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CN-121994878-A - Microfluidic biosensor for detecting citrus yellow dragon disease

CN121994878ACN 121994878 ACN121994878 ACN 121994878ACN-121994878-A

Abstract

The invention discloses a microfluidic biosensor for detecting yellow-tailed bacteria of citrus and a detection method thereof, belonging to the technical field of biological sensing. The sensor integrates a microfluidic chip of a separation zone, a concentration zone and an electrochemical detection zone. The separation area is a spiral microchannel, the concentration area is provided with an interdigital working electrode array for capturing and enriching target bacteria through dielectrophoresis force, the electrochemical detection area is provided with a sensing working electrode, and the surface of the working electrode is sequentially modified with an MXene@AgNPs nano composite material layer, a nucleic acid aptamer layer and a sealing agent layer, wherein the nucleic acid aptamer layer specifically binds to the outer membrane protein OmpA of the yellow-dragon bacteria. After the sample is separated and enriched during detection, the target bacteria and the aptamer are combined to cause electrochemical impedance change, and the result is output after treatment. The invention integrates sample pretreatment, target enrichment and high-sensitivity detection, realizes rapid, simple and high-specificity field detection of the yellow-dragon bacteria, and is suitable for large-scale field screening.

Inventors

  • LIU HAIBO
  • XIE CHUNLIU
  • WANG JING

Assignees

  • 广西大学

Dates

Publication Date
20260508
Application Date
20260203

Claims (10)

  1. 1. A microfluidic biosensor for detecting a citrus yellow shoot pathogen, comprising: the microfluidic chip is provided with an integrally formed separation area, a concentration area and an electrochemical detection area which are sequentially in fluid communication; Wherein the separation zone is a spiral microchannel provided with at least one sample inlet, at least one sheath flow inlet, at least one waste outlet, and at least one target liquid outlet to the concentration zone; The concentration zone is provided with an interdigital working electrode array for generating dielectrophoresis force, the inlet of the interdigital working electrode array is communicated with a target liquid outlet of the separation zone, and the outlet of the interdigital working electrode array is communicated with the electrochemical detection zone; An electrochemical sensing working electrode is arranged in the electrochemical detection area, and the working electrode surface of the electrochemical sensing working electrode is sequentially modified with an MXene@AgNPs nano composite material layer, a nucleic acid aptamer layer and a sealing agent layer, wherein the nucleic acid aptamer layer specifically binds to the outer membrane protein OmpA of the yellow dragon germ; And the signal processing module is electrically connected with the electrochemical sensing working electrode and is used for detecting and processing electrochemical signal changes generated by the combination of the nucleic acid aptamer and the OmpA protein and outputting detection results related to the existence of the yellow dragon bacteria.
  2. 2. The microfluidic biosensor according to claim 1, wherein the nucleic acid aptamer is a sequence capable of specifically binding to the outer membrane protein OmpA of yellow-tailed bacteria, which is selected from one of the sequences of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4.
  3. 3. The microfluidic biosensor of claim 2, wherein the nucleic acid aptamer is a sequence shown in SEQ ID No. 3.
  4. 4. The microfluidic biosensor of claim 1, wherein the working electrode is prepared by the following method: (1) Preparation of MXene@AgNPs nanocomposite: Premixing LiF and HCl, stirring, gradually adding Ti 3 AlC 2 powder, etching, centrifuging, collecting the obtained product, adding 1M HCl to remove LiF until the supernatant is colorless, washing with ultrapure water until the pH of the supernatant is more than or equal to 6, performing ultrasonic treatment on the precipitate by using nitrogen and ice water bath, centrifuging, collecting the supernatant, and freeze-drying for 48 hours to obtain MXene powder for later use; Dispersing MXene powder in water, adding silver nitrate solution for reaction, centrifuging, washing and drying to obtain an MXene@AgNPs nanocomposite; (2) And then dropwise adding a 6-mercapto-1-hexanol solution, incubating, and washing after incubation is completed to obtain the working electrode.
  5. 5. The microfluidic biosensor of claim 1, wherein the electrochemical sensing working electrode is a screen printed working electrode, the working electrode is a carbon working electrode, and the blocking agent is 6-mercapto-1-hexanol.
  6. 6. The microfluidic biosensor of claim 1, wherein the substrate of the microfluidic chip is polydimethylsiloxane.
  7. 7. The microfluidic biosensor according to claim 1, wherein the signal processing module comprises a wireless transmission unit, and is configured to send the detection result to an intelligent terminal for display.
  8. 8. The application of the nucleic acid aptamer in preparing a sensor or a detection reagent for detecting the yellow-tailed bacteria is characterized in that the sequence of the nucleic acid aptamer is shown as SEQ ID NO. 1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO. 4.
  9. 9. A detection method for rapidly screening citrus yellow-tailed bacteria in a field, characterized in that the microfluidic biosensor according to any one of claims 1 to 7 is adopted, comprising the following steps: (1) Taking the leaf or leaf stem of citrus, grinding, mixing with buffer solution, filtering to obtain sample liquid to be detected, injecting the sample liquid to be detected into the sample inlet, and simultaneously injecting sheath fluid at a higher flow rate, wherein the sample liquid to be detected and the sheath fluid flow in a spiral separation area, and the injection speed ratio of the sample liquid to be detected to the sheath fluid injection speed is 1:5; (2) In the separation zone, large particle impurities in the sample liquid are discharged to an outer waste liquid outlet by utilizing an inertial focusing effect, and bacteria-containing liquid flows through the target liquid outlet and enters the concentration zone; (3) In the concentration area, through applying alternating voltage 6V to the interdigital working electrode array, dielectrophoresis force is generated after the frequency is 6 MHz, and the flowing bacteria-containing liquid is captured and enriched to the center line of the micro-channel, so that concentrated bacteria liquid is formed and conveyed to the electrochemical detection area; (4) In the electrochemical detection area, concentrated bacterial liquid enters the detection area, if the concentrated bacterial liquid contains yellow dragon bacteria, ompA protein on the surface of the yellow dragon bacteria is combined with the nucleic acid aptamer on the working electrode, so that interface electron transfer impedance is increased; (5) And detecting the impedance change through the signal processing module, and comparing the impedance change with a pre-established standard curve or threshold value to give qualitative judgment and semi-quantitative analysis results.
  10. 10. A citrus yellow shoot germ detection system is characterized by comprising the microfluidic biosensor of any one of claims 1 to 7, a microinjection pump for driving a sample liquid, an intelligent terminal for receiving, displaying and/or analyzing the detection result and a matched application program.

Description

Microfluidic biosensor for detecting citrus yellow dragon disease Technical Field The invention relates to the technical field of biosensors, in particular to a microfluidic biosensor for detecting citrus yellow dragon disease. Background Citrus yellow dragon disease (Huanglongbing, HLB) is a devastating disease caused by phloem-specific parasitic bacteria Candidatus Liberibacter spp, called "citrus cancer", which is predominantly Candidatus Liberibacter asiaticus (CLas) in China. The affected plants usually decay or lose fruiting capacity within 3-5 years, and the pathogens can spread rapidly to the whole orchard and even the whole producing area through the propagation of diaphorina citri, thus forming a serious threat to the citrus industry. At present, no effective cure method exists for the disease, so early, accurate and convenient detection is important for disease prevention and control. However, field diagnosis of yellow dragon disease faces a number of difficulties. The surface symptoms of leaf yellowing, mottle and the like are very similar to trace element deficiency diseases such as zinc, manganese and the like, and misjudgment is easy to cause. In the aspect of laboratory detection, the pathogen can not realize artificial pure culture so far, and the development of a traditional detection method based on culture is limited. Historically, iodine-starch chromogenic methods, serological assays, electron microscopy, and the like have been used, but these methods have been rarely used in practice either because of low accuracy or because of complicated and time-consuming procedures. Currently, the mainstream detection technology at home and abroad depends on real-time fluorescent quantitative polymerase chain reaction (qPCR). Although the sensitivity of the technology is higher, the application of the technology has the limitations that the primer design is complex, the reaction system is complicated to optimize, the technology particularly relies on a precise thermal cycle instrument to carry out precise temperature control, has high professional requirements on operators, and is not suitable for field and orchard on-site rapid detection scenes. Therefore, developing a novel detection technology with high sensitivity, high specificity, simple operation and convenient carrying so as to meet the urgent need of rapid on-site diagnosis of the yellow dragon disease has become the current research focus. Finding stable detection targets is the basis for developing new methods. CLas of the outer membrane protein OmpA is highly conserved among different strains, is abundant in the outer membrane, and is a specific detection target with great potential. In the aspect of recognition element, the aptamer is used as a chemical antibody obtained by screening through a SELEX technology, can be folded to form a specific space structure to be combined with a target with high affinity and high specificity, has the advantages of good stability, easiness in synthesis and modification and the like, and is widely applied to detection and analysis of various targets. The microfluidic chip technology integrates the steps of sample preparation, reaction, separation, detection and the like into a micro-channel network, and provides an ideal solution for constructing a portable field detection platform by virtue of the advantages of low sample consumption, high analysis speed, high integration level, easiness in automation and the like. The microfluidic chip is combined with the electrochemical sensing system, and a three-working-electrode detection unit can be integrated on the chip, so that integrated closed-loop detection of 'sample in-out' is realized. The microfluidic environment can strengthen mass transfer efficiency, and the detection sensitivity can be remarkably improved by combining nano material modified working electrodes (such as silver nano particles, MXene and the like). In addition, the microfluidic chip can integrate a sample pretreatment module based on the principles of inertia force, magnetophoresis and the like, and directly separate and enrich target objects from complex matrixes, so that direct, rapid and quantitative analysis of actual samples such as citrus leaf extract is realized. In summary, aiming at the defects of the existing yellow cron disease detection technology, particularly the qPCR method in the aspects of portability and field application, the molecular recognition technology based on the specific nucleic acid aptamer is combined with an integrated and low-consumption microfluidic electrochemical sensing platform, and a novel integrated detection method which is easy to operate, rapid, accurate and suitable for field fields is hopefully developed. Disclosure of Invention The invention aims to provide a microfluidic biosensor for detecting citrus yellow croaker, which is constructed based on screened yellow croaker outer membrane A protein (OmpA) nucleic acid aptamer and is suitable f