A Nucleic Acid Paper-Based Microfluidic POCT Product and Its Application in Point-of-Site Testing on PCB77

Abstract

This invention discloses a nucleic acid paper-based microfluidic POCT product and its application in the on-site point-of-care testing of PCB77. Based on nano-DNA elements and combined with paper-based microfluidic technology, this invention develops a paper-based microfluidic POCT platform for the on-site point-of-care testing of PCB77. A foldable paper-based microfluidic chip is constructed using a paper-based microfluidic chip as a carrier for the on-site point-of-care testing of PCB77. By modifying the test strip with TMB, Hemin, and CHA-G4 based on the foldable paper-based microfluidic chip, rapid and visual identification of PCB77 can be achieved. It exhibits good detection performance in the range of 20-100 ng / L, with a detection limit as low as 20 ng / L, and high anti-interference ability and stability. This invention enriches the means of visual identification of PCB77, and the use of nucleic acid-functionalized paper-based microfluidic technology for sensitive and rapid detection and analysis of POPs provides protection for environmental protection and food safety.

Description

Technical Field

[0001] This invention relates to the field of electrochemical sensing analysis and detection and pollutant detection technology, and more specifically, to a nucleic acid paper-based microfluidic POCT product and its application in on-site real-time detection of PCB77. Background Technology

[0002] Point-of-care testing (POCT) refers to a testing method that uses portable analytical instruments and reagents to rapidly obtain test results at the sampling site. It offers advantages such as speed, simplicity, and convenience, and is a research hotspot in analytical chemistry. Common POCT technologies include flurometric immunoassay, microfluidic chips, dry chemistry test strips, and paper-based microfluidics, demonstrating broad application prospects and significant value in disease diagnosis, food safety, and environmental monitoring. Among these, paper-based microfluidics, with its low cost, biodegradability, ease of operation, lack of external power, and high portability, is widely used in analytical chemistry.

[0003] Although paper-based microfluidics performs excellently in routine analysis, there are still technical gaps in the trace detection of POPs. The contradiction between the low concentration of POPs in environmental matrices and the insufficient sensitivity of existing paper-based sensors restricts the progress of related research. Currently, paper-based microfluidics relies on the preparation and functionalization of paper-based microfluidic chips. However, the preparation of paper-based microfluidic chips is limited by cost and accuracy. Currently, commonly used chromatographic analysis methods for POPs (such as GC, HPLC, GC-MS, HPLC-MS, etc.) require high levels of expertise in detection equipment and skilled personnel, and have long detection cycles. Antibody-based immunoassay methods still have shortcomings in terms of detection accuracy, antibody preparation, and preservation. For typical POPs such as 3,3',4,4'-tetrachlorobiphenyl (PCB77), the developed biosensor detection methods still have deficiencies in terms of stability, repeatability, detection limit, and portability. Therefore, there is an urgent need to develop more products and methods for the rapid, stable, efficient, and real-time testing of PCB77. This invention application is thus filed. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art. The present invention provides a nucleic acid paper-based microfluidic POCT product and its application in on-site real-time detection on PCB77.

[0005] The first objective of this invention is to provide a nano-DNA element for detecting PCB77.

[0006] A second objective of this invention is to provide a biosensor.

[0007] A third objective of this invention is to provide applications for biosensors.

[0008] The fourth objective of this invention is to provide a PCB77 testing product.

[0009] The fifth objective of this invention is to provide a method for detecting PCB77 in seawater.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a nano-DNA element for detecting PCB77, comprising the following sequence: Apt: 5'-GGCGGGGCTACGAAGTAGTGAAAAAAACCGATGGCCCGTG-3'; S: 5'-CTCTTCTCGTAGCCCGCC-3'; H1: 5'-GGGATGGCGGGGCTACGAGAAGAGTAGCCCCCTCTTCTACTCTTCTCGTAGCC-3'; H2: 5'-TAGAAGAGGGGGCTACTCTTCTCGTAGCCCCTCCTCTTCT-3'; G4: 5'-GGGTAGGGCGGGTTGGGTTAGAAGAGGAGGCCATCCCACCC-3'.

[0011] This invention combines aptamer technology, CHA, and DNAzyme, leveraging the advantages of DNA nanotechnology to functionalize DNA onto a paper-based microfluidic chip. A trifold design is used to modify the test strip with TMB, Hemin, and CHA-G4 systems, achieving functional modification of the paper-based microfluidic chip. This chip exhibits good detection performance in the 20-100 ng / L range, with a detection limit of 20 ng / L for actual samples. Furthermore, it demonstrates good anti-interference ability and stability in complex samples. A paper-based microfluidic POCT platform has been developed for the on-site, point-of-care testing of PCB77, providing a theoretical basis for environmental monitoring technology development and food safety testing.

[0012] The specific detection principle is as follows: When PCB77 is absent in the sample, sequence S is blocked by Apt and cannot react with H1, thus failing to induce CHA amplification. When PCB77 is present, its stronger binding force to Apt displaces sequence S. After the sample solution is added to the binding pad, the free sequence S activates the CHA reaction, generating a large amount of double-stranded complex H1 / H2. H1 / H2 further unlocks the hairpin sequence G4, forming a triple-helix DNA complex H1 / H2 / G4. This complex contacts Hemin, promoting the generation of the G4 DNAzyme. Then, it forms a "sandwich structure" with TMB. Finally, adding H2O2 solution induces TMB color development. After the reaction, the sample solution is qualitatively identified by comparing the color change of the TMB-modified wells, thus completing the detection of PCB77.

[0013] The present invention provides a biosensor comprising the above-described DNA element.

[0014] Preferably, the biosensor further contains Hemin, TMB, and H2O2.

[0015] This invention provides the application of a biosensor in detecting PCB77.

[0016] This invention provides the application of a biosensor in the preparation of products for detecting PCB77.

[0017] This invention provides a PCB77 detection product containing a DNA element or a biosensor.

[0018] Preferably, the product is a paper-based microfluidic chip.

[0019] Preferably, the product further comprises a binding pad, a paper-based hydrophilic network, and a biological substrate sequentially connected; the binding pad is modified with H1, H2, and G4 sequences; and the two ends of the paper-based hydrophilic network are modified with Hemin and TMB, respectively.

[0020] The present invention also provides a method for using the above-mentioned product: after adding the sample solution to be tested to the conjugate pad, the paper-based hydrophilic network portion modified with Hemin is folded in half and then brought into contact with the conjugate pad to promote the generation of G4 DNAzyme; then the paper-based hydrophilic network portion modified with TMB is folded in half and placed on top of the paper-based hydrophilic network portion modified with Hemin, and then H2O2 is added and reacted for 10-15 minutes, and the color development can be observed.

[0021] As a more preferred embodiment, the paper-based hydrophilic network provided by the present invention can be prepared by 3D printing, and its size can be reasonably adjusted. Preferably, a laser printer and original toner are used to print the hydrophilic network. After the paper-based hydrophilic network is constructed, it is divided into three equal parts, and an open circle with a diameter of 6 mm is punched in the middle. Different substances (TMB, Hemin) are modified at both ends by physical adsorption modification, and the middle part is connected to the binding pad.

[0022] Preferably, the hydrophilic network construction conditions for the paper-based microfluidic chip are heating at 150-200℃ for 30-60 minutes.

[0023] Preferably, the optimal procedure for constructing the hydrophilic network of the paper-based microfluidic chip is to heat at 170°C for 45 minutes.

[0024] Further, the functionalization modification method of the paper-based microfluidic chip is as follows: TMB is dissolved in DMSO to prepare a standard solution 1 containing 100 μM TMB. Hemin is dissolved in DMSO to prepare a standard solution 2 containing 5 μM Hemin. The H1 (1 μM), H2 (1 μM), and G4 (2 μM) sequences are placed in a water bath at 95°C for 10 minutes to denature, and then naturally cooled to room temperature to form a hairpin structure. In addition, the Apt sequence (2 μM) and S sequence (1.5 μM) are incubated at 95°C for 5 minutes, and then cooled to room temperature to promote the formation of the Apt / S complex. Then, standard solutions 1 and 2 are added dropwise to the corresponding wells on the paper-based microfluidic chip and allowed to dry naturally for later use. Simultaneously, H1, H2, and G4 are added dropwise to the binding pad and dried at room temperature for later use. After mixing the Apt / S complex with the sample solution to be tested, it can be added dropwise to the paper-based microfluidic chip for detection.

[0025] This invention provides a method for detecting PCB77 in seawater, using a biosensor or the aforementioned product for detection.

[0026] The present invention has the following beneficial effects: This invention develops a paper-based microfluidic POCT platform for the on-site, real-time detection of PCB77, based on nano-DNA elements and combined with paper-based microfluidic technology. It combines isothermal enzyme-free nucleic acid amplification technology with DNAzyme, using a paper-based microfluidic chip as a carrier to construct a folded paper-based microfluidic chip for POCT detection of PCB77. Based on the trifolded paper-based microfluidic chip, TMB, Hemin, and CHA-G4 systems are modified onto the test strip to achieve rapid and visual identification of PCB77. It exhibits good detection performance in the 20-100 ng / L range, with a detection limit as low as 20 ng / L, and demonstrates good anti-interference ability and stability in complex samples. This invention enriches the means of visual identification of PCB77, and the nucleic acid-functionalized paper-based microfluidic technology holds promise for sensitive and rapid detection and analysis of POPs, providing safeguards for environmental protection and food safety. Attached Figure Description

[0027] Figure 1 This describes the fabrication and detection principles of paper-based microfluidic chips.

[0028] Figure 2 Assembly of paper-based microfluidic chips.

[0029] Figure 3 a) Secondary structure of CHA-G4 DNAzyme; b) Hybridization of H1+H2+G4.

[0030] Figure 4 a) Comparison of hydrophilic network preparation methods; b) Color development under different conditions.

[0031] Figure 5 Temperature optimization for hydrophilic network construction.

[0032] Figure 6 Optimize the construction time for hydrophilic networks.

[0033] Figure 7 This serves as a validation of the specificity of the paper-based microfluidic detection platform.

[0034] Figure 8 This is for sensitivity verification of the paper-based microfluidic detection platform.

[0035] Figure 9 This serves to verify the stability of the paper-based microfluidic testing platform.

[0036] Figure 10 This demonstrates the actual sample testing performance of the paper-based microfluidic detection platform. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0038] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0039] In the examples, 3,3',4,4'-tetrachlorobiphenyl (PCB77), 2,2',3,5'-tetrachlorobiphenyl (PCB44), 2,4,5-trichlorobiphenyl (PCB29), and 2,2',4,5,5'-pentachlorobiphenyl (PCB101), as well as hydrochloric acid, Tris, ethylenediaminetetraacetic acid (EDTA), hemin chloride, 3,3',5,5'-tetramethylbenzidine (TMB), ammonium persulfate, boric acid, and tetramethylethylenediamine were all purchased from Shanghai Maclean Biotechnology Co., Ltd. PVC substrate (DB-6) and conjugation pad (GL-B02-II) were purchased from Shanghai Jieyi Biotechnology Co., Ltd. 20 cm x 20 cm square filter paper was purchased from Suzhou Baoweide Environmental Protection Technology Co., Ltd., and relevant DNA sequences (Table 1) were purchased from Sangon Biotech (Shanghai) Co., Ltd. Ultrapure water was prepared using an ultrapure water system. All reagents were used directly without further purification.

[0040] Example 1: Preparation and Construction Principle of Paper-based Microfluidic Detection Platform 1. Sequence Design Using the publicly available PCB77 aptamer (Apt) sequence (FAN L, ZHANG C, SHI H, et al. Design of a simple and novel photoelectrochemical aptasensor for detection of 3,3′,4,4′-tetrachlorobiphenyl [J]. Biosensors and Bioelectronics, 2019, 124-125: 8-14.), and based on Catalytic Hairpin Assembly (CHA) technology, two hairpin-type DNA probes (commonly referred to as H1 and H2), along with the S strand and hairpin sequence G4, were designed. The most suitable sequence was optimized using free energy calculations, and the primer sequences are shown in Table 1.

[0041] Table 1 DNA Sequence Information

[0042] When the target PCB77 is absent, sequence S is blocked by Apt and cannot react with H1, thus failing to trigger CHA amplification. When PCB77 is present, it binds to Apt, displacing sequence S. The free sequence S activates the CHA reaction, opening the hairpin sequence H1. The free sticky ends of H1 further open the hairpin sequence H2, generating a large number of double-stranded complexes H1 / H2. H1 / H2 then further open the hairpin sequence G4, forming a triple-helix DNA complex H1 / H2 / G4.

[0043] Example 2: Fabrication and Functional Modification of Paper-Based Microfluidic Chips 1. Fabrication of paper-based microfluidic chips First, the hydrophilic network of the paper-based microfluidic chip was designed using CAXA CAD Electronic Drawing Board 2020 software, with the printing parameters set to a 1:1 mapping relationship and the print quality set to high. A square filter paper was placed in the printer's paper feed slot, and the print offset was adjusted to ensure the printed pattern was centered on the filter paper. The hydrophilic network was printed using a laser printer and original toner, and then heated in a blower drying oven to form the paper-based microfluidic chip.

[0044] The paper-based microfluidic chip consists of three components. For example... Figure 1 As shown in diagram a, the components are a binding pad, a paper-based hydrophilic network, and a bio-plate, which are assembled together using the bio-plate. After the paper-based hydrophilic network is constructed, a 6 mm diameter open circular hole is punched in the center of the chip. The paper-based hydrophilic network is divided into three equal parts, and a hole is punched in the middle of each part, with a length three times that of the binding pad. Simultaneously, the binding pad is precisely cut into 6 mm diameter circles using a punch. The binding pad, paper-based hydrophilic network, and bio-plate are assembled layer by layer as shown in the diagram to obtain the paper-based microfluidic chip.

[0045] Based on the designed paper-based microfluidic chip, each component was fabricated step by step, and the prepared physical object is shown below. Figure 2 As shown. First, cut the bio-based substrate to one-third the size of the test strip, ensuring it is the same width as the test strip, and then adhere the bio-based substrate to the test strip. The purpose of the bio-based substrate is to ensure that the back of the paper-based microfluidic chip does not directly contact the table surface during use, causing leakage of the reaction solution and resulting in false positives / false negatives. It also provides adhesive holes for bonding the conjugate pads. Then, attach the cut circular conjugate pads to the substrate, corresponding to the pre-cut holes in the center of the paper substrate, completing the assembly of the paper-based microfluidic chip. Direct bonding of the conjugate pads to the bio-based substrate avoids the use of other adhesives and prevents interference from foreign components in sample detection.

[0046] This embodiment provides a 3D paper-based microfluidic chip, which uses the stacking and folding of paper-based materials to construct a three-dimensional hydrophilic network to achieve complex detection functions.

[0047] Then, a paper-based microfluidic detection platform was prepared through functional modification. Specific modification strategies are detailed in [link to relevant documentation]. Figure 1 b. This scheme modifies the paper-based chip by physically adsorbing and loading TMB onto the pores above and Hemin onto the pores below, and then modifies the bonding pads with hairpin sequences H1, H2, and G4.

[0048] 2. Functional modification of paper-based microfluidic chips First, TMB was dissolved in DMSO to prepare standard solution 1 containing 100 μM TMB. Hemin was dissolved in DMSO to prepare standard solution 2 containing 5 μM Hemin. The H1 (1 μM), H2 (1 μM), and G4 (2 μM) sequences were denatured in a water bath at 95°C for 10 minutes, then allowed to cool naturally to room temperature to form hairpin structures. Additionally, the Apt (2 μM) and S (1.5 μM) sequences were incubated at 95°C for 5 minutes, followed by cooling to room temperature to promote the formation of the Apt / S complex. Then, standard solutions 1 and 2 were added dropwise to the corresponding wells on a paper-based microfluidic chip and allowed to air dry for later use. Simultaneously, H1, H2, and G4 were added dropwise to the binding pad and allowed to air dry at room temperature for later use.

[0049] Example 3: Paper-based microfluidic chip testing process Add 10 μL of samples containing different concentrations of PCB77 to 10 μL of detection solution containing Apt / S (100 nM) and react for 15 minutes at room temperature. Then, transfer 10 μL of the reaction solution and add it to the conjugate pad, reacting for 15 minutes. Next, fold the paper-based test strip, first contacting the modified well containing Hemin with the conjugate pad, and continue reacting for 15 minutes. Then, fold the modified well containing TMB and contact it with the modified well containing Hemin. Finally, add 5 μL of 3% H2O2 and react for 15 minutes, observing the color development.

[0050] The specific detection principle is as follows: Figure 1 As shown in Figure c, when PCB77 is absent in the sample solution, sequence S is blocked by Apt and cannot react with H1, thus failing to induce CHA amplification. When PCB77 is present in the sample solution, its stronger binding affinity to Apt displaces sequence S. Upon adding the sample solution to the binding pad, the free sequence S activates the CHA reaction, generating a large amount of the double-stranded complex H1 / H2. Further, H1 / H2 unlocks the hairpin sequence G4, forming a triple-helix DNA complex H1 / H2 / G4. A folding step brings the modified well containing Hemin into contact with the binding pad, promoting the generation of the G4 DNAzyme. Then, another folding step folds the modified well containing TMB to form a "sandwich structure." Then, as... Figure 1As shown in diagram d, H₂O₂ solution was added to the back of the TMB-modified wells to induce TMB color development. After the reaction was complete, the sample solution was qualitatively identified by comparing the color changes in the TMB-modified wells.

[0051] Example 4: Feasibility Analysis of Paper-Based Microfluidic Chips To investigate whether paper-based CHA amplification products can open G4 hairpins, thermodynamic stability simulations were performed. The free energies of the Apt and S sequences, as well as the binding energy of the EDC process, were calculated using NUPACK software (https: / / www.nupack.org / ). Unless otherwise specified, the analysis interface was set to DNA as the nucleic acid type, 25℃ as the reaction temperature, and the maximum complex number as the actual chain number. The free energies of the reactants, product concentrations, and product free energies were recorded, and the combinatorial efficiency was calculated for further analysis.

[0052] The simulation results are as follows Figure 3 As shown, the double-stranded product generated by the CHA self-assembly reaction forms a stable complex with the G4 hairpin sequence, with a ΔG value of -80.22 kcal / mol. Figure 3 a) This result indicates that the CHA product can further open the G4 hairpin sequence, freeing the G-rich base sequence. Since the free G-rich bases can react with Hemin to generate a G4 DNAzyme, the binding of CHA to a G4 DNAzyme is theoretically feasible. Simultaneously, the equilibrium probability of the reaction product approaches 1.0 (3b), indicating that the H1, H2, and G4 DNA sequences can stably bind, forming a triple-stranded complex. These results demonstrate that the CHA product's ability to open the G4 hairpin is practically feasible.

[0053] To further verify the feasibility of the paper-based microfluidic chip designed and fabricated in this study, we first compared its fabrication precision with that of a hand-drawn chip. Figure 4 a) Although the hand-drawing process uses a circular mold, the hydrophobic ink diffuses rapidly on the filter paper, making it difficult to precisely control the shape of the hydrophobic boundary, which is detrimental to the fabrication of fine microfluidic chips. Furthermore, hand-drawing is slow, hindering mass production. In contrast, the laser printing method used in this invention offers advantages in terms of high precision and speed compared to hand-drawing. Both methods can achieve the construction of hydrophilic networks. To avoid human error and improve fabrication efficiency, laser printing is the optimal solution for fabricating microfluidic chips.

[0054] Furthermore, to verify the practical feasibility of using paper-based microfluidic chips in sample testing, samples containing PCB77 were analyzed, with pure water used as a control group. The test results are as follows: Figure 4As shown in b, the sample containing PCB77 ultimately exhibited a distinct blue signal on the paper-based microfluidic chip, while the pure water group showed no significant color change. In conclusion, this method is practically feasible.

[0055] Example 5: Condition Optimization To further optimize the construction of the hydrophilic network for paper-based microfluidic chips, the heating temperature was controlled, and different heating times were set. After heating, ultrapure water was added to the hydrophilic network, and the performance was evaluated by observing the integrity of the hydrophobic boundary and the diffusion area of ​​the water-pure network.

[0056] The results are as follows Figure 5 As shown, at 120-140 °C, the hydrophobic boundary of the paper-based chip did not exhibit a hydrophobic effect, and the surface hydrophilic network construction failed. This is related to the failure of the organic components of the laser printing toner to fuse with the filter paper. Further increasing the reaction temperature revealed that the hydrophobic boundary of the paper chip initially showed hydrophobicity at 150 °C, but this was disrupted after one minute, insufficient to maintain the operation of the hydrophilic network. This indicates that the heating temperature is related to the effect of the organic components of the toner fusing with the filter paper.

[0057] Meanwhile, heating to 150 °C was insufficient to allow the organic components of the toner to fully penetrate the filter paper. Further increasing the temperature to 160 °C strengthened the hydrophobic boundary and prolonged its duration, but some solution still leaked, insufficient to meet the application requirements of microfluidic chips. This indicates that the organic components of the toner did not penetrate the filter paper evenly. Finally, when the temperature was increased to 170 °C, the hydrophobicity of the paper-based chip was significantly improved, and the solution was confined within a hydrophilic network, meeting the fabrication requirements of 3D paper-based microfluidic chips.

[0058] Furthermore, heating time has a significant impact on the penetration of toner into filter paper. To further optimize the fabrication process of the hydrophilic network for the paper-based microfluidic chip, the heating time was controlled at the optimal heating temperature of 170℃. The results are as follows: Figure 6 As shown, the hydrophobic effect of the paper-based microfluidic chip becomes increasingly pronounced with prolonged heating time, leading to more effective preparation of the hydrophilic network. Furthermore, the hydrophilic network can be maintained for an extended period after 45 minutes of heating. In conclusion, the optimal procedure for constructing the hydrophilic network of the paper-based microfluidic chip is heating at 170℃ for 45 minutes.

[0059] Example 6: Characteristic Analysis of Paper-Based Microfluidic Chips 1. Specificity analysis To evaluate the specificity of the paper-based microfluidic detection platform, interference tests were conducted using homologues of PCB77 (PCB101, PCB44, and PCB29). The sample liquid detection steps and result analysis were consistent with those in Example 3.

[0060] The results are as follows Figure 7 As shown, when directly measuring PCB77, PCB101, PCB44, and PCB29, only the PCB77 sample showed color development on the paper-based microfluidic platform, while other homologues showed no obvious phenomenon, indicating the high specificity of this detection strategy. Furthermore, the color development was still observed in mixed samples, demonstrating the applicability of this strategy under complex environments.

[0061] 2. Sensitivity Analysis To verify the sensitivity of the paper-based microfluidic detection platform, samples containing different concentrations of PCB77 were analyzed. The sample liquid detection steps and result analysis were the same as in Example 3.

[0062] Test results as follows Figure 8 As shown, the colorimetric signal intensity increases with increasing PCB77 concentration. When the target concentration reaches 20 ng / L, the paper-based microfluidic chip produces a visible blue color, indicating that the qualitative detection limit of this platform is 20 ng / L, and semi-quantitative analysis can be performed based on the color intensity.

[0063] 3. Stability Analysis The paper-based microfluidic chip was placed in a sealed bag and stored at room temperature. Every 7 days, a sample of the paper-based microfluidic test paper was taken out for testing to evaluate its stability. The sample liquid testing procedure and result analysis were the same as in Example 3.

[0064] To evaluate the performance changes of paper-based microfluidic chips under long-term storage conditions and ensure their reliability and effectiveness in practical applications, long-term stability testing was conducted on the paper-based microfluidic platform. The test results are as follows: Figure 9 As shown, the paper-based microfluidic platform maintained signal integrity and reliability during storage, and no significant performance degradation was observed.

[0065] Example 7: Seawater Sample Detection and Analysis DMSO solutions containing different concentrations of PCB77 were added to seawater collected from Zhanjiang Bay to prepare sample solutions. The sample solution detection steps and result analysis were the same as in Example 3, and qualitative analysis was performed.

[0066] Test results as follows Figure 10 As shown, the platform has a signal response to concentrations above 20 ng / L, indicating that the platform has excellent detection performance and can be used for qualitative identification of samples containing PCB77 in practical applications.

[0067] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A nano-DNA device for detecting PCB77, characterized in that, Includes the following sequences: Apt: 5'-GGCGGGGCTACGAAGTAGTGAAAAAAACCGATGGCCCGTG-3'; S: 5'-CTCTTCTCGTAGCCCGCC-3'; H1: 5'-GGGATGGCGGGGCTACGAGAAGAGTAGCCCCCTCTTCTACTCTTCTCGTAGCC-3'; H2: 5'-TAGAAGAGGGGGCTACTCTTCTCGTAGCCCCTCCTCTTCT-3'; G4: 5'-GGGTAGGGCGGGTTGGGTTAGAAGAGGAGGCCATCCCACCC-3'.

2. A biosensor characterized by, Contains the DNA element as described in claim 1.

3. The biosensor according to claim 2, characterized in that, It also contains Hemin, TMB, and H2O2.

4. The application of the biosensor of claim 2 in detecting PCB77.

5. The application of the biosensor of claim 2 in the preparation of products for detecting PCB77.

6. A PCB77 testing product, characterized in that, Contains the DNA element of claim 1 or the biosensor of claim 2.

7. The product according to claim 6, characterized in that, The product is a paper-based microfluidic chip.

8. The product according to claim 7, characterized in that, The product also contains a binding pad, a paper-based hydrophilic network, and a biological substrate sequentially linked together; the binding pad is modified with H1, H2, and G4 sequences; and the two ends of the paper-based hydrophilic network are modified with Hemin and TMB, respectively.

9. The method of using the product according to claim 8, characterized in that, After adding the test sample solution to the conjugate pad, fold the paper-based hydrophilic network portion modified with Hemin in half and bring it into contact with the conjugate pad to promote the generation of G4 DNAzyme. Then, fold the paper-based hydrophilic network portion modified with TMB in half above the paper-based hydrophilic network portion modified with Hemin, and then add H2O2 and react for 10-15 minutes to observe the color development.

10. A method for detecting PCB77 in seawater, characterized in that, The detection is performed using the biosensor described in claim 2 or the product described in claim 7.

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