CN110564607B

CN110564607B - Full-automatic nucleic acid extraction amplification micro-fluidic chip dynamic quantitative detection integrated device

Info

Publication number: CN110564607B
Application number: CN201910859499.4A
Authority: CN
Inventors: 司远; 徐仕强; 吕洋; 褚春旭
Original assignee: Changchun Jite Bio Tech Co ltd
Current assignee: Changchun Jite Bio Tech Co ltd
Priority date: 2019-09-11
Filing date: 2019-09-11
Publication date: 2022-10-28
Anticipated expiration: 2039-09-11
Also published as: CN110564607A

Abstract

The invention relates to a full-automatic nucleic acid extraction amplification micro-fluidic chip dynamic quantitative detection integrated device, which comprises: a nucleic acid automated extraction module for automated lysis and purification of nucleic acids in one or more samples; a nucleic acid amplification and hybridization module comprising a microfluidic chip capable of receiving the purified nucleic acid from the nucleic acid automated extraction module and performing PCR amplification and hybridization; and the dynamic quantitative detection module is used for scanning and imaging the microfluidic chip through a CCD camera to detect. The invention can utilize a mechanical arm to complete the continuous sample loading function of a plurality of sample containers, realize the automatic detection of large samples, and the CCD camera can carry out scanning imaging when finishing one round of PCR amplification, realize the dynamic quantitative detection and then integrate the whole amplification and hybridization processes to carry out quantitative analysis on the samples, thereby improving the precision of the digital PCR technology.

Description

Full-automatic nucleic acid extraction amplification micro-fluidic chip dynamic quantitative detection integrated device

Technical Field

The invention relates to the field of biological detection and molecular diagnosis, in particular to a full-automatic integrated device for dynamic quantitative detection of a nucleic acid extraction amplification micro-fluidic chip.

Background

In 1953, watson and Crick propose a double helix structure model of deoxyribonucleic acid, which is a milestone achievement and lays a solid theoretical foundation for the development of molecular biology and the progress of molecular diagnosis technology. The development of molecular diagnosis goes through four stages, including molecular hybridization technology, PCR technology, biochip technology, and second generation sequencing technology. In recent years, molecular diagnosis is rapidly becoming one of the most effective ways for human disease diagnosis, wherein the biochip technology solves the problems of complex, low automation, small number of target molecules to be detected and low flux of the traditional nucleic acid blotting hybridization technology. And the problems of complex second-generation sequencing, high cost, long time, strong specialty and the like are also solved. Can be applied to various fields such as clinical disease diagnosis, blood transfusion safety, forensic medicine identification, nucleic acid sample library establishment, environmental microorganism detection, food safety detection, molecular biology research and the like.

The development trend of molecular diagnosis technology is developing towards high-end, intelligent and integrated development. However, the current international molecular diagnosis including berle, ABI, BD, roche and other products are basically a real-time fluorescence quantitative PCR technology and a digital PCR technology, most of the nucleic acid sample treatment, PCR and nucleic acid detection machines are completed by a plurality of split machines, and the split systems are complex in operation, low in intelligence degree, high in cost, easy to produce pollution and easy to cause human errors due to the operation of a plurality of instruments. At present, only the Xpert RIF product has high integration of nucleic acid sample extraction, PCR and detection in an all-in-one machine, but is very expensive. China has no related mature technology and products. In addition, the product has a disadvantage in that it can only detect the final PCR result, and cannot perform real-time fluorescent quantitative analysis.

The invention integrates a sample nucleic acid extraction and purification technology module, a nucleic acid PCR amplification and hybridization module and a biochip detection module into an integrated product. The method has the advantages of realizing intelligence, automation and simplification, reducing cost and improving accuracy and detection efficiency.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a novel full-automatic detection device and a using method thereof by combining a micro-fluidic chip with a CCD imaging technology, a sample does not need to be taken out in the whole process, the possibility of biological pollution and sample pollution is greatly reduced, the matching of instruments does not need to be cleaned, and the cost is reduced. In addition, the real state of the sample can be visually seen through the scanned picture or image in the PCR process, and the information of the sample can be more accurately evaluated by combining the analysis and detection of upper computer software, so that real-time monitoring, detection and analysis are realized. In addition, the invention can realize the automatic and continuous completion of nucleic acid sample treatment, nucleic acid amplification, nucleic acid hybridization and dynamic quantitative detection, can reduce the technical threshold and the cost, and is favorable for the popularization of the technology to the basic level.

In short, the object of the present invention is achieved by the following technical solutions:

in a first aspect, the present invention provides an integrated apparatus for dynamic quantitative detection of a full-automatic nucleic acid extraction amplification microfluidic chip, comprising:

a nucleic acid automated extraction module for automated lysis and purification of nucleic acids in one or more samples;

a nucleic acid amplification and hybridization module comprising one or more microfluidic chips capable of receiving purified nucleic acid from the nucleic acid automated extraction module and performing PCR amplification and hybridization; and

and the dynamic quantitative detection module is used for detecting the micro-fluidic chip by scanning and imaging the micro-fluidic chip through a CCD camera.

In one embodiment, the automated nucleic acid extraction module comprises:

one or more sample containers for receiving the sample and lysis buffer, an

A microplate for housing reagents required for nucleic acid purification for purification,

the one or more microfluidic chips are arranged on the microfluidic chip loading platform, and the sample container, the microporous plate and the microfluidic chip loading platform are sequentially arranged on the horizontal guide rail and can slide along the horizontal guide rail,

preferably, the sample container, the microplate and the microfluidic chip loading platform are integrally combined into a reagent cartridge module and integrally slide on the horizontal guide rail.

In one embodiment, the horizontal guide rail is provided with a magnet, preferably a cylindrical magnet,

the sample container is provided with magnetic beads, when the magnet is driven to move, the magnetic beads in the sample container are driven by magnetic force to vibrate so as to lyse cells in the sample,

preferably, the magnet is arranged 2-5 cm from the guide rail plane and 2-10 cm from the sample container.

In one embodiment, the automated nucleic acid extraction module further comprises a heating bath capable of heating the sample in one or more sample containers to provide a temperature required for nucleic acid extraction.

In one embodiment, the sample container is a sample lysis tube; and/or the microplate is a 96-well plate.

In one embodiment, the apparatus further comprises:

the automatic pipetting module is positioned above the horizontal guide rail and can pipette the cracked sample in the sample container to a micro-porous plate with a nucleic acid purification reagent for one or more times of extraction and elution so as to purify the sample; and/or pipetting the purified sample in the microplate to a chip loading well of the microfluidic chip.

In one embodiment, the device comprises a PCR control module for controlling PCR reactions in the one or more microfluidic chips.

In one embodiment, the PCR control module is disposed adjacent to the other end of the guide rail away from the sample container and is capable of making sufficient contact with the microfluidic chip of the microfluidic chip loading platform while the microfluidic chip loading platform slides out of the other end.

In one embodiment, the dynamic quantitative detection module comprises an imaging system and a motion module capable of moving the imaging system along a direction perpendicular to the horizontal guide rail, wherein the imaging system comprises one or more excitation light sources and a CCD camera, so that one or more samples on the microfluidic chip can be detected simultaneously, and particularly, each round of PCR can be detected.

In one embodiment, the objective lens for the CCD camera is arranged to be at least 6cm from the microfluidic chip during operation

In one embodiment, the automated pipetting module comprises a linear motor motion module, a stepper motor motion module, and an extraction motion module,

the stepping motor motion module drives the pipetting module to move along the vertical direction vertical to the surface of the horizontal guide rail

The linear motor motion module drives the liquid transfer module to mount and dismount the liquid transfer tube, and the tail end of the linear motor comprises one or more butt joints which are in butt joint with the liquid transfer tube;

the extraction motion module drives the pipette to extract or expel liquid from a sample container or microplate and contains a photodiode sensor, preferably a correlation photodiode sensor, arranged to be able to read the status of liquid extraction or expulsion from the pipette.

In one embodiment, the pipette has two filter elements, an aerosol filter element at the top of the pipette prevents the filter element from falling into a nucleic acid pollution source and sucking back pollution, and the filter element is 1-3 cm away from the top of the pipette; a nucleic acid adsorbing filter element at the bottom of the pipette for adsorbing nucleic acid, the filter element being located at a distance of 1-3 cm from the bottom of the pipette, preferably the nucleic acid adsorbing filter element is selected from the group consisting of silica gel, diatomaceous earth, glass powder, more preferably a glass powder filter element, in particular a quartz glass or borate glass powder filter element.

In one embodiment, the microfluidic chip has a three-dimensional array of microspheres with fluorescent probes on the surface capable of specifically binding to biomolecules.

In one embodiment, the PCR control module comprises:

a heating plate capable of sufficiently contacting and heating the microfluidic chip fixed on the microfluidic chip loading platform;

a semiconductor cooling device, such as a peltier device, disposed below the heating plate;

a heat dissipation plate; and

a heat dissipation fan.

In one embodiment, the apparatus further comprises

An automatic loading module capable of automatically providing samples to the nucleic acid automated extraction module in sequence by a robotic arm.

In a second aspect, the present invention provides an integrated apparatus for dynamic quantitative detection of a full-automatic nucleic acid extraction amplification microfluidic chip, comprising: the kit module, the automatic pipetting module and the dynamic quantitative detection module are arranged on the horizontal guide rail and can slide along the horizontal guide rail;

one end of the horizontal guide rail is provided with a magnet;

from the one end of horizontal guide rail that is provided with magnet, the kit module includes in proper order:

one or more sample containers for receiving a sample and a lysis buffer and containing magnetic beads;

a microplate for holding reagents required for nucleic acid purification for purification, and

one or more microfluidic chips fixed on a microfluidic chip loading platform, wherein the microfluidic chips can receive purified nucleic acid from the nucleic acid automatic extraction module and perform PCR amplification and hybridization;

the automatic pipetting module is positioned above the horizontal guide rail, is fixed on the fixed frame, and can pipette the cracked sample in the sample container to a micro-porous plate with a nucleic acid purification reagent for one or more times of extraction and elution so as to purify the sample; and/or pipetting the purified sample in the microplate to the chip loading wells of one or more microfluidic chips,

the dynamic quantitative detection module is positioned above the horizontal guide rail, is fixed on the support frame, and scans and images the microfluidic chip through a CCD camera to perform detection.

In one embodiment, the cartridge module further comprises a heating well capable of heating the sample in one or more sample containers.

In one embodiment, the sample container is a sample lysis tube; and/or

The microplate is a 96-well plate.

In one embodiment, the magnet is a cylindrical magnet, rotatable around a center and coaxial with a drive motor, and the magnet is arranged 2-5 cm from the guide rail plane and 2-10 cm from the sample container.

In one embodiment, the device comprises a PCR control module for controlling a PCR reaction in the microfluidic chip.

In one embodiment, the PCR control module is disposed adjacent to the other end of the guide rail away from the sample container and is capable of making sufficient contact with one or more microfluidic chips of the microfluidic chip loading platform while the microfluidic chip loading platform slides out of the other end

In one embodiment, the PCR control module includes:

a heating plate capable of being brought into sufficient contact with and heating one or more microfluidic chips fixed on the microfluidic chip loading platform;

a heat dissipation plate; and

a heat dissipation fan.

the stepping motor moving module drives the liquid-transferring module to move along the vertical direction vertical to the surface of the horizontal guide rail;

the extraction motion module drives the pipette to extract or expel liquid from a sample container or microplate and contains a photodiode sensor, preferably a correlation photodiode sensor, arranged to read the state of liquid extraction or expulsion from the pipette.

The pipette is provided with two filter elements, an aerosol filter element positioned at the top of the pipette prevents from falling into a nucleic acid pollution source and suck back pollution, and the distance between the filter element and the top of the pipette is 1-3 cm; a nucleic acid adsorbing filter at the bottom of the pipette for adsorbing nucleic acid, the filter being located at a distance of 1 to 3cm from the bottom of the pipette, preferably the nucleic acid adsorbing filter is selected from the group consisting of silica gel, diatomaceous earth, glass powder, more preferably a glass powder filter, especially a quartz glass or borate glass powder filter.

In one embodiment, the objective lens for the CCD camera is arranged to be at a distance of more than 6cm from the microfluidic chip in operation.

In one embodiment, the apparatus further comprises:

an automated loading module capable of automatically providing samples to sample containers in the cartridge module in sequence via a robotic arm.

In a preferred embodiment, all modules in the device are integrated in a closed container.

The invention has the advantages of

Compared with the automatic device in the prior art, the invention, especially the Xpert RIF product, has the following advantages:

(1) The device can realize the automatic and continuous completion of the integrated machine of nucleic acid sample treatment, nucleic acid amplification and hybridization and dynamic quantitative detection and analysis by using special pipettes with filter elements, biological reagents, microfluidic chips and other consumables.

(2) The open type consumables are adopted, so that the samples and the reagents can be conveniently replaced; the process of extracting, amplifying and analyzing nucleic acid is fully automatically realized by combining a micro-fluidic chip; the application is flexible, and the simultaneous analysis of single or multiple samples is met; compared with the traditional analysis method, the method obviously reduces the consumption of samples, reagents and detection time, and saves the samples.

(3) The integrated machine integrates the nucleic acid extraction, amplification and microfluidic chip detection into a high degree. The operation is automatic, the analysis speed is fast, the flux is high, the error is small, and the precision is high.

(4) The system is integrated in a closed container, so that the inspection pollution of a sample and a reagent is avoided, and the accuracy is improved. The system is flexible and can simultaneously analyze single or multiple samples. The system reduces consumption of samples and reagents by using the microfluidic chip, and is beneficial to saving cost.

(5) The invention can utilize a mechanical arm to complete the continuous sample loading function of a plurality of sample containers, realize the automatic detection of large samples, and the CCD camera can carry out scanning imaging when finishing one round of PCR amplification, realize the dynamic quantitative detection and then integrate the whole amplification and hybridization processes to carry out quantitative analysis on the samples, thereby improving the precision of the digital PCR technology.

In conclusion, the full-automatic nucleic acid amplification microfluidic chip analysis device provided by the invention has the advantages of less reagent sample consumption, high precision, high flux, automatic operation and high analysis speed, and is particularly suitable for medical units and field rapid detection application. The device obviously reduces the purchase and operation cost of the equipment, is beneficial to improving the medical service quality, and can better meet the medical needs of the masses.

Drawings

FIG. 1 is a perspective view of a full-automatic nucleic acid amplification microfluidic chip dynamic quantitative detection device. As shown in fig. 1, the apparatus includes: the device comprises a power module 1, an automatic pipetting module 2, a dynamic quantitative detection module 3 and a kit module 4.

FIG. 2 is a reagent cartridge module, which is an enlarged view of the reagent cartridge module of FIG. 1. As shown in fig. 2, the module includes: a heat sink 20, a micro-well plate 21, a sample container 22, a chip loading well 232, a chip loading platform 23 with a micro-fluidic chip 231, and a horizontal guide rail HG. The heating tank 20 fastens one side of the microporous plate 21, the sampling holes 232 fasten the other side of the microporous plate 21, and the chip loading platform 23 fastens the chip sampling holes 232.

FIG. 3 is an extraction section in an automated pipetting module. As shown in fig. 3, the portion includes: the device comprises a first stepping motor 5, a synchronous belt 6, a first push rod motor 7, a fixing frame 9, a pipette 10 and a photoelectric pair tube 11. The first stepping motor 5 is used for fastening a fixing frame 9, the synchronous belt 6 is used for fastening the upper part of the fixing frame 9, the photoelectric pair tube 11 is used for fastening the fixing frame 9, the pipette 10 is used for fastening a screw rod slide block and is connected with an air pipe (not shown), and the upper part of the screw rod is connected with the first push rod motor 7. The air tube connects the special pipette and the pressure supply section as described below, and specifically, the air tube connects the special pipette and the cavity of the pressure supply section.

FIG. 4 is a pressure supply section in an automated pipetting module. As shown in fig. 4, the portion includes: the device comprises a cavity 12, a push rod 13, a baffle, a photoelectric pair tube 11, a fixing frame 9, a second stepping motor 17, a conversion valve 18 and a second push rod motor 19. An air pipe (not shown) is fixedly connected with the cavity 12, the cavity 12 is fixedly connected with the conversion valve 18, the other side of the conversion valve 18 is fixedly connected with one side of the push rod 13, the second stepping motor 17 is fixedly connected with the fixed frame 9, the baffle plate is fixedly connected above the push rod 13, the photoelectric pair tube 11 is fixedly connected with the fixed frame 9, and the second push rod motor 19 is fixedly connected with the fixed frame 9.

FIG. 5 is a single excitation light source dynamic quantitative detection module. As shown in fig. 5, the module includes: led light source 25, CCD camera 26, light source lens barrel 27, camera lens barrel 28, reflector 29, objective lens 30, and support frame 31. The LED light source 25 is fastened with the support frame 31, the light source lens barrel 27 is fastened below the LED light source 25, the reflector 29 is fixed below the light source lens barrel 27, the camera 26 is fastened with the support frame 31, the camera lens barrel 28 is fastened below the camera 26, and the objective lens 30 is fastened below the camera lens barrel 28. It should be noted that this example only exemplifies a dynamic quantitative detection module using a single excitation light source. It is also possible to use dual excitation light sources and more excitation light sources to simultaneously detect multiple fluorescence images.

FIG. 6 is a PCR control module. As shown in fig. 6, the heat sink includes a heating plate 61 which is in sufficient contact with the chip, a peltier 62 having heating and cooling functions, a heat sink 63, and a heat sink fan 64.

Fig. 7 is a borate glass filter cartridge as an example of a nucleic acid adsorbing filter cartridge in a special pipette.

Detailed Description

The invention is described in more detail below with reference to the accompanying drawings.

As described above, the integrated device for dynamic quantitative detection of a full-automatic nucleic acid extraction amplification microfluidic chip of the present invention may include an automatic sample loading module (optional module), a nucleic acid automatic extraction module, a nucleic acid amplification and hybridization module, and a dynamic quantitative detection module. The modules are integrated on an analyzer, and the lysis, extraction and purification, nucleic acid amplification and detection of the nucleic acid sample are performed on the integrated analyzer.

In addition, the full-automatic nucleic acid extraction amplification microfluidic chip dynamic quantitative detection integrated device further comprises an automatic pipetting module, a PCR control module and other auxiliary modules.

The modules of the present invention are described in more detail below.

As a specific example, the integrated device for dynamic quantitative detection of a full-automatic nucleic acid extraction amplification microfluidic chip of the invention can be the device shown in FIG. 1. The device includes: the device comprises a power module 1, an automatic pipetting module 2, a dynamic quantitative detection module 3 and a kit module 4. Wherein, the nucleic acid automatic extraction module, the nucleic acid amplification and hybridization module are integrated into a kit module 4 in the embodiment. The cartridge module 4 comprises one or more sample containers 22, a microplate 21 and a microfluidic chip loading platform 23 with microfluidic chips 231. Reference may be made in particular to the detailed description hereinafter.

1. Automatic sample loading module

The automatic sample loading module is used for loading samples, kits and the like by operators and placing the samples, the kits and the like in a designated waiting area, and the mechanical arm automatically provides the samples for the nucleic acid extraction module in sequence, so that the continuous sample loading function of a plurality of sample containers is completed, and the automatic detection of large samples is realized. The automatic loading module is an optional module of the present invention, not shown in the figures, which can use an automatic loading device in existing instruments. For example, in one embodiment, after the sample is prepared in the automated loading device, the instrument is activated, the sample is moved by the conveyor belt to the robot arm, the robot arm is tightened, pulled, and moved along the guide rail to the sample container of the nucleic acid automated extraction module described below, the robot arm is opened, the sample is mounted, and returned to the sample conveyor belt. When the first sample is purified and sent to the PCR module (nucleic acid amplification and hybridization module), the second sample moves to the manipulator. And repeating the steps to automatically sample.

However, in practice, the sample can be loaded manually or automatically. From the viewpoint of automation and standardization, it is preferable to use an automatic loading module.

It should be noted that, no matter the automatic sample loading module automatically loads the sample or the operator manually loads the sample, the full-automatic integrated device for dynamic quantitative detection of the nucleic acid extraction amplification microfluidic chip can complete the detection of all samples as long as the sample to be detected is added to the sample container, and the capability of workstation type cycle work is embodied.

2. Automatic extraction module for nucleic acid

The nucleic acid automatic extraction module is a module for automatically extracting nucleic acid in a sample, and can extract nucleic acid in the sample by physical lysis, chemical lysis, and chemical reagent extraction elution, for example.

In one embodiment, it may be part of a cartridge module as shown in fig. 2. Which comprises one or more sample containers 22, a microplate 21 and a magnet (in particular a cylindrical magnet M).

The sample container 22 is an area for receiving a sample and containing a lysis buffer, which is adjacent to the microplate 21 and fixed on the horizontal guide rail HG. The sample container 22 may carry one or more samples, and magnetic substances, such as magnetic beads, may be first added to move under the driving of a magnetic field to physically lyse the sample, and then a lysis buffer may be added to chemically lyse the sample. Both the magnetic beads and the lysis buffer can be obtained from a commercially available nucleic acid extraction kit (DNA extraction kit or RNA extraction kit)

The microplate 21 is a region where nucleic acid purification is performed, and may contain reagents necessary for nucleic acid extraction, for example, reagents related to a nucleic acid extraction kit (DNA extraction kit or RNA extraction kit). In operation, the lysed suspension (containing nucleic acids) from the sample container is extracted and eluted with reagents from each well of the microplate by pipettes in a pipetting module as described below until the desired nucleic acids are obtained that are suitable for performing PCR.

A cylindrical magnet M adjacent to and external to the sample container 22, the cylindrical magnet M rotating about its circular center; the magnet is horizontally vertical to the horizontal guide rail HG, two sides of the magnet are fixed on the outer side of the horizontal guide rail HG along the bus direction, the cylindrical magnet M is driven by a motor (not shown), the motor is coaxial with the circular surface of the cylindrical magnet M, the cylindrical magnet M rotates around the center of the circular surface of the cylindrical magnet M, and then the magnetic beads in the sample container 22 are driven to rotate through a magnetic field so as to lyse cells; the molecule of interest for lysing the cells is purified using a filter that binds specifically to the target molecule. The cylindrical magnet M is disposed 2 to 10cm from the sample container containing a magnetic substance such as magnetic beads, and at a height of 2 to 5cm from the bottom of the sample container. The sample container may be provided with a heating bath 20 on one side thereof to provide a temperature required for nucleic acid extraction, and one or more samples may be heated at the same time.

Preferably, the microplate is a multi-well plate, such as 48-well plate, 64-well plate, 96-well plate, etc., and the sample container is a sample lysis tube.

Note that the cylindrical magnet M located on the right side in fig. 2 is actually located on the left side in fig. 1. For the purpose of exposing the heating bath 20, it is described upside down.

3. Automatic liquid transfer module

The automatic pipetting module is a module for transferring liquid from one position to another position in the full-automatic integrated device for the dynamic quantitative detection of the nucleic acid extraction amplification microfluidic chip. This function can be accomplished using automated robotic arms of the prior art.

In a preferred embodiment, however, the automated apparatus of the present invention shown in fig. 3 and 4 is used as the automated pipetting module of the present invention.

In this embodiment, as shown in fig. 1, the automated pipetting module 2 is located above the horizontal guide rail HG and is capable of pipetting the lysed sample in the sample container 22 to the microplate 21 having the nucleic acid purification reagent for one or more extractions and elutions for purification; and/or pipetting the purified sample in microwell plate 21 to chip loading wells 232 of microfluidic chip 231 located on microfluidic chip loading platform 23.

The automated pipetting module can be divided into an extraction section and a pressure supply section, as shown in fig. 3 and 4, respectively, which are connected by a gas line to pneumatically control the aspiration and release of the liquid.

In general, the automated pipetting module comprises a first stepper motor 5, a synchronous belt 6, a first push rod motor 7, an air tube (not shown), a fixed frame 9, a pipette 10 (special pipette), and a photoelectric pair tube 11. The first stepping motor 5 is fastened on a fixing frame 9, the synchronous belt 6 is fastened above the fixing frame 9, the photoelectric pair tube 11 is fastened on the fixing frame 9, the pipette 10 is fastened on a screw rod sliding block and connected with an air pipe, and the upper portion of the screw rod is connected with the first push rod motor 7. A cavity 12, a push rod 13, a baffle plate (not shown), a photoelectric switch, a second stepping motor 17, a conversion valve 18 and a second push rod motor 19. The air pipe is tightly connected with the cavity 12, the conversion valve 18 is tightly fixed on the cavity 12, the other side of the conversion valve 18 is tightly fixed on one side of the push rod 13, the second stepping motor 17 is tightly fixed on the fixing frame, the baffle plate is tightly fixed above the push rod 13, the photoelectric switch is tightly fixed on the fixing frame 9, and the second push rod motor 19 is tightly fixed on the fixing frame 9.

As shown in fig. 3, the automated pipetting module may carry one or more pipettes 10. The automated pipetting module 2 is fixed to the holder 9 adjacent to and above the sample container 22 and the microplate 21. The automated pipetting module 2 may have integrated therein a pipette 10 and a motion module, which moves the pipette 10 in a vertical direction perpendicular to the horizontal guide HG.

More specifically, the automated pipetting module may comprise a linear motor motion module (controlled by the first pusher motor 7), a stepper motor motion module (controlled by the first stepper motor 5) and an extraction motion module (implemented by the pipette 10), the stepper motor motion module driving the pipetting device to move in a vertical direction perpendicular to the horizontal guide rail HG; the linear motor motion module drives the liquid transfer device to mount and dismount the pipette 10, and the tail end of the linear motor comprises a special butt joint which is butted with the pipette 10, wherein the number of the butt joints can be one or more; the extraction motion module drives the pipetting device to extract or discharge liquid from the sample container 22 or the micro-porous plate 21 and comprises photodiode sensors (photoelectric pair tubes 11), wherein the photodiodes are arranged on two sides of the vertical motion track of the pipette and are in a correlation mode, and the extraction or discharge state of the liquid on the special pipette can be read.

The pipette 10 is a special pipette specially designed for the device and provided with two filter elements, an aerosol filter element positioned at the top of the special pipette prevents pollution sources such as nucleic acid and the like and suck-back pollution, and the filter element is 1-3 cm away from the top of the special pipette; the nucleic acid adsorption filter element at the bottom of the special pipette adsorbs nucleic acid, and the distance between the filter element and the bottom of the special borate is 1-3 cm.

The nucleic acid-adsorbing filter is not particularly limited as long as it can adsorb nucleic acid. Because the nucleic acid contains phosphoric acid residue and is negatively charged, the adsorption filter element is usually positively charged, and adsorbs the nucleic acid according to the acidity or alkalinity of the nucleic acid, so that the purification of the nucleic acid in a pipette is realized, and then the adsorbed nucleic acid is separated by using buffer solutions with different pH values in a micropore plate, and the circulation is carried out, so that the aim of purifying the nucleic acid is fulfilled. As the material of the nucleic acid adsorbing filter, silica gel, diatomaceous earth, glass powder, or the like can be used. Preference is given to using filter elements made of glass material. The glass may be silicate glass, potassium glass, quartz glass, borate glass, etc., and preferably quartz glass or borate glass. The glass filter element can be in the forms of glass fiber, glass paper, glass fiber membrane and the like. Particularly preferred is a cartridge formed by heating and compressing a glass powder (e.g., a powder of quartz glass or borate glass), as shown in FIG. 7, which has a large contact area with nucleic acids, thereby achieving large sample amount purification and reducing the loss of nucleic acids, for example, in such a manner that the recovery rate thereof can be 80% or more, more preferably 90% or more.

4. Nucleic acid amplification and hybridization modules

The nucleic acid amplification and hybridization module is a region where PCR amplification and hybridization are performed.

In one embodiment, as shown in fig. 2, the nucleic acid amplification and hybridization module is a microfluidic chip loading platform 23 on which a microfluidic chip 231 is fixed, which is a part of the reagent cartridge module 4 in fig. 1, and constitutes the reagent cartridge module 4 together with the sample container 22 and the micro well plate 21.

Specifically, the micro-fluidic chip tray (the micro-fluidic chip loading platform 23) may be integrated with the micro-fluidic chip 231, and may carry 1 to a plurality of micro-fluidic chips 231, and perform PCR amplification simultaneously, and the micro-fluidic chip tray is designed to have an effect of fixing the micro-fluidic chip 231, so that the micro-fluidic chip 231 is in full contact with the heating plate 61 in the PCR control module shown in fig. 6.

The microfluidic chip 231 includes one or more chip loading wells 232 (injection wells) through which one or more samples subjected to nucleic acid lysis and purification can be introduced from the microplate 21, and the chip includes a minute space capable of satisfying nucleic acid amplification and hybridization, and a substance capable of binding to a specific molecule, such as a fluorescent probe, etc. The substance is stored in a special array of the chip, the microfluidic chip is provided with a liquid injection hole, the chip can simultaneously comprise one or more liquid injection holes, the microfluidic chip loading platform 23 is adjacent to the microporous plate 21, and the microfluidic chip 231 is fixed on the chip loading platform 23 through a mounting hole position. The micro-fluidic chip loading platform 23 is made of a firm heat-insulating material. The effective contact area of the heating plate 61 and the microfluidic chip 231 in the PCR control module shown in FIG. 6 is not smaller than the area of the chip itself.

For example, the microfluidic chip may have a three-dimensional microsphere array, a fluorescent probe specifically bound to a biomolecule, etc. disposed in a specific three-dimensional microsphere array, so as to detect more biological characteristics in an integrated manner.

Of course, the microfluidic chip may also be a tiled microfluidic chip, such as a detection droplet tiled generation and detection integrated chip previously disclosed in CN109107624A by the present inventors.

Microfluidic chips comprising a three-dimensional microsphere array of microspheres are preferred for throughput, sensitivity, signal-to-noise, etc.

And during detection, the nucleic acid amplification and hybridization module is moved to the position below the dynamic quantitative analysis module, so that dynamic quantitative analysis is realized.

It should be noted that since the nucleic acid extraction time is generally about 1 hour and 40 minutes, and the PCR requires about 6 hours, it is preferable that the number of the microfluidic chips on the microfluidic chip tray (the microfluidic chip loading platform 23) is plural, for example, 2 to 12, preferably 4 to 8. In one embodiment, after the nucleic acid automatic extraction module finishes loading the required number of microfluidic chips, the PCR reaction and the detection are performed simultaneously.

PCR control Module

The PCR control module is used for controlling PCR reaction and realizes the aim by controlling the temperature of the PCR reaction area. A common temperature control device may be used in the present invention.

In one embodiment of the present invention, the PCR control module is disposed adjacent to the other end of the horizontal guide rail HG far away from the sample container 22, and can be in sufficient contact with the microfluidic chip 231 of the microfluidic chip loading platform 23 while the microfluidic chip loading platform 23 slides out of the other end of the horizontal guide rail HG, so as to control the PCR reaction in the microfluidic chip.

Alternatively, the PCR control module may be provided integrally with the microfluidic chip loading platform 23.

As an example of the PCR control module, as shown in fig. 6, the PCR control module includes: a heating plate 61 capable of being brought into sufficient contact with the microfluidic chip and heating it; a semiconductor cooling device 62, such as a peltier device, disposed below the heating plate; a heat dissipation plate 63; and a heat radiation fan 64. The heat radiating plate 63 and/or the heat radiating fan 64 are used to radiate heat to the heating plate and the semiconductor cooling device. As a preferred embodiment, when the microfluidic chip loading platform 33 is moved over the PCR module, it can be fastened to ensure sufficient contact of the heating plate with the microfluidic chip.

The PCR control module may also preferably include a movement module (not shown) that drives the PCR control module, for example, in a direction perpendicular to the sliding direction of the rail in the horizontal rail plane, thereby better adjusting the relative positions of the heating plate 61 and the microfluidic chip 231.

6. Dynamic quantitative detection module

And the dynamic quantitative detection module scans and images the microfluidic chip through a CCD camera to carry out real-time monitoring, detection and analysis. In the invention, scanning imaging can be carried out when each round of PCR amplification is finished, so that the quantitative analysis of the sample is realized by integrating the whole amplification and hybridization processes, and the precision of the digital PCR technology is improved.

In one embodiment, the dynamic quantitative detection module is fixed above the horizontal guide rail HG and fixed on a support frame (not shown), and the micro-fluidic chip 231 is scanned and imaged by a CCD camera for detection.

In one embodiment, as shown in FIG. 5, the dynamic quantitative detection module comprises one or more excitation light sources 25, a high resolution photo imaging system (CCD camera) 26 and necessary optical systems. The excitation light source 25 and the high resolution photographing system 26 are adjacent and fixed on an axis perpendicular to the plane of the horizontal guide rail HG above the adjacent position of the end of the guide rail remote from the sample container 22. The optical system is, for example, an objective lens 30, a mirror 29, or the like that cooperates with the CCD camera. For example, the objective lens 30 may include a plurality of lenses having a particular magnification (0.6) and field of view (compatible with the camera). In the PCR reaction process, the dynamic quantitative detection module can be adjusted to be located above the microfluidic chip 231 to scan and image the microfluidic chip in real time. The led light source 25 may be a single excitation light source, such as a green light source, or may be a multi-excitation light source.

Specifically, the dynamic quantitative detection module comprises: led light source 25, camera 26, light source barrel 27, camera barrel 28, reflector 29, objective lens 30, and support frame 31. The led light source 25 is fastened on a support frame 31, the light source lens barrel 27 is fastened below the led light source 25, the reflective mirror 29 is fixed below the light source lens barrel 27, the camera 26 is fastened on the support frame 31, the camera lens barrel 28 is fastened below the camera 26, and the objective lens 30 is fastened below the camera lens barrel 28.

It should be noted that this embodiment is merely exemplary of a dynamic quantitative detection module using a single excitation light source. It is also possible to use dual excitation light sources and more excitation light sources to detect multiple fluorescence images simultaneously.

Through real-time scanning imaging, the dynamic quantitative detection module can detect one to a plurality of PCR processes in the microfluidic chip. The detection method of the microfluidic chip is a scanning photographing imaging mode. The excitation light source is designed with HEX, FAM, ROX, CY3, CY5 and VIC at present, can be selected according to specific sample requirements, and can also be designed into other waveband excitation light sources according to requirements.

The dynamic quantitative detection module further comprises a motion module (not shown). The motion module drives the imaging system to move along an axis perpendicular to the plane of the horizontal guide rail, and the distance between the imaging system (including the objective lens) and the microfluidic chip 231 is adjusted, so that the imaging is clearer and more accurate.

In one embodiment, the imaging system includes a multi-band excitation light source, a high resolution camera (CCD camera). The multiband excitation light source can excite substances on the chip, the optical system is arranged in front of the high-resolution camera, and the imaging system can simultaneously detect one or more samples on the chip.

Examples

The present invention will be described in further detail with reference to examples.

Example 1

This example illustrates an example of detection of tuberculosis patients using the integrated apparatus for dynamic quantitative detection of full-automatic nucleic acid extraction amplification microfluidic chip of the present invention.

The operation process comprises the following steps:

(1) A liquid sample suspension containing sputum (or other samples) and lysate of a tuberculosis patient is heated in a water bath at 56 ℃ for about 20 minutes to clarify the solution.

(2) 5ml of the artificial sputum was added to 400. Mu.l of the liquefaction buffer, vortex mixed for 15 seconds, and subjected to a 56 ℃ water bath for 20 minutes.

(3) Clean desktop and experimental article and device.

(4) The cracking tube and the micropore plate are placed at the designated position on the horizontal guide rail of the device.

(5) 380. Mu.l of buffer was added to the heating tube.

(6) Vortex the treated artificial sputum for 15-30 seconds and take 500. Mu.l/tube.

(7) The lysis tube, the reagent kit and the special pipette are moved to ensure that the horizontal distance between the lysis tube and the cylindrical magnet is 2-10 cm.

(8) The motor drives the cylindrical magnet to rotate for 5-10 minutes, and the magnetic force drives the magnetic beads in the cracking tube to vibrate, so that the liquid sample suspension is promoted to be physically cracked.

(9) Adding lysis buffer to the physically lysed suspension for chemical lysis for 5-20 min.

(10) And continuously extracting the suspension liquid subjected to chemical cracking with a reagent on a micropore plate through a special pipette, and eluting to obtain the purified nucleic acid.

The operation mode is as follows: the nucleic acid is transferred from the microporous plate to the microfluidic chip by the movement of the horizontal guide rail, a nucleic acid solution is injected through the sample adding hole of the chip microfluidic chip, and in order to combine the nucleic acid with substances in the chip, the microfluidic chip is driven to a heating plate of the PCR control module along the horizontal guide rail, so that proper temperature is provided for the microfluidic chip, and the nucleic acid is promoted to finish amplification and substance combination in the microfluidic chip. And the dynamic quantitative detection module works, the CCD camera scans and images the microfluidic chip to perform real-time monitoring, detection and analysis, and particularly can scan and image when each round of PCR amplification is completed, so that the dynamic quantitative analysis is realized.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. The whole device of dynamic quantitative detection of micro-fluidic chip of full-automatic nucleic acid extraction amplification, the device includes:

a dynamic quantitative detection module for scanning and imaging the microfluidic chip by a CCD camera to perform detection,

the automated nucleic acid extraction module comprises:

one or more sample containers for receiving the sample and lysis buffer, an

A microplate for holding reagents required for nucleic acid purification for purification,

wherein the one or more microfluidic chips are arranged on a microfluidic chip loading platform, and the sample container, the microplate and the microfluidic chip loading platform are sequentially arranged on a horizontal guide rail and can slide along the horizontal guide rail,

the sample container, the microporous plate and the microfluidic chip loading platform are integrated into a reagent box module and integrally slide on the horizontal guide rail,

the horizontal guide rail is provided with a magnet at one end close to the sample container,

wherein the apparatus further comprises:

the automatic pipetting module is positioned above the horizontal guide rail and can pipette the cracked sample in the sample container to a micro-porous plate with a nucleic acid purification reagent for one or more times of extraction and elution so as to purify the sample; and/or pipetting the purified sample in the microplate to chip loading wells of one or more microfluidic chips; and

a PCR control module for controlling PCR reaction in the one or more microfluidic chips;

the dynamic quantitative detection module comprises an imaging system and a motion module capable of enabling the imaging system to move along a direction perpendicular to the horizontal guide rail, wherein the imaging system comprises one or more excitation light sources and a CCD camera, so that one or more samples on the microfluidic chip can be detected simultaneously.

2. The apparatus of claim 1, wherein,

the magnet is arranged 2-5 cm from the guide rail plane and 2-10 cm from the sample container.

3. The apparatus of claim 1, wherein,

the automated nucleic acid extraction module further comprises a heating bath capable of heating the sample in one or more sample containers to provide a temperature required for nucleic acid extraction.

4. The apparatus of claim 1, wherein,

the sample container is a sample lysis tube; and/or

The microplate is a 96-well plate.

5. The apparatus of claim 1, wherein,

the magnet is a cylindrical magnet.

6. The apparatus of claim 1, wherein,

the PCR control module is arranged to be adjacent to the other end of the guide rail, which is far away from the sample container, and can be fully contacted with the microfluidic chip of the microfluidic chip loading platform when the microfluidic chip loading platform slides out of the other end.

7. The apparatus of claim 1, wherein,

the imaging system is capable of detecting each round of PCR.

8. The apparatus of claim 1, wherein,

the objective lens for the CCD camera is set to be more than 6cm away from the microfluidic chip during working.

9. The apparatus of claim 1, wherein,

the automatic pipetting module comprises a linear motor motion module, a stepping motor motion module and an extraction motion module,

the stepping motor movement module drives the liquid-transfering module to move along the vertical direction vertical to the surface of the horizontal guide rail

the extraction motion module drives the pipette to extract or discharge liquid from a sample container or a microplate and contains a photodiode sensor arranged to be able to read the state of liquid extraction or discharge in the pipette.

10. The apparatus of claim 9, wherein,

the photodiode sensor is a correlation photodiode sensor.

11. The apparatus of claim 9, wherein,

the pipette is provided with two filter elements, an aerosol filter element positioned at the top of the pipette prevents from falling into a nucleic acid pollution source and suck back pollution, and the distance between the filter element and the top of the pipette is 1-3 cm; the nucleic acid adsorption filter element is arranged at the bottom of the pipette and used for adsorbing nucleic acid, and the distance between the filter element and the bottom of the pipette is 1-3 cm.

12. The apparatus of claim 11, wherein,

the nucleic acid adsorption filter element is selected from the group consisting of silica gel, diatomite and glass powder.

13. The apparatus of claim 11, wherein,

the nucleic acid adsorption filter element is a glass powder filter element.

14. The apparatus of claim 13, wherein,

the glass powder filter element is a quartz glass or borate glass powder filter element.

15. The apparatus of claim 1, wherein,

the microfluidic chip is provided with a three-dimensional microsphere array, and the surface of each microsphere is provided with a fluorescent probe capable of being specifically combined with biomolecules.

16. The apparatus of claim 1, wherein,

the PCR control module comprises:

a semiconductor refrigerating device disposed below the heating plate and having heating and cooling functions;

a heat dissipation plate; and

a heat dissipation fan.

17. The apparatus of claim 16, wherein,

the semiconductor cooling device is a peltier device.

18. The apparatus of claim 1, wherein,

the device further comprises:

an automated loading module capable of automatically providing samples to the nucleic acid automated extraction module in sequence via a robotic arm.

19. The device of claim 1, wherein all modules in the device are integrated within a closed container.