Paper-based sensor device based on deoxyribozyme probe and application

Abstract

The invention discloses a paper-based sensor device based on a deoxyribozyme probe and application, and belongs to the technical field of analysis and detection. The paper-based sensor based on the deoxyribozyme probe by using the fluorescence signal is constructed, complex treatment on a sample is not needed, only a bacterial lysis buffer needs to be added into the sample for lysis, the deoxyribozyme is enabled to cut the deoxyribozyme probe by the generated escherichia coli CIM specificity, so that a sequence fragment containing an FAM group modified on the deoxyribozyme probe falls off, and the detection result is accurate. A complementary sequence modified at a detection line is used for capturing, a fluorescence signal is read to realize the detection purpose, and the whole reaction process can be completed within 30 minutes. The method integrates the processes of bacterial lysis, reaction sampling, bacterial detection and the like, avoids the complex and time-consuming operation process of a traditional method, has the analysis performance of rapidness, simplicity, rapidness, low cost and the like, and has potential application value in analysis and detection of various complex liquid bacteria in the clinical medicine field.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of analytical detection, and particularly relates to a paper-based sensor device based on a deoxyribozyme probe and application. BACKGROUND

[0002] In the field of clinical medicine and public health, rapid diagnosis of bacterial infection is of great significance for early intervention of diseases, rational use of drugs, and control of the spread of infectious diseases. Escherichia coli, as one of the common gram-negative bacteria in the human intestine, can cause urinary tract infection, septicemia, diarrhea and other diseases in some serotypes, and plays an important role in hospital infections. Therefore, developing a technology that can rapidly and sensitively detect Escherichia coli in complex biological samples such as urine has wide clinical application value.

[0003] Traditional bacterial detection methods mainly rely on culture method, immunological detection or molecular biology technology. Among them, although the culture method has high accuracy, it is complicated to operate and usually needs a culture period of more than 24 hours, which is difficult to meet the needs of point-of-care testing (POCT); immunological methods such as enzyme-linked immunosorbent assay (ELISA) have certain sensitivity and specificity, but still need complex sample pretreatment and special instrument support; and nucleic acid detection methods based on PCR amplification have high sensitivity, but have high requirements for experimental environment and technical personnel, and are difficult to promote in primary medical institutions.

[0004] In recent years, with the development of new recognition elements such as aptamer, DNAzyme and nanomaterials, paper-based microfluidic analysis platform has gradually become an important research direction of portable biosensing technology. Paper-based sensors are widely used in food safety, environmental monitoring and clinical diagnosis due to their low cost, portability, and the ability to achieve visual reading without complex equipment. Especially combined with fluorescence labeling, colorimetric analysis or smartphone image acquisition, paper-based sensors show good prospects in improving detection sensitivity and quantitative ability.

[0005] Deoxyribozyme is a kind of catalytically active DNA molecule that can cleave RNA substrates in the presence of specific metal ions. Recent studies have found that some deoxyribozymes can recognize specific protein or small molecule targets by design, thereby achieving indirect detection of pathogenic microorganisms. Biosensors constructed using deoxyribozyme probes not only have good stability, but also are easy to synthesize and modify chemically, and are suitable for the design of on-site rapid detection systems.

[0006] Deoxyribozyme probes have been reported for the detection of heavy metal ions, inflammatory factors, tumor markers, etc., but their application in bacterial detection is still in the initial exploration stage. In particular, there is no mature and commercialized paper-based sensor based on deoxyribozyme probes for the detection of common pathogenic bacteria such as Escherichia coli. There is also a lack of an integrated detection scheme that integrates sample lysis, reaction, capture, and signal reading in the prior art, resulting in most detection processes still relying on laboratory environment and professional equipment.

[0007] In addition, traditional paper-based sensors mostly use antibody-antigen reactions or DNA hybridization mechanisms, which are susceptible to cross-reactions and have certain limitations in sensitivity. The introduction of deoxyribozyme probes can amplify the signal response through their specific cleavage mechanism, improve the accuracy and sensitivity of detection, and reduce non-specific interference. Therefore, paper-based sensors based on deoxyribozyme probes are expected to overcome many defects in the prior art and provide a new rapid detection tool for clinical applications.

[0008] In summary, there is an urgent need for an Escherichia coli detection method that is simple in structure, easy to operate, high in sensitivity, and suitable for various complex liquid samples. SUMMARY

[0009] To solve the problems existing in the prior art, the purpose of the present application is to provide a paper-based sensor device based on deoxyribozyme probes and its application. The present application integrates bacterial lysis, reaction sampling, and bacterial detection processes, enabling the detection of Escherichia coli in urine. It is simple to operate and does not require professional personnel to spend time on operation, avoiding the complex and time-consuming operation process of traditional methods. It has the analysis performance of rapid, simple, fast, and low cost, fills the gap in the prior art, promotes the development of portable bacterial detection technology towards higher efficiency, lower cost, and easier popularization, and has potential application value for the analysis and detection of various complex liquid bacteria in the field of clinical medicine.

[0010] To achieve the above purpose, the present application provides the following technical solutions:

[0011] In a first aspect, the present application provides a paper-based sensor device based on deoxyribozyme probe, which mainly comprises a sample adding area device, a labeled reagent storage area device and a water absorption pad connected in sequence, the sample adding area device is connected with the labeled reagent storage area device and there is an overlapping area between them; the labeled reagent storage area device is connected with the water absorption pad and there is an overlapping area between them, the labeled reagent storage area is provided with a detection line (T line) and a control line (C line), wherein the detection line is close to the side of the sample adding area device, the control line is close to the side of the water absorption pad, the detection line is coated with streptavidin and the complex of the sequence complementary to the sequence released after the deoxyribozyme probe is cut and containing FAM group, and the control line is coated with streptavidin and the complex of another complementary sequence; the structure of the deoxyribozyme probe is: F-CTATGAACTGACTRTGACCTCACTACCAAGCAGGTCCATCGAGTGGTAGGATG CGGCGGTCAGTCGCACTGCTCCTTTTTTTTTTT-B, B is biotin, R is RNA base A (rA), and F is T base containing fluorescent group; the structure of the complementary sequence coated on the detection line is: B-TTTTTTTTTTTAGTCAGTTCATAGGATCATCCAG, B is biotin; and the structure of the complementary sequence coated on the control line is: ACCTTCCTCCGCAATACTCCCCCAGGTTTTTT-B, B is biotin.

[0012] Further, the sample adding area device comprises a filter paper JY-BX108 GL-b01 of Shanghai Jieyi Biotechnology Co., Ltd.; the labeled reagent storage area device comprises a Whatman Grade 1 nitrocellulose membrane; and the water absorption pad comprises a filter paper JY-X101 H5076 of Shanghai Jieyi Biotechnology Co., Ltd.

[0013] Further, the sample adding area device, the labeled reagent storage area device and the water absorption pad are in the shape of rectangle.

[0014] Further, the length of the sample adding area device is 10-20 mm, the length of the labeled reagent storage area device is 20-30 mm, the length of the water absorption pad is 10-30 mm, the length of the overlapping area is 1-5 mm, and the width of the sample adding area device, the labeled reagent storage area device and the water absorption pad is 1-5 mm.

[0015] Further, the paper-based sensor device is assembled in a plastic shell as a whole.

[0016] Further, the deoxyribozyme probe has specific recognition and cutting ability for E. coli CIM, and has a cuttable RNA connection site; the deoxyribozyme probe is modified with a fluorescent marker FAM, and one end is fixed with an agarose bead; a detection line (T line) is fixed with a DNA sequence complementary to the sequence fragment containing the FAM group released after the deoxyribozyme probe is cut; when the deoxyribozyme probe cuts the RNA connection site under the action of E. coli CIM, a sequence fragment containing the FAM group is released, and binds with the complementary sequence fixed at the detection line, thereby generating a detectable fluorescent signal. The reading of the fluorescent signal can be scanned and quantified by Image J software to determine the presence or absence of E. coli in the sample and the concentration level of E. coli.

[0017] In a second aspect, the application provides the use of the above-mentioned paper-based sensor device for detecting whether there is E. coli in the liquid sample to be tested and the concentration of E. coli.

[0018] Further, the liquid sample to be tested includes urine.

[0019] Further, the use includes the following steps:

[0020] (1) Lysis of bacteria in the liquid sample to be tested, and adding the obtained lysis solution, deoxyribozyme probe fixed with agarose beads and buffer solution into a test tube, reacting at room temperature for 10-30 min, and adding 10-40 ul of the reaction mixture to the sample addition area of the paper-based sensor device;

[0021] (2) After the liquid completely flows through the water-absorbing pad, the fluorescent signal and the fluorescent intensity at the detection line are scanned and read using Image J software.

[0022] Further, in step (1), the liquid sample to be tested is mixed with a bacterial lysis solution for bacterial lysis, and the bacterial lysis solution is an SDS lysis solution.

[0023] Further, in step (1), the reaction time is 20 min, and the amount of the reaction mixture added is 25 ul.

[0024] Further, in step (1), the agarose bead and the deoxyribozyme probe are fixed by streptavidin connection.

[0025] Further, in step (1), the volume ratio of the lysis solution, the deoxyribozyme probe fixed with agarose beads and the buffer solution is 1-2: 1-2: 1-3.

[0026] Further, the buffer in step (1) is composed of 50 mM sodium citrate, 150 mM NaCl, 50 mM KCl, 20 mM EDTA, 0.01% Tween 20, pH 6-7.

[0027] Further, the content of E. coli in the liquid sample to be tested in step (1) is 10 2 ~ 10 7 CFU / mL.

[0028] Further, in step (2), if E. coli exists in the liquid sample to be tested, the E. coli CIM released by lysis makes the deoxyribozyme specific cleavage of the deoxyribozyme probe, resulting in the shedding of the sequence fragment containing the FAM fluorescent group into the sample addition zone device, and the deoxyribozyme probe is fixed in the sample addition zone device under the action of the agarose beads, the shed FAM-labeled cleavage fragment is captured by the complementary sequence at the detection line (T line), generating a detectable fluorescent signal, and the presence or absence of E. coli in the sample and the concentration level are determined by the fluorescence intensity.

[0029] Compared with the prior art, the beneficial effects of the present application are:

[0030] The present application integrates the processes of bacterial lysis, reaction sampling and bacterial detection, can realize the detection of E. coli in urine, is simple to operate, does not require professional personnel to operate for a long time, avoids the complex and time-consuming operation process of the traditional method, has the analysis performance of rapidness, simplicity, speediness and low cost, and has potential application value for the analysis and detection of various complex liquid bacteria in the field of clinical medicine. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is a detection principle schematic diagram of the paper-based sensor based on the deoxyribozyme probe in Example 2.

[0032] Figure 2 It is a feasibility analysis diagram of the paper-based sensor based on the deoxyribozyme probe in Example 3, wherein A is the detection result of the paper-based sensor, and B is the fluorescence signal intensity.

[0033] Figure 3 It is a condition optimization analysis diagram of the paper-based sensor based on the deoxyribozyme probe in Example 4, wherein A is the pH condition optimization, B is the total volume optimization of the reaction system, and C is the reaction time optimization.

[0034] Figure 4 It is a specificity analysis diagram of the paper-based sensor based on the deoxyribozyme probe in Example 5 for testing different pathogenic bacteria.

[0035] Figure 5 It is a sensitivity analysis diagram of the paper-based sensor based on the deoxyribozyme probe in Example 6 for detecting E. coli. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the specific implementation methods of the present invention are further described in detail below in conjunction with embodiments.

[0037] Table 1: Nucleic acid sequences used in the present invention

[0038]

[0039]

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0042] Example 1 Preparation of paper-based sensor based on DNAzyme probe

[0043] The paper-based sensor consists primarily of a sample pad (JY-BX108 GL-b01 filter paper, Shanghai Jieyi Biotechnology Co., Ltd.), a nitrocellulose membrane (NC membrane, Whatman Grade 1 nitrocellulose membrane), and an absorbent pad (JY-X101 H5076 filter paper, Shanghai Jieyi Biotechnology Co., Ltd.). The sample pad, nitrocellulose membrane (NC membrane), and absorbent pad are 15 mm, 25 mm, and 20 mm long, respectively, and 3 mm wide. During assembly, the sample pad overlaps the NC membrane by 2 mm, and the NC membrane overlaps the absorbent pad by 2 mm to ensure fluid continuity. The sample pad and absorption pad do not need to be treated. The NC membrane needs to be modified with T-line and C-line in advance. Take 25ul of a sequence with a concentration of 1uM (DCP in Table 1 is selected for T-line modification, and ACP in Table 1 is selected for C-line modification) and 50ul of 1uM streptavidin and resuspend them in 200ul of 1×PBS solution. Incubate at room temperature for 30min, then transfer to a 30K ultrafiltration tube and centrifuge and wash twice at 13000r / min. The final product is resuspended in 200ul of 1×PBS and then coated on the designated positions of the NC membrane using a BioDot dot sprayer, namely the test line (T line) and the control line (C line). Then assemble them into the detection shell to complete the preparation of the paper-based sensor.

[0044] Example 2 Detection principle of paper-based sensor based on DNAzyme probe

[0045] When E. coli exists in the sample, 1 ml of the sample is centrifuged at 8000 r / min for 10 min, and the precipitate is added into 200 ul of SDS lysis solution, and shaken for 1 min to prepare E. coli CIM. The deoxyribozyme probe used in the application has specific recognition and cutting ability for E. coli CIM, and has a cuttable RNA connection site, and the deoxyribozyme probe is modified with a fluorescent marker FAM, and the other end is fixed with an agarose bead. A DNA sequence complementary to the fragment released after the deoxyribozyme probe is cut is fixed on the test line (T line). When the deoxyribozyme probe is cut under the action of E. coli CIM, the sequence fragment modified with the FAM fluorescent group will flow away with the liquid, and will be combined with the complementary sequence fixed on the test line to generate a detectable fluorescent signal. The reading of the fluorescent signal can be scanned and quantified by Image J software to determine the presence or absence of E. coli in the sample and the concentration level.

[0046] Example 3 Feasibility analysis of detecting E. coli based on the deoxyribozyme probe paper-based sensor

[0047] Take 100 ul of streptavidin-modified agarose beads and centrifuge at 3500 r / min with 500 ul of 1xPBS for 3 times, and shake and incubate 1 nmol of linked aRCD-EC1FS with the agarose beads for 1 h. Take 20 ul of the fixed aRCD-EC1FS and 20 ul of the prepared E. coli CIM and react in 20 ul of 1xRB buffer solution (50 mM sodium citrate, 150 mM NaCl, 50 mM KCl, 20 mM EDTA, and 0.01% Tween 20, pH 7.0) at room temperature for 30 min. Take 25 ul of the reacted solution and drop it onto the sample pad. After the test strip is completely flowed through, the fluorescent signal intensity is scanned and read by Image J software. At the same time, aRCD-EC1FS only, target E. coli CIM only, no aRCD-EC1FS and target E. coli CIM control groups are set respectively.

[0048] The results are shown in Figure 2 It can be seen that there is obvious signal intensity when the target and the probe are added at the same time.

[0049] Example 4 Condition optimization analysis of detecting E. coli based on the deoxyribozyme probe paper-based sensor

[0050] Take 100 ul of streptavidin-modified agarose beads and centrifuge at 3500 r / min with 500 ul of 1xPBS for 3 times, and shake and incubate 1 nmol of linked aRCD-EC1FS with the agarose beads for 1 h.

[0051] pH condition: Take 20ul fixed aRCD-EC1FS and 20ul E. coli CIM, respectively, and mix with 40ul 1x RB buffer solution (pH 5.5, 6.0, 6.5, 7.0, 7.5) at RT for 30 minutes. Then take 30ul of the reaction solution and add it to the sample pad. After the test strip is completely flowed through, use Image J software to scan and read the fluorescence signal intensity. It is found that the fluorescence signal is strongest at pH 6.5 Figure 3 A).

[0052] Reaction system total volume condition: Take 20ul fixed aRCD-EC1FS and 20ul E. coli CIM, and mix with 40ul 1x RB buffer solution (pH 6.5) at RT for 30 minutes. Then take different volumes of the reaction solution (5, 10, 15, 20, 25, 30ul) and add it to the sample pad. After the test strip is completely flowed through, use Image J software to scan and read the fluorescence signal intensity. It is found that the fluorescence signal reaches the strongest at 25ul volume Figure 3 B).

[0053] Reaction time condition: Take 20ul fixed aRCD-EC1FS and 20ul E. coli CIM, and mix with 40ul 1x RB buffer solution (pH 6.5) at RT for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes and 30 minutes. Then take 25ul of the reaction solution and add it to the sample pad. After the test strip is completely flowed through, use Image J software to scan and read the fluorescence signal intensity. It can be seen that the signal intensity increases significantly with the increase of reaction time, and reaches the maximum signal intensity value at 20min Figure 3 C).

[0054] Example 5 Detection of E. coli specificity analysis based on deoxyribozyme probe paper-based sensor

[0055] Take 100ul streptavidin modified agarose beads and centrifuge at 3500r / min for 3 times with 500ul 1x PBS. Incubate 1nmol of linked aRCD-EC1FS with agarose beads for 1h. Take 20ul fixed aRCD-EC1FS and 40ul 1x RB buffer solution (pH 6.5), and then add 20ul E. coli CIM, Staphylococcus aureus CIM, Pseudomonas cocovenenans CIM, Vibrio cholera CIM, Enteropathogenic Escherichia CIM, and V. anguillarum CIM, respectively, and incubate at RT for 30 minutes. Then take 25ul of the reaction solution and add it to the sample pad. After the test strip is completely flowed through, use Image J software to scan and read the fluorescence signal intensity.

[0056] The results are as followsFigure 4 As shown, it can be seen that there is obvious signal intensity when E. coli CIM is added, while there is no obvious signal intensity in the other groups.

[0057] Example 6: Sensitivity analysis of the paper-based sensor based on DNAzyme probe for detecting Escherichia coli

[0058] 100ul of streptavidin-modified agarose beads were centrifuged at 3500r / min with 500ul of 1×PBS and washed three times. 1nmol of linked aRCD-EC1FS was incubated with the agarose beads and shaken for 1h. 20ul of immobilized aRCD-EC1FS was mixed with 40ul of 1×RB buffer solution (pH 6.5), and then 20ul of Escherichia coli CIM (10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 and 10 7 CFU / mL) and incubated at room temperature for 30 minutes. Then, 25 μl of the reaction solution was added dropwise to the sample pad. After it completely flowed through the test strip, the fluorescence signal intensity was scanned and read using Image J software.

[0059] The results are as follows Figure 5 As shown, it can be seen that when the concentration of E. coli is 10 2 ~10 7 The signal intensity can be detected at 10 CFU / mL.

Claims

1. A paper-based sensor device based on a DNAzyme probe, characterized in that: The device mainly includes a sample adding area device, a labeling reagent storage area device and a water absorbent pad connected in sequence. The sample adding area device is connected to the labeling reagent storage area device and there is an overlapping area; the labeling reagent storage area device is connected to the water absorbent pad and there is an overlapping area. A detection line and a control line are provided on the labeling reagent storage area, wherein the detection line is close to the sample adding area device side, and the control line is close to the water absorbent pad side. The detection line is coated with a complex of streptavidin and a sequence containing a FAM group sequence complementary to the sequence released after the deoxyribozyme probe is cut, and the control line is coated with a complex of streptavidin and another complementary sequence; the structure of the deoxyribozyme probe is: F-CTATGAACTGACTRTGACCTCACTACCAAGCAGGTCCATCGAGTGGTAGGA TGCGGCGGTCAGTCGCACTGCTCCTTTTTTTTTTT-B, where B is biotin, R is RNA base A (rA), and F is T base containing a fluorescent group; the complementary sequence structure of the test line coating is: B-TTTTTTTTTTTAGTCAGTTCATAGGATCATCCAG, where B is biotin; the complementary sequence structure of the control line coating is: ACCTTCCTCCGCAATACTCCCCCAGGTTTTTT-B, where B is biotin.

2. The paper-based sensor device according to claim 1, characterized in that: The sample addition area device includes JY-BX108 GL-b01 filter paper from Shanghai Jieyi Biotechnology Co., Ltd.; the labeling reagent storage area device includes Whatman Grade 1 nitrocellulose membrane; and the absorbent pad includes JY-X101 H5076 filter paper from Shanghai Jieyi Biotechnology Co., Ltd.

3. The paper-based sensor device according to claim 1, characterized in that: The sample adding area device, the labeling reagent storage area device and the water absorbing pad are rectangular in shape.

4. The paper-based sensor device according to claim 3, characterized in that: The length of the sample adding area device is 10-20 mm, the length of the labeling reagent storage area device is 20-30 mm, and the length of the absorbent pad is 10-30 mm; the length of the overlapping area is 1-5 mm, and the width of the sample adding area device, the labeling reagent storage area device and the absorbent pad is 1-5 mm.

5. The paper-based sensor device according to claim 1, characterized in that: The paper-based sensor device is integrally assembled in a plastic housing.

6. Use of the paper-based sensor device according to any one of claims 1 to 5, characterized in that: Used to detect whether Escherichia coli is present in the liquid sample to be tested and the concentration of Escherichia coli.

7. The use according to claim 6, characterized in that The liquid sample to be tested includes urine.

8. The use according to claim 6, characterized in that The application comprises the following steps: (1) Lysing the bacteria in the liquid sample to be tested, adding the resulting lysate, the DNAzyme probe immobilized with agarose beads, and a buffer solution into a test tube, reacting at room temperature for 10 to 30 minutes, and then dropping 10 to 40 μl of the reaction mixture into the sample addition area of ​​the paper-based sensor device; (2) After the liquid has completely flowed through the absorbent pad, use Image J software to scan and read the fluorescence signal and fluorescence intensity.

9. The use according to claim 8, characterized in that The reaction time in step (1) is 20 minutes, and the amount of the reaction mixture added is 25 μl; the agarose beads and the DNA enzyme probe are connected and fixed by streptavidin; the volume ratio of the lysate, the DNA enzyme probe fixed to the agarose beads, and the buffer solution is 1-2:1-2:1-3; the composition of the buffer solution is: 50 mM sodium citrate, 150 mM NaCl, 50 mM KCl, 20 mM EDTA, 0.01% Tween 20, pH 6-7; the Escherichia coli content in the liquid sample to be tested is 10 2 ~10 7 CFU / mL.

10. The use according to claim 8, characterized in that In step (2), if E. coli is present in the liquid sample to be tested, the E. coli CIM released by lysis causes the DNAzyme to specifically cut the DNAzyme probe, resulting in the fall-off of sequence fragments containing the FAM fluorescent group, which are then added to the sample application area device. The DNAzyme probe is fixed to the sample application area device under the action of agarose beads, and the fallen FAM-labeled cleavage fragments are captured by the complementary sequence at the detection line, generating a detectable fluorescence signal. The presence of E. coli in the sample and the concentration level are determined by the fluorescence intensity.

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