus aureus and Shigella (in a 1:1 ratio);

[0197] 7) Contains a concentration of 10 5 Sterilized rabbit whole blood containing CFU / mL Bacillus subtilis and Salmonella typhimurium (in a 1:1 ratio);

[0198] 8) Contains a concentration of 10 5 Sterilized rabbit whole blood containing CFU / mL Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium (in a ratio of 1:1:1);

[0199] 9) Contains a concentration of 10 5 Sterilized rabbit whole blood containing CFU / mL of Staphylococcus aureus, Bacillus subtilis, and Shigella (in a ratio of 1:1:1).

[0200] The steps for analyzing whole blood samples using a magnetic sensor device based on DNA binding force are as follows:

[0201] 1) Immobilize TP-AP-FP double-stranded probes (concentration of 3 μM) that specifically respond to different bacteria on an aldehyde-modified glass substrate in equal proportions, block them, couple them with magnetic beads and magnetize them;

[0202] 2) Mix WS-Aptamer double strands (3uM concentration) that specifically recognize different bacteria in equal proportions, and add exonuclease III to form mixed solution A;

[0203] 3) Add mixed solution A to the above sample groups 1)-9) to simulate the whole blood sample to be tested;

[0204] 4) Add the mixed solution obtained in step 3) to the magnetic sensor constructed in step 1) and incubate at 37°C for 80 minutes;

[0205] 5) Apply F0 (26pN) to the magnetic sensor in step 4) to remove the unreacted TP1-AP1-FP1 double strand, and record the magnetic signal at this time as B0;

[0206] 6) Different binding forces of TP-FP double strands are applied to the sensor sequentially, and the magnetic signal is recorded after each force is applied. The type of bacteria in the sample can be determined by the change in magnetic signal ΔB.

[0207] The results are as follows Figure 9 and 10 As shown, after adding blank sample 1, the magnetic sensor showed very little change in magnetic signal under different external forces, indicating that the simulated blood sample was free of bacteria. In sample 2, the magnetic sensor responded significantly under an external force of 38 pN, with the largest change in magnetic signal, indicating that the simulated blood sample contained Staphylococcus aureus, consistent with the pre-set parameters. When testing sample 3, Figure 9 The darkest color corresponds to Shigella medusata. Figure 10 The magnetic signal change was greatest corresponding to Shigella, indicating the presence of Shigella in the simulated blood sample. Furthermore, this magnetic sensing device can simultaneously detect multiple target bacteria. For example, when detecting sample 4, the magnetic signal of the sensor significantly decreased under external forces of 38 pN and 67 pN, indicating the presence of Staphylococcus aureus and Escherichia coli in the blood sample. Figure 9 and 10 As shown, when the magnetic sensor detected sample 5, the colors corresponding to Bacillus subtilis and Escherichia coli were darker, and the magnetic signal changes were larger, indicating that the blood sample contained Bacillus subtilis and Escherichia coli. When detecting sample 6, the magnetic sensor showed the largest changes in magnetic signal under external forces of 38 pN and 102 pN. Through thermograms and statistical analysis, the bacterial species in the blood sample were identified as Staphylococcus aureus and Shigella. When the magnetic sensor detected sample 7, by comparison... Figure 9 and Figure 10The system can confirm the presence of Bacillus subtilis and Salmonella typhimurium in the blood sample. When testing sample 8, the magnetic sensor accurately identified Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium in the blood sample. Similarly, the magnetic sensor can detect Staphylococcus aureus, Bacillus subtilis, and Shigella in sample 9.

[0208] Based on the above test results, the magnetic sensor can accurately detect different combinations of single and multiple bacteria in simulated blood samples, corresponding to pre-set experiments. Therefore, the DNA-binding magnetic sensor device has very high accuracy in multiplex detection of bacteria in whole blood, capable of detecting all bacterial species in the blood. This indicates that the sensor device has great potential in guiding decision-making in various clinical situations.

[0209] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A device for analyzing biological samples, including a magnetic sensor, a DNA walking chain (WS), a nucleic acid aptamer specific to the biological sample to be tested, and an exonuclease III; The magnetic sensor is an array containing N types of double-stranded DNA molecules named TP-AP-FP fixed on a substrate and a magnetic solid modified with streptavidin. The double-stranded DNA molecules of TP-AP-FP are composed of single-stranded DNA molecules named template probe TP, auxiliary probe AP and binding force probe FP, respectively. The 5' end of the template probe TP is modified with a biotin molecule, and the 3' end has m1 deoxythymidine nucleotides. The first m2 nucleotides from the 5' end are complementary to the auxiliary probe AP. The auxiliary probe AP pairs complementaryly with the template probe TP for the first m2 nucleotides starting from the 5' end, and pairs complementaryly with the DNA strand WS for the first m3 nucleotides starting from the 3' end, forming a blunt 3' end with the DNA strand WS; the DNA strand WS pairs complementaryly with the nucleic acid aptamer specific to the biological sample to be tested; The binding force probe FP has a group modified at its 5' end for immobilization on the substrate, and has m1 deoxythymidine nucleotides at its 3' end; the binding force probe FP is immobilized on the substrate; the binding force probe FP is complementary to the template probe TP, and the complementary pairing region of FP and TP overlaps with the complementary pairing region of AP and TP; the number of bases in the overlapping region is 3 or more. N is a natural number greater than 1; The m1 is a natural number greater than or equal to 4; The m2 is a natural number greater than or equal to 6; The m3 is a natural number greater than or equal to 12.

2. The apparatus according to claim 1, characterized in that: The magnetic sensor also contains N types of double-stranded DNA molecules named TP-FP immobilized on the substrate. The TP-FP double-stranded DNA molecules are formed by linking the template probe TP and the binding force probe FP single-stranded DNA molecules.

3. The apparatus according to claim 1, characterized in that: The biological sample to be tested is Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhimurium and / or Shigella, or the biological sample to be tested is a sample containing Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhimurium and / or Shigella.

4. The apparatus according to claim 1, characterized in that: The nucleic acid aptamer specific to the biological sample to be tested is a nucleic acid aptamer specific to Staphylococcus aureus, a nucleic acid aptamer specific to Bacillus subtilis, a nucleic acid aptamer specific to Escherichia coli, a nucleic acid aptamer specific to Shigella, or a nucleic acid aptamer specific to Salmonella typhimurium.

5. A method for detecting microbial samples for non-disease diagnostic and therapeutic purposes, comprising the following steps: 1) Add mixed solution A and the sample to be tested to the magnetic sensor in the device of claim 1 or 2 and incubate, wherein the mixed solution A contains DNA walking chain WS-Aptamer double-stranded probe solution and exonuclease III, wherein the DNA walking chain WS-Aptamer double-stranded probe solution is prepared by mixing one or more DNA walking chain solutions WS and complementary nucleic acid aptamer solutions Aptamer and annealing at 95°C for 5 to 10 minutes; 2) After incubation, external mechanical force is applied to the magnetic sensor. The composition of the sample to be tested is determined by the binding force of the DNA double-stranded probe and the decrease in magnetic signal.

6. The apparatus of claim 1 or 2, and the method of claim 5, for the detection of proteins, small molecules, metal ions, or cells; said application is for purposes other than disease diagnosis and treatment.

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