Centrifugal multiple microorganism detection paper chip and detection device

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to a centrifugal multiple microbiological detection paper chip and a detection device. The multiple microbiological detection paper chip mainly comprises a micro-fluidic chip upper layer, a micro-fluidic chip lower layer and a chromatographic test strip arranged between the micro-fluidic chip upper layer and the micro-fluidic chip lower layer, a liquid flowing path is etched on the upper layer of the micro-fluidic chip; a liquid flowing path is etched on the micro-fluidic chip lower layer, detection areas for arranging a chromatography test strip group are distributed along the circumferential direction, and when the micro-fluidic chip upper layer and the micro-fluidic chip lower layer are mounted and attached, the detection areas are isolated from each other; the chromatographic test strip group comprises a sample pad and at least one piece of chromatographic test paper connected to the sample pad.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and in particular relates to a centrifugal multiple microbial detection paper chip and a detection device. Background Art

[0002] Outbreaks of infectious diseases caused by pathogenic microorganisms pose a threat to global public health. Early detection of these pathogenic microorganisms can provide timely treatment and prevent further spread of infectious diseases. Nucleic acid detection technology (NAT) enables early diagnosis of pathogens and is essential for the development of accurate, rapid, and on-site detection methods to screen suspected patients and diagnose pathogen infections. Compared with polymerase chain reaction detection of pathogens, nucleic acid isothermal amplification does not require thermal cycling, making nucleic acid testing faster and more convenient. As pathogen infections become more and more harmful to society, portable detection devices with complete "sample in-result out" functions are essential to achieve on-site detection and have become a hot topic in the current field of molecular testing. Among them, the detection method of microorganisms (especially pathogenic microorganisms) based on the combination of microfluidic chips and nucleic acid isothermal amplification technology has received widespread attention.

[0003] Microfluidic chips have been widely developed, mainly including centrifugal microfluidic chips, pressure-driven microfluidic chips, magnetic-driven microfluidic chips and emerging paper chips. Centrifugal chips have the advantages of high analytical throughput, simple fluid control, no need for external pumps and valves, simple parallel processing, high integration and compact platform. They provide a disc-type experimental platform for efficient biochemical analysis, which can achieve high throughput and multiple detection. Lateral flow test strips also show great potential in the field of instant diagnosis. They have the advantages of light weight, portability, storage of dry reagents, and fluid flow controlled by the capillary force of the substrate without the need for other external pumps. They can significantly reduce the need for external instruments and provide user-friendly result reading. Based on different paper materials or combining different processes with paper-based materials, nucleic acid extraction, amplification and detection are completed on paper. The development of nucleic acid lateral flow test strips can create low-cost nucleic acid sensors, which has become an important detection method in the field of molecular detection technology. Combining test strips with disc-type centrifugal microfluidic chips can increase detection throughput and achieve the goal of multiple detection.

[0004] Nucleic acid detection technologies mainly include PCR, isothermal amplification technology and CRISPR technology. Isothermal amplification technology simplifies the amplification process, has low instrument dependence, and can quickly and efficiently replicate nucleic acid sequences at a constant temperature without a thermal cycler, making it very suitable for rapid detection. The diagnostic method based on the clustered regularly interspaced short palindromic repeat sequence CRISPR / Cas system has also attracted interest in the field of infectious disease diagnosis. After the specific recognition of the target sequence by CRISPR RNA (crRNA), the Cas protein can activate the ability to non-specifically cut the nucleic acid reporter gene, thereby generating a large number of fluorescent signals, which can improve the sensitivity and specificity of the detection.

[0005] (1) Traditional test strips have low detection sensitivity and usually require more accurate technology for subsequent result confirmation;

[0006] (2) Test strips can usually only test one sample at a time and lack the potential for multiple testing;

[0007] (3) The degree of automation of the test is low and requires professional operators;

[0008] (4) The heating equipment required for the reaction and the equipment required for detection are usually separate, which interrupts automation and may introduce contamination due to manual handling of the microfluidic chip placed on another platform, which may limit its development as a point-of-care diagnostic device. Summary of the invention

[0009] In view of this, in order to achieve multi-target detection of the same sample and improve detection efficiency, a centrifugal microfluidic chip and a test strip are combined. The present invention provides a centrifugal multiple microorganism detection paper chip and a detection device.

[0010] The technical solution for implementing the present invention is as follows:

[0011] In the first aspect, the embodiment of the present application is a centrifugal multiple microorganism detection paper chip, which is mainly composed of a microfluidic chip upper layer, a microfluidic chip lower layer, and a chromatography test strip built in between the two;

[0012] A liquid flow path is etched on the upper layer of the microfluidic chip;

[0013] The lower layer of the microfluidic chip is etched with a liquid flow path, and detection areas for setting the chromatography test strip group are distributed along the circumference. When the upper layer of the microfluidic chip is installed and attached to the lower layer of the microfluidic chip, the detection areas are isolated from each other;

[0014] The chromatography test strip set comprises a sample pad and at least one chromatography test paper connected to the sample pad.

[0015] Furthermore, the chromatographic test paper of the present invention is composed of a connecting pad, a reaction zone and a water absorbent pad. The sample pad is pre-prepared with a lysis solution for lysing microorganisms and releasing nucleic acids therein. The connecting pad is used to connect the sample pad and the reaction zone. The water absorbent pad is placed downstream of the reaction pad and connected to the reaction pad through the connecting pad.

[0016] Furthermore, the liquid flow path etched on the upper layer of the microfluidic chip described in the present invention is composed of a pre-liquid separation chamber, a sample addition hole and a capillary valve, wherein the pre-liquid separation chamber is a connecting channel scattered along the circumferential direction, and the capillary valve is arranged at the top of the pre-liquid separation chamber channel and is aligned with the sample pad on the chromatography test strip group; the sample addition hole is connected to the pre-liquid separation chamber, and the surface of the capillary valve is coated with a hydrophobic reagent for hydrophobic modification.

[0017] Furthermore, the number of the detection zones of the present invention is N, and the pre-liquid separation chamber is a centrally symmetrical channel with N corners.

[0018] Furthermore, the liquid flow paths etched on the upper layer and the lower layer of the microfluidic chip of the present invention are multiple groups of sample addition holes, and each group of sample addition holes corresponds to a reaction area on the built-in chromatography test strip group.

[0019] Furthermore, the detection area on the lower layer of the microfluidic chip of the present invention is fan-shaped, and an isolation zone is provided between adjacent detection areas.

[0020] Furthermore, the upper layer and the lower layer of the microfluidic chip of the present invention are both made of polymethyl methacrylate.

[0021] Furthermore, the present invention also includes a sealing layer, the material of the sealing layer is 3M glue, and the injection well is sealed with a PCR plate sealing film after the reagent is loaded.

[0022] Furthermore, the functional area of ​​the present invention has a diameter of 3.5 mm and a depth of 2.0 mm, the upper pre-liquid separation area has a width of 2.0 mm and a depth of 5.0 mm, and can accommodate 1.2 mL of liquid, and the lower pre-liquid separation area has a width of 2.0 mm and a depth of 3.0 mm, and can accommodate 0.8 mL of liquid.

[0023] In a second aspect, an embodiment of the present application provides a centrifugal multi-microorganism detection device, including a biological detection paper chip, a centrifugal drive module, a temperature control module, a fluorescence detection module, and a data processing module;

[0024] The temperature control module is attached to the top of the biological detection paper chip and is used to uniformly heat the paper chip;

[0025] The centrifugal driving module is used to drive the paper chip to rotate to provide centrifugal force;

[0026] The fluorescence detection module is used to detect the fluorescence signal generated by carboxyfluorescein FAM;

[0027] The data processing module is used to process the fluorescence signal detected by the fluorescence detection module to obtain the detection result.

[0028] Beneficial effects:

[0029] First, the centrifugal microfluidic chip contains multiple sealed functional chambers, which can significantly increase the number of detection targets, improve detection efficiency and detection throughput, and reduce detection time.

[0030] Second, the entire process is carried out on fully sealed chips and equipment, avoiding excessive chip handling steps and eliminating the need to repeatedly pick up the chips, reducing the risk of contamination.

[0031] Third, it can realize portable detection of microorganisms with strong usability and no need for complex and bulky temperature control equipment and detection equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 This is a schematic diagram of a multiple detection chip according to an embodiment;

[0034] Figure 2 This is a schematic diagram of a high-throughput detection chip according to another embodiment;

[0035] Figure 3 is a schematic diagram of the detection device;

[0036] Figure 4 This is a schematic diagram of the detection chip installation;

[0037] Figure 5 Schematic diagram of the detection device.

[0038] Among them, 1-capillary valve; 2-pre-liquid separation chamber; 3-reaction area; 4-connection pad; 5-water absorption pad; 6-sample pad; 7-sample loading hole; 8-lower chip; 9-upper chip; 10-sealing film; 11-heat cover; 12-heat dissipation hole; 13-switch; 14-display screen; 15-fixed knob; 16-chip; 17-heating film; 18-fluorescence detector. DETAILED DESCRIPTION

[0039] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0040] It should be noted that the following embodiments and features in the embodiments may be combined with each other in the absence of conflict; and, based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in the field without making any creative work are within the scope of protection of the present disclosure.

[0041] It should be noted that various aspects of the embodiments within the scope of the appended claims are described below. It should be apparent that the aspects described herein may be embodied in a wide variety of forms, and any specific structure and / or function described herein is merely illustrative. Based on the present disclosure, it should be understood by those skilled in the art that an aspect described herein may be implemented independently of any other aspect, and two or more of these aspects may be combined in various ways. For example, any number of aspects described herein may be used to implement the device and / or practice the method. In addition, other structures and / or functionalities other than one or more of the aspects described herein may be used to implement this device and / or practice this method.

[0042] In the first aspect, the present application embodiment provides a centrifugal multiple microbial detection paper chip, such as Figure 1 As shown, it is mainly composed of an upper layer of a microfluidic chip, a lower layer of a microfluidic chip, and a chromatography test strip built in between the two;

[0043] A liquid flow path is etched on the upper layer of the microfluidic chip;

[0044] The lower layer of the microfluidic chip is etched with a liquid flow path, and detection areas for setting the chromatography test strip group are distributed along the circumference. When the upper layer of the microfluidic chip is installed and attached to the lower layer of the microfluidic chip, the detection areas are isolated from each other;

[0045] The chromatography test strip set comprises a sample pad and at least one chromatography test paper connected to the sample pad.

[0046] The multiple centrifugal detection chip of the embodiment of the present application injects samples from the upper liquid flow path and then discharges samples from the lower liquid flow path. During this process, after the chip is acted upon by centrifugal force, the liquid is thrown into multiple detection areas. Therefore, this design can realize multiple groups of measurements.

[0047] In another embodiment of the present application, the liquid flow path etched on the upper layer of the microfluidic chip and the lower layer of the microfluidic chip consists of a pre-liquid separation chamber, a sample addition hole and a capillary valve, wherein the pre-liquid separation chamber is a connecting channel scattered along the circumferential direction, and the capillary valve is arranged at the top of the pre-liquid separation chamber channel and is aligned with the sample pad on the chromatography test strip group; the sample addition hole is connected to the pre-liquid separation chamber.

[0048] like Figure 1As shown, in the specific implementation of this embodiment, the chip is made of polymethyl methacrylate (PMMA) with an outer dimension of 136mm×136mm×6mm. The sealing layer uses 3M glue. After the reagent is loaded, the injection well is sealed with a PCR sealing film. Under the premise of not affecting amplification and detection, the leakage of reagents can be effectively prevented. The two layers of PMMA are engraved with designated liquid flow paths on the top and bottom surfaces. Among them, the upper chip is engraved with a pre-liquid separation chamber, a sample well (one inlet and one outlet) and a capillary valve, and the lower chip is engraved with a pre-liquid separation chamber, a sample well (one inlet and one outlet), a capillary valve and eight parallel functional areas. The functional area can be used to place a chromatography test strip to achieve simultaneous detection of 24 targets. The surface of the capillary valve is coated with a hydrophobic reagent for hydrophobic modification. The diameter of the functional area is 3.5mm and the depth is 2.0mm. The width of the upper pre-liquid separation area is 2.0mm and the depth is 5.0mm, which can accommodate 1.2mL of liquid. The width of the lower pre-liquid separation area is 2.0mm and the depth is 3.0mm, which can accommodate 0.8mL of liquid. The centrifugal microfluidic platform can simply use the motor-driven centrifugation to control and divide the liquid into equal parts without the need for a complex pump and valve system for fluid control. The centrifugal detection chip of the embodiment of the present application only needs one sample addition, from the sample addition to the sample hole to the sample outlet. After the liquid fills the pre-liquid separation channel, only one centrifugation process is required. The 8 equal parts of liquid in the pre-liquid separation chamber break through the corresponding capillary valves and are thrown onto the reaction pads of the 8 chambers, so multiple detections can be achieved.

[0049] like Figure 2 As shown, in another embodiment of the present application, the liquid flow path etched on the lower layer of the microfluidic chip is a plurality of groups of sample addition holes, and each group of sample addition holes corresponds to the reaction area on the built-in chromatography test strip group. Compared with the above embodiment, each group of sample addition holes in this embodiment corresponds to the reaction area on the built-in chromatography test strip group, and after multiple sample additions, multiple groups can be measured simultaneously.

[0050] In another embodiment of the present application, each chromatographic test paper is composed of a connecting pad, a reaction zone and a water-absorbing pad. The sample pad is pre-prepared with a lysis solution for lysing microorganisms and releasing the nucleic acids therein. The connecting pad is used to connect the sample pad and the reaction zone. The water-absorbing pad is placed downstream of the reaction pad and is connected to the reaction pad through the connecting pad, which can promote the flow of liquid and promote the flow of nucleic acids from the sample pad to the reaction zone. Excess water and impurities can be absorbed by the water-absorbing pad.

[0051] In the specific implementation of this embodiment, the built-in test strip mainly includes 4 parts, namely, a sample pad, a connection pad, a reaction zone and a water-absorbing pad. The sample pad is prefabricated with a lysate, which can lyse microorganisms and release the nucleic acid therein. The connection pad is used to connect the sample pad and the reaction zone. The reaction zone is stacked with four layers of circular paper pads, which are, from bottom to top, a chitosan pad for nucleic acid enrichment, a Tris-HCl pad for nucleic acid elution, a LAMP reaction pad for amplification reaction, a CRISPR reaction pad for signal amplification and fluorescence detection, and a water-absorbing pad for promoting the movement of nucleic acids. Before the chip is sealed, the test strip with the prefabricated reaction system is preloaded into the functional area of ​​the chip.

[0052] In addition, according to the fixed size of the adapter device, while supporting multiple tests, the design can be changed to design a disc for multi-sample testing. By increasing the number of sample holes and the number of sample additions, the number of samples tested can be increased, and multiple samples can be tested simultaneously to meet the needs of higher throughput.

[0053] like Figure 3-5 As shown, a centrifugal multi-microorganism detection device according to an embodiment of the present application includes a biological detection paper chip, a centrifugal drive module, a temperature control module, a fluorescence detection module and a data processing module;

[0054] The temperature control module is attached to the top of the biological detection paper chip and is used to uniformly heat the paper chip;

[0055] The centrifugal driving module is used to drive the paper chip to rotate to provide centrifugal force;

[0056] The fluorescence detection module is used to detect the fluorescence signal generated by carboxyfluorescein FAM;

[0057] The data processing module is used to process the fluorescence signal detected by the fluorescence detection module to obtain the detection result.

[0058] When this embodiment is implemented, the size is 230mm×200mm×150mm, and it is mainly composed of multiple functional modules such as centrifugal drive, temperature control, fluorescence detection and main control module. Temperature control part: The heating film is above the paper pad, which can heat the reaction area non-contact, quickly and evenly, and the heating film is on the same side as the detector to avoid evaporation of water and the additional operation steps and pollution caused by repeated chip removal. The NTC temperature sensor is used to monitor the temperature in real time so that the reaction chamber of the chip is heated at a certain temperature within a specified time. Centrifugal drive module: The high-speed rotation of the microfluidic chip is precisely controlled by a DC motor, and the power of the centrifugal motor is controlled by a PWM wave to achieve different centrifugal speeds. The direction of rotation is switched by converting the direction signal. At the same time, a photoelectric switch is placed above the motor to position the chip. Fluorescence detection module: A fluorescence detector with excitation and emission wavelengths of 470nm and 525nm, respectively, detects the fluorescence signal generated by FAM. Data processing module: The fluorescence detector transmits the collected signal to the Raspberry Pi through serial communication. The Raspberry Pi stores the data and calculates and processes the signal. The processed data is displayed on the screen or connected to the computer through the network port. The instrument also integrates a display and provides a touch screen user interface that can display the software interface and analyze the detection data. It is used to monitor the workflow of nucleic acid detection and view the fluorescence signal of the reaction unit in real time. The embedded software is developed using Python on the Raspberry Pi for automatic execution of programs and analysis of data. The system is powered by a mains supply or an external battery pack.

[0059] The centrifugal force generated by the rotation of the centrifugal chip and the capillary force on the test strip inserted in the chip are used to control the flow of fluids, so as to achieve uniform pre-liquidation of the added sample and enhance the fluorescent signal generated by the nucleic acid amplification reaction on the lateral flow test strip, and finally achieve multiple detection, overcoming the shortcomings of single detection of the test strip. The basic liquid processing steps, such as sample equalization, nucleic acid movement and reaction, are achieved by optimizing key parameters such as centrifugal speed, rotation direction and pre-liquidation channel design.

[0060] The specific operation steps of this device are as follows: first, collect samples from the patient's nasopharyngeal swab and mix them evenly with the buffer solution, and inject the mixed solution into the pre-liquid separation chamber of the lower chip; inject another set of buffer solution into the pre-liquid separation chamber of the upper chip, and seal the injection well with a PCR plate sealing film. Then centrifuge at 2030rpm for 5s, and the mixed solution infiltrates the sample pad through centrifugation, completes the lysis of pathogenic microorganisms and the release of nucleic acids on the sample pad; finally, centrifuge at 2320rpm for 60s, and the buffer solution infiltrates the sample pad through centrifugation and promotes the release of lysis nucleic acids to move to the reaction area through the capillary action of paper, incubate at 52℃ for 30min, and detect the fluorescence signal of the amplified product.

[0061] Compared with the existing measurement methods, this device has the following specific effects:

[0062] (1) Constructing portable instruments to overcome the limitations of fixed locations and fixed equipment, with strong usability, adaptability to long-distance transportation, and meeting the testing needs outside the laboratory environment;

[0063] (2) The advantages of nucleic acid chromatography test strips, such as simplicity, convenience, low cost, and pump-free transportation, were utilized, and the simple fluid control capabilities of the centrifugal microfluidics platform were applied to improve the detection performance, avoiding external pumps and valves, and constructing a low-cost automated platform;

[0064] (3) The fully enclosed and automated microfluidic system effectively avoids aerosol contamination caused by isothermal nucleic acid amplification, and does not require manual transfer of active ingredients, which can prevent the active ingredients from being contaminated during the transfer process, improve the accuracy of the test results, and avoid contamination and inconsistent operations caused by human operations. It is suitable for pathogen detection outside diagnostic laboratories;

[0065] (4) Only two centrifugations are required, and the power consumption of the entire device is low;

[0066] (5) Due to the symmetry of its functional units, at least eight sets of reactions can be performed simultaneously, with the potential for higher throughput and multiplex detection;

[0067] (6) High degree of automation improves response speed.

[0068] In summary, the above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A centrifugal multi-microorganism detection paper chip, characterized in that: It is mainly composed of an upper layer of a microfluidic chip, a lower layer of a microfluidic chip, and a chromatography test strip built in between the two; A liquid flow path is etched on the upper layer of the microfluidic chip; The lower part of the microfluidic chip is etched with a liquid flow path, and detection areas for setting the chromatography test strip group are distributed along the circumference. When the upper centrifugal microfluidic layer and the lower centrifugal microfluidic layer are installed and attached, the detection areas are isolated from each other; The chromatography test strip set comprises a sample pad and at least one chromatography test paper connected to the sample pad.

2. The centrifugal multi-microorganism detection paper chip according to claim 1, characterized in that: The chromatographic test paper consists of a connection pad, a reaction zone and a water absorbent pad. The sample pad is pre-prepared with a lysis solution for lysing microorganisms and releasing nucleic acids therein. The connection pad is used to connect the sample pad and the reaction zone. The water absorbent pad is placed downstream of the reaction pad and connected to the reaction pad through the connection pad.

3. The centrifugal multi-microorganism detection paper chip according to claim 1, characterized in that: The liquid flow path etched on the upper layer of the microfluidic chip consists of a pre-liquid separation chamber, a sample addition hole and a capillary valve, wherein the pre-liquid separation chamber is a connecting channel scattered along the circumferential direction, and the capillary valve is arranged at the top of the pre-liquid separation chamber channel and is aligned with the sample pad on the chromatography test strip group; the sample addition hole is connected to the pre-liquid separation chamber, and the surface of the capillary valve is coated with a hydrophobic reagent for hydrophobic modification.

4. The centrifugal multi-microorganism detection paper chip according to claim 3, characterized in that: The number of the detection zones is N, and the pre-liquid separation chamber is a centrally symmetrical channel with N corners.

5. The high-throughput microbial detection paper chip according to claim 1, characterized in that: The liquid flow paths etched on the upper layer of the microfluidic chip and the lower layer of the microfluidic chip are multiple groups of sample addition holes, and each group of sample addition holes corresponds to a reaction area on the built-in chromatography test strip group.

6. The centrifugal multi-microorganism detection paper chip according to claim 1, characterized in that: The detection area on the lower layer of the microfluidic chip is fan-shaped, and an isolation zone is provided between adjacent detection areas.

7. The centrifugal multi-microorganism detection paper chip according to claim 1, characterized in that: The upper layer and the lower layer of the microfluidic chip are both made of polymethyl methacrylate.

8. The centrifugal multi-microorganism detection paper chip according to claim 1, characterized in that: It also includes a sealing layer, the material of which is 3M glue, and the injection well is sealed with a PCR plate sealing film after the reagent is loaded.

9. The centrifugal multi-microorganism detection paper chip according to claim 1, characterized in that: The functional area has a diameter of 3.5 mm and a depth of 2.0 mm. The upper pre-liquid separation area has a width of 2.0 mm and a depth of 5.0 mm, and can accommodate 1.2 mL of liquid. The lower pre-liquid separation area has a width of 2.0 mm and a depth of 3.0 mm, and can accommodate 0.8 mL of liquid.

10. A centrifugal multi-microorganism detection device, characterized in that: A bioassay paper chip comprising any one of claims 1 to 9, a centrifugal drive module, a temperature control module, a fluorescence detection module and a data processing module; The temperature control module is attached to the top of the biological detection paper chip and is used to uniformly heat the paper chip; The centrifugal driving module is used to drive the paper chip to rotate to provide centrifugal force; The fluorescence detection module is used to detect the fluorescence signal generated by carboxyfluorescein FAM; The data processing module is used to process the fluorescence signal detected by the fluorescence detection module to obtain the detection result.

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