Multi-pathogen combined detection microfluidic chip and method for pet constant-temperature nucleic acid detection

Abstract

The application discloses a multi-pathogen joint detection micro-fluidic chip and method for constant-temperature nucleic acid detection of pets, belongs to the technical field of biomedical detection, and integrates a high-persistence polymerase subjected to directional evolution and a dynamically controlled micro-fluidic network. The chip adopts mutant Bst DNA polymerase (such as Bst-M1), improves the strand displacement capacity through saturation mutation of key sites, and significantly enhances the tolerance to inhibitors (such as mucopolysaccharide) in pet samples. The micro-fluidic architecture is designed as a multi-chamber parallel network, precise sample distribution is realized through a laser-driven burst valve, each chamber is preloaded with specific pathogen primers (such as bronchial septic Bordetella, canine parvovirus) and orthogonal fluorescent probes. The integrated time-resolved fluorescence sensor combines a noise elimination algorithm, and realizes a detection sensitivity of 0.1 copy / μL. The chip is seamlessly connected with a sample processing module (50 μm filter hole serpentine channel) and a PID temperature control unit (65±0.2°C), supports synchronous detection of 10 kinds of pathogens, and the specificity reaches 95%.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically a microfluidic chip and method for multi-pathogen joint detection of pet isothermal nucleic acid detection. Background Technology

[0002] Rapid and accurate detection of pet pathogens is crucial for animal health management and the prevention and control of zoonotic diseases. Traditional nucleic acid detection methods, such as polymerase chain reaction (PCR), rely on thermal cyclers, making them unsuitable for on-site testing. In recent years, isothermal nucleic acid amplification technologies, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), have become ideal for portable testing due to their elimination of the need for complex temperature control equipment. These technologies have been successfully applied to the detection of pet pathogens, such as single-target detection of canine parvovirus and feline coronavirus.

[0003] Microfluidic chip technology has further advanced the development of portable testing by integrating sample processing, amplification, and detection functions. Existing research has achieved parallel design of multiple reaction chambers and fluid control of burst valves, supporting multiplex detection. For example, digital RPA technology and staged amplification strategies have significantly improved detection throughput. However, pet clinical samples (such as saliva or feces) often contain inhibitory components such as mucopolysaccharides and particulate matter, which leads to the easy inactivation or decreased amplification efficiency of existing polymerases in a microfluidic environment.

[0004] To address the issue of enzyme stability, directed evolution techniques (such as error-prone PCR and site saturation mutations) have been used to modify BstDNA polymerase, but its ability to sustain synthesis in complex samples remains insufficient.

[0005] Limitations of existing technology:

[0006] Poor sample adaptability: Traditional polymerases are sensitive to inhibitors in pet samples;

[0007] Multiple detection bottlenecks: Existing microfluidic chips mostly use a single amplification chamber, which is prone to primer cross-interference;

[0008] Insufficient portability: Existing devices rely on external temperature control modules. Summary of the Invention

[0009] The purpose of this invention is to provide a microfluidic chip and method for multi-pathogen joint detection of pet isothermal nucleic acid detection, so as to solve the technical problems of poor sample adaptability, multiple detection bottleneck and insufficient portability mentioned in the background art.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] A multi-pathogen co-detection microfluidic chip for portable isothermal nucleic acid detection in pets includes:

[0012] An engineered mutant Bst DNA polymerase, obtained through directed evolution, exhibits enhanced strand substitution ability and tolerance to inhibitors in pet samples;

[0013] A multi-chamber parallel microfluidic reaction network, comprising multiple physically isolated reaction chambers, each preloaded with specific primers for different pet pathogens;

[0014] Microfluidic sample dispensing structure for dispensing the same test sample into each reaction chamber;

[0015] The isothermal reaction structure is used to amplify nucleic acids in each reaction chamber under constant temperature conditions.

[0016] The detection module is used to detect the amplification signals in each reaction chamber separately, thereby realizing the joint detection of multiple pathogens.

[0017] The microfluidic chip unit, serving as the core carrier of the system, is fabricated using multilayer soft lithography and includes an inlet, a serpentine filter channel, a burst valve array, and parallel reaction chambers. After the sample is injected through the inlet, it first passes through a 50μm pore size serpentine filter channel to remove particulate impurities, and then, under the precise control of laser-triggered burst valves, is distributed to 4-10 independent reaction chambers. Each chamber is pre-embedded with primer pairs and fluorescent probes for specific pathogens (such as FAM-labeled canine distemper virus probes and HEX-labeled feline calicivirus probes), forming a physically isolated multiplex detection environment.

[0018] The temperature-controlled optical detection unit is integrated at the bottom of the chip, comprising a PID temperature control system and a multi-band fluorescence sensor. The temperature control system maintains a constant temperature environment of 65±0.2℃ in the reaction chamber through embedded Peltier elements, and its heat-conducting layer uses aluminum nitride ceramic material to ensure temperature uniformity. The optical detection section is equipped with a 405 / 488 / 640nm tri-color LED excitation source and a high-sensitivity photodiode, and monitors the amplification signal of each chamber in real time through time-resolved fluorescence acquisition technology.

[0019] The sample pretreatment unit, as an upstream module, includes a lysis buffer storage chamber and a magnetic bead nucleic acid extraction component. Pet samples (such as saliva or fecal suspensions) are lysed, and nucleic acids are purified using magnetic bead adsorption. Finally, 50-100 μL of purified solution is output to the microfluidic chip inlet. All modules of the system are connected via flexible circuit boards, and a built-in microprocessor coordinates temperature control timing, valve control commands, and signal acquisition, achieving a fully automated "sample in - result out" detection process.

[0020] A mutant Bst-M1 DNA polymerase was obtained using directed evolution technology. This enzyme underwent systematic modification of key amino acid residues (E658, Q461) through site-specific saturation mutagenesis, significantly enhancing its strand substitution activity and inhibitor resistance. Its kinetic properties can be described by the following equation:

[0021] in: The extension rate of polymerase (nt / s) directly affects amplification efficiency.

[0022] The catalytic constant reflects the efficiency of enzyme-substrate binding and conversion.

[0023] It is the Michaelis constant of dNTPs, which decreases by 30% after mutation (verification value: 0.12 mM→0.08 mM).

[0024] Quantitative analysis of the effect of mutation on the ability of chain synthesis (experimentally measured to be an increase of 2.3 kcal / mol in the mutant).

[0025] This is a scaling factor (empirical value 0.7).

[0026] The enzyme retained 85% activity in a simulated pet saliva sample containing 1% mucopolysaccharide, compared to only 28% in the wild type. Its thermal stability is measured by its melting temperature (…). Evaluation, mutant Increased by 4.2℃ (wild type 62.5℃ vs mutant 66.7℃) to ensure no degradation during prolonged reaction at 65℃.

[0027] Multi-chamber microfluidic network The chip employs a polydimethylsiloxane (PDMS)-glass heterojunction structure and includes the following key components:

[0028] Bursting Valve Fluid Control System Sample allocation is controlled by a laser-activated burst valve array, with an opening threshold energy density of:

[0029]

[0030] Traffic of each channel Following the modified Hagen-Poiseuille equation:

[0031]

[0032] In the formula:

[0033] For the first Channel radius (design value: 150±5 μm);

[0034] It is the driving pressure difference (generated by the chip tilt angle of 10°, approximately 17 Pa).

[0035] The viscosity of the sample-enzyme mixture is 1.2 mPa·s (measured).

[0036] It is a step function. It is the first Laser triggering time of the valve.

[0037] Parallel reaction chamber design Each chamber (2 μL volume) contains:

[0038] Pre-lyophilized reagent layer: pathogen-specific primers (e.g., canine parvovirus VP2 gene primers: 5'-FAM-ACAGGTGGTCAAGTTGA-3'), dNTPs, and stabilizers (trehalose / BSA).

[0039] Probe functional regions: orthogonally fluorescently labeled molecular beacons (such as HEX-labeled feline calicivirus probes, stem-loop structure Tm=68℃);

[0040] Hydrophilic coating: Polyacrylamide grafted surface (contact angle <15°) to ensure automatic liquid filling.

[0041] Cross-contamination rate between chambers <0.01% (Verification method: Load positive and negative samples respectively, and detect signals in adjacent chambers).

[0042] Time-resolved fluorescence detection system Signal acquisition employs a three-wavelength excitation / emission configuration:

[0043]

[0044] Parameter description:

[0045] : No. Original fluorescence intensity of the channel (ADC resolution 20 bits);

[0046] Channel-specific background correction factor (calibrated by template-free control experiment);

[0047] Integral window (optimized value: 30 seconds).

[0048] The system detection limit (LOD) is 0.1 copies / μL (linear range 10²–10⁷ copies / μL). >0.99), positive results are determined by dynamic threshold algorithm (signal slope >5 RFU / min for 3 consecutive samplings).

[0049] Temperature control subsystem Proportional-integral-derivative (PID) control is used to maintain temperature stability.

[0050]

[0051] in:

[0052] (Set value 65℃);

[0053] The parameters were tuned using the Ziegler-Nichols method. , , );

[0054] Actual control accuracy ±0.2℃ (NTC sensor sampling rate 10Hz).

[0055] Sample preprocessing interface The fluid resistance model for the serpentine filter channel is as follows:

[0056]

[0057] Design parameters:

[0058] aperture μm;

[0059] Total length cm;

[0060] Number of bends ;

[0061] The measured pressure drop was 3.2 kPa (at a flow rate of 5 μL / s).

[0062] This module can remove >90% of particles larger than 50 μm (validation sample: saliva simulant containing 10% fecal suspension).

[0063] A method for combined detection of multiple pathogens in pets, using the aforementioned microfluidic chip, includes the following steps:

[0064] The sample is injected into the chip inlet after being lysed and purified by the pretreatment module;

[0065] Laser-triggered burst valves distribute samples to each reaction chamber;

[0066] Isothermal amplification was performed at a constant temperature of 65±0.2℃.

[0067] The amplification signal is monitored in real time using a time-resolved fluorescence detection system;

[0068] Output a multi-pathogen joint detection report.

[0069] A portable pet nucleic acid testing device includes the aforementioned microfluidic chip and a corresponding temperature control module and signal detection module.

[0070] Compared with the prior art, the beneficial effects of the present invention are:

[0071] 1. Engineered high-persistence polymerase

[0072] The wild-type Bst DNA polymerase used in existing technologies is easily inactivated in complex pet samples (such as saliva or feces containing mucopolysaccharides), leading to decreased detection sensitivity. This invention utilizes a mutant Bst-M1 polymerase obtained through directed evolution, whose mutations in key amino acid residues (E658, Q461) significantly enhance strand substitution ability and inhibitor tolerance. Specifically, the mutant retains 85% activity in simulated pet samples containing 1% mucopolysaccharides (compared to only 28% for the wild-type), and exhibits improved thermostability (…). The temperature was increased by 4.2℃, overcoming the limitations of traditional isothermal amplification technology in pet samples.

[0073] 2. Multi-chamber microfluidic network design

[0074] Existing microfluidic multiplex detection systems mostly employ a single amplification chamber, which is prone to false positives or missed detections due to primer cross-interference. This invention achieves simultaneous and independent detection of multiple pathogens through physically separated parallel reaction chambers (4-10) and precise fluid control via laser-driven burst valves. Each chamber is pre-loaded with specific primers and orthogonal fluorescent probes, resulting in a cross-contamination rate of <0.01%. This structural design significantly reduces the risk of cross-reactions in multiplex detection, a level of physical isolation that cannot be achieved with existing technologies.

[0075] 3. Dynamic temperature control and noise cancellation algorithm

[0076] Traditional portable testing devices rely on external temperature control modules, which have insufficient temperature control accuracy (±1℃). This invention integrates a PID algorithm ( , , The embedded temperature control system keeps temperature fluctuations within ±0.2℃. Furthermore, the time-resolved fluorescence detection system utilizes a noise reduction algorithm (…). The formula dynamically corrects for background interference, bringing the detection limit down to 0.1 copies / μL, a 10-fold improvement over existing technologies. These innovations collectively address the issue of detection stability in portable devices under complex environments. Attached Figure Description

[0077] Figure 1 This is a schematic diagram of the overall system architecture of the present invention;

[0078] Figure 2 This is a detailed structural diagram of the microfluidic chip of the present invention. Detailed Implementation

[0079] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0080] Figure 1 middle

[0081] Sample processing module: responsible for sample lysis and nucleic acid extraction.

[0082] Lysis Buffer Chamber: A storage chamber for lysing sample cells to release nucleic acids.

[0083] Nucleic Acid Extraction Unit: This unit uses magnetic beads for nucleic acid extraction and outputs purified nucleic acid solutions.

[0084] Isothermal amplification module: Maintains an isothermal environment and performs nucleic acid amplification.

[0085] Heating Element: PID temperature-controlled heating element, maintaining a constant temperature environment of 65±0.2℃.

[0086] Proposed Microfluidic Chip: The core microfluidic chip of this invention

[0087] Microfluidic chip: integrating engineered polymerase and multi-chamber detection system

[0088] Engineered Polymerase: A mutant Bst-M1 polymerase with high persistence and inhibitor tolerance.

[0089] Multi-Chamber Network: A parallel multi-reaction chamber network that enables simultaneous detection of multiple pathogens.

[0090] Time-Resolved Fluorescence Sensor: A time-resolved fluorescence sensor combined with noise cancellation algorithms.

[0091] Control and Data Processing Unit: The Core of System Control

[0092] Microcontroller: A microcontroller that coordinates temperature control, fluid control, and signal acquisition.

[0093] Display Interface: Results display interface, outputs test report.

[0094] Figure 2 middle

[0095] Engineered polymerase systems: High-performance polymerase preparation systems

[0096] Mutant Bst Polymerase: Mutant Bst-M1 polymerase with the critical site D215G / A485T mutation.

[0097] Processivity Optimization: Continuous synthesis capability optimized through directed evolution.

[0098] Multi-chamber structure: parallel detection reaction unit

[0099] Burst Valves: Laser-driven burst valves for precise sample dispensing control

[0100] Reaction Chambers: Independent reaction chambers pre-loaded with pathogen-specific primers.

[0101] Capillary Flow Channels: Enables automated liquid delivery

[0102] Detection system: High-sensitivity signal acquisition unit

[0103] Fluorescent Probes: Multicolor fluorescent molecular beacons for orthogonal detection

[0104] Noise-Canceling Algorithm: A dynamic noise cancellation algorithm that improves the signal-to-noise ratio.

[0105] Example 1

[0106] This embodiment provides a modular microfluidic chip structure with a core feature: a detachable reaction chamber module. Each reaction chamber is an independent PDMS unit, interfaced with the main control chip via a standard interface (1.5mm diameter magnetic fluid connector). Users can freely combine 4-10 different pathogen detection modules according to their detection needs, for example:

[0107] Basic combination: a three-in-one module containing canine parvovirus (CPV), canine distemper virus (CDV), and feline calicivirus (FCV);

[0108] Expanded combination: Added detection modules for 7 pathogens, including Leptospira and Bordetella bronchiseptica.

[0109] Each module has a built-in pre-lyophilized reagent (shelf life 12 months, 4℃). When replacing, simply press the old module to release the latch and insert the new module. The system will automatically identify the module ID (via RFID tag) and load the corresponding testing program.

[0110] Example 2

[0111] For passive fluid control without power supply in the field, this embodiment proposes a passive microfluidic system driven by capillary force and gravity:

[0112] Sample introduction structure: A trapezoidal cross-section sample introduction channel (800μm wide at the top and 200μm wide at the bottom) is used to achieve self-filling by utilizing the surface tension of the liquid;

[0113] Distribution control: Hydrophilic and hydrophobic patterns with different contact angles (15° contact angle in the hydrophilic region / 110° contact angle in the hydrophobic region) guide the sample flow to a specific chamber;

[0114] Waste liquid treatment: Each chamber is connected to an absorbent material (nitrocellulose membrane) at the end to automatically absorb excess liquid and prevent backflow.

[0115] This design, combined with a specially designed manual centrifuge device (which generates 50g of centrifugal force after 3 rotations), can complete the entire testing process without the need for an external power source.

[0116] Example 3

[0117] This embodiment of the multimodal detection integrated chip integrates an immunochromatographic detection unit on the basis of standard nucleic acid detection function to realize joint nucleic acid-protein analysis.

[0118] Upper structure: Retains the multi-chamber isothermal amplification module for pathogen nucleic acid detection;

[0119] Lower layer structure: Adds transverse flow immunochromatographic bands to detect pathogen antigens (such as canine parvovirus VP2 protein).

[0120] Shared interface: Samples are split through the same inlet, with the nucleic acid portion entering the microfluidic channel and the protein portion migrating to the detection line through the lateral flow channel.

[0121] The results show that by integrating a reflective photoelectric sensor (to detect the nano-gold labeled signal) and outputting the fluorescence signal simultaneously, a dual verification report of "nucleic acid positive + antigen positive" is generated.

[0122] Example 4

[0123] To simplify the operation process, this embodiment provides a fully enclosed disposable test kit:

[0124] Integrated sample processing: The sealed capsule with built-in pre-filled lysis buffer (press to break and release) allows the sample to be directly injected into the injection port and automatically completes lysis;

[0125] Extraction-free design: The chip filter channel integrates a diatomaceous earth purification membrane (0.5μm pore size), enabling "filtration-amplification" to be completed in one step;

[0126] Visualized Results: Each reaction chamber has a colorimetric window at the top. The amplification product reacts with a pH-sensitive dye (such as phenol red) to produce a visible color change (positive results turn yellow / negative results remain red). This design is particularly suitable for home users, as no professional operation is required throughout the testing process, and results can be interpreted by observing the color within 15 minutes.

[0127] Example 5

[0128] This embodiment develops a flexible wearable detection device to address the needs of continuous health monitoring for pets:

[0129] Base material: A patch made of medical-grade polyurethane film (150μm thick) to fit the pet's skin;

[0130] Microfluidic network: A serpentine channel (100 μm wide) is formed by laser engraving, with four micro-reaction chambers (0.5 μL in volume) connected to the ends.

[0131] Sampling method: A microneedle array (200μm in height) is set on the inner side of the patch to penetrate the pet's epidermis and draw tissue fluid into the detection chamber;

[0132] Wireless transmission: Integrated Bluetooth Low Energy module, which sends detection data to mobile APP in real time.

[0133] This patch can work continuously for 24 hours and automatically sample and test every 2 hours, making it particularly suitable for postoperative infection monitoring or vaccination efficacy evaluation.

[0134] This invention constructs a highly efficient and portable platform suitable for the joint detection of multiple pathogens in pets through the synergistic innovation of engineered polymerase and intelligent microfluidic system.

[0135] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A microfluidic chip for combined detection of multiple pathogens in portable isothermal nucleic acid testing for pets, characterized in that, include: An engineered mutant Bst DNA polymerase, obtained through directed evolution, exhibits enhanced strand substitution ability and tolerance to inhibitors in pet samples; A multi-chamber parallel microfluidic reaction network, comprising multiple physically isolated reaction chambers, each preloaded with specific primers for different pet pathogens; Microfluidic sample dispensing structure for dispensing the same test sample into each reaction chamber; The isothermal reaction structure is used to amplify nucleic acids in each reaction chamber under constant temperature conditions. The detection module is used to detect the amplification signals in each reaction chamber separately, thereby realizing the joint detection of multiple pathogens.

2. The microfluidic chip according to claim 1, characterized in that, The mutant Bst DNA polymerase was obtained by mutation at key amino acid sites, and it still maintains higher amplification activity than the wild-type polymerase in pet samples containing mucopolysaccharides.

3. The microfluidic chip according to claim 1, characterized in that, The multi-chamber parallel microfluidic reaction network includes 4–10 independent reaction chambers, and each reaction chamber is physically isolated to avoid primer cross-interference.

4. The microfluidic chip according to claim 1, characterized in that, The microfluidic sample dispensing structure includes laser-driven burst valves, which are opened sequentially under predetermined conditions to achieve precise sample dispensing.

5. The microfluidic chip according to claim 1, characterized in that, Each reaction chamber contains lyophilized amplification reaction reagents.

6. The microfluidic chip according to claim 1, characterized in that, The detection module is a fluorescence detection module, and different fluorescent labels are used in different reaction chambers to achieve differentiation and detection.

7. The microfluidic chip according to claim 1, characterized in that, The isothermal reaction structure maintains the reaction temperature within the range of 60–70°C.

8. The microfluidic chip according to claim 1, characterized in that, The chip further includes a sample preprocessing channel for filtering or releasing nucleic acid from pet samples.

9. A method for combined detection of multiple pathogens in pets, characterized in that, The microfluidic chip according to any one of claims 1-8 comprises the following steps: The sample is injected into the chip inlet after being lysed and purified by the pretreatment module; Laser-triggered burst valves distribute samples to each reaction chamber; Isothermal amplification was performed at a constant temperature of 65±0.2℃. The amplification signal is monitored in real time using a time-resolved fluorescence detection system; Output a multi-pathogen joint detection report.

10. A portable pet nucleic acid testing device, characterized in that, It includes the microfluidic chip as described in any one of claims 1–8, and the accompanying temperature control module and signal detection module.

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