CN110004024A - A kind of micro-fluidic chip and portable infrared isothermal PCR platform - Google Patents
A kind of micro-fluidic chip and portable infrared isothermal PCR platform Download PDFInfo
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- CN110004024A CN110004024A CN201910247499.9A CN201910247499A CN110004024A CN 110004024 A CN110004024 A CN 110004024A CN 201910247499 A CN201910247499 A CN 201910247499A CN 110004024 A CN110004024 A CN 110004024A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
- B01L3/50851—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates specially adapted for heating or cooling samples
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
- B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/02—Adapting objects or devices to another
- B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
- B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
Abstract
The invention discloses a kind of micro-fluidic chips and portable infrared isothermal PCR platform.The chip includes the lower cover being arranged from bottom to top, MWNT heating layer, PCR room floor and upper cover；It is arranged in the PCR room floor and has family, it further includes temperature-controlled chamber that the room, which includes passing sequentially through channel attached peroxidating hydrogen room, the sample room PCR and the room PCR,.The platform is matched with the micro-fluidic chip.The present invention has high sensitivity in terms of detecting a variety of sexually transmitted diseases, can carry out multiple pathogen diagnosis simultaneously, and easy to carry, cheap, is suitable for volume production.
Description
Technical field
The present invention relates to detection field more particularly to a kind of micro-fluidic chips and portable infrared isothermal PCR platform.
Background technique
Sexually transmitted disease (STDs) seriously affects human health, is the main original of non-gonococcal urethritis (NGU) and stranguria syndrome
Cause.Venereal disease causes the pelvic inflammation of female patient, infertility, cervicitis, salpingitis, premature labor, miscarriage and cervical carcinoma and male
The prostatitis of property patient, urethritis and epididymitis.Further, it is also possible to infect newborn by birth canal, lead to baby's conjunctivitis,
Pneumonia etc..In recent years, the disease incidence one of the urogenital infections as caused by Ureaplasma urealyticum (UU) and chlamydia trachomatis (CT)
Directly rising.And the secret situation of this disease hinders patient and seeks medical treatment before being in a bad way.Therefore, it is a kind of fixed to establish
It is extremely important that phase rapidly and accurately detects the method that these are infected with screening.Early diagnosis venereal disease is early intervention and treatment patient
Key.
The latest developments of Protocols in Molecular Biology provide various detection methods for early diagnosis and extensive STD screening,
Such as enzyme linked immunological (ELISA) strand displacement amplification polymerase chain reaction (PCR) molecular beacon.
Summary of the invention
In order to improve the function of miniaturization instrument, demand point is tested the analytical equipment with microfluidic chip technology by the present invention
It combines, provides a kind of micro-fluidic chip and portable infrared isothermal PCR platform, realized especially by following technical scheme:
A kind of micro-fluidic chip, including lower cover, MWNT heating layer, PCR room floor and the upper cover being arranged from bottom to top；The room PCR
Setting is had family on layer, and it further includes temperature control that room, which includes passing sequentially through channel attached peroxidating hydrogen room, the sample room PCR and the room PCR,
Room processed.
Further, MWNT heating layer includes mistake corresponding with peroxidating hydrogen room, temperature-controlled chamber and the matching of the room PCR respectively
Aoxidize hydrogen room MWNT heating element, temperature-controlled chamber MWNT heating element and the room PCR MWNT heating element.
It further, further include that the extraction chamber RNA and RNA extract chamber cap；MWNT heating layer further includes matching with the extraction chamber RNA
The corresponding extraction chamber RNA MWNT heating element；The extraction chamber RNA is arranged on the extraction chamber RNA MWNT heating element.
Further, the upper surface of lower cover is provided with the first double faced adhesive tape；Be provided on first double faced adhesive tape respectively with RNA
Extraction chamber, peroxidating hydrogen room, the sample room PCR, temperature-controlled chamber, the corresponding hollow out in the room PCR；The upper surface of PCR room floor is provided with
Second double faced adhesive tape；Be provided on second double faced adhesive tape respectively with peroxidating hydrogen room, the sample room PCR, temperature-controlled chamber, the room PCR
And the corresponding hollow out in channel；Upper cover is provided with peroxidating hydrogen room through-hole corresponding with peroxidating hydrogen room and the sample room PCR respectively
With the sample room PCR through-hole；Micro-fluidic chip further include peroxidating hydrogen room through-hole double faced adhesive tape, the sample room PCR through-hole double faced adhesive tape,
Peroxidating hydrogen room through-hole lid and the sample room PCR through-hole lid；
MWNT heating layer is made of the mixture of MWNT and heat-conductive bonding agent, and wherein MWNT accounts for 4-10 mass parts, thermally conductive viscous
Mixture accounts for 100 mass parts.Preferably, MWNT accounts for 6.6 mass parts, and heat-conductive bonding agent accounts for 100 mass parts.Preferably, the number of the room PCR
Amount is 3.
The present invention also provides a kind of portable infrared isothermal PCR platforms to match with micro-fluidic chip as described above.
Further, including thermal module component, fluorescence detection modular assembly and locating module component；Thermal module component
Including infrared LED, thermocouple, cold junction compensation circuit and LED circuit；Infrared LED is electrically connected with LED circuit；Thermocouple
It is electrically connected with cold junction compensation circuit；
Fluorescence detection modular assembly includes blue-ray LED, bandpass filter, the first camera lens, the second camera lens, Si-PIN photoelectricity two
Pole pipe and radiator；It is glimmering in PCR sample to excite by the first camera lens that fluorescence detection module is arranged to blue led sending light
Light, fluorescence capture light intensity signal by Si-PIN photodiode after bandpass filter by the second camera lens；
Locating module includes micro-fluidic chip bracket, transmission shaft, stepper motor and shading box；The setting of micro-fluidic chip bracket
In shading box, stepper motor is connect by transmission shaft with micro-fluidic chip bracket；
The lower section of micro-fluidic chip bracket is arranged in infrared LED；The top of shading box is arranged in fluorescence detection modular assembly.
Further, fluorescence detection modular assembly further includes printed circuit board, and light intensity signal is turned by printed circuit board
It is changed to voltage signal；Gear teeth track is provided on micro-fluidic chip bracket, the transmission gear of transmission shaft is configured to
It is rotated on gear teeth track to control the rotation angle and direction of micro-fluidic chip bracket；It is provided on micro-fluidic chip bracket
Heat transfer hollow out is so that the energy that infrared LED generates can be placed on micro-fluidic chip bracket by heat transfer hollow out arrival
Micro-fluidic chip.
The present invention also provides a kind of production methods of micro-fluidic chip as described above, comprising the following steps:
Step A, the first double faced adhesive tape, the second double faced adhesive tape, peroxidating hydrogen room through-hole double faced adhesive tape, the sample room PCR are made
Through-hole double faced adhesive tape；Chamber cap is extracted using PCR layers of PMMA production, upper cover, lower cover, the extraction chamber RNA and RNA；
Step B, by PCR layer that step A makes, upper cover, lower cover, the extraction chamber RNA and RNA extract chamber cap ethyl alcohol and go from
Sub- water rinses, and dry with nitrogen before assembling；
Step C, after step B, peroxidating hydrogen room, the room PCR, temperature-controlled chamber and the extraction chamber RNA bottom appearance
Face applies MWNT heating layer, and after electroconductive binder is dry, the extraction chamber RNA and PCR room floor are placed on the first double faced adhesive tape；The
The another side of one double faced adhesive tape is pasted onto the upper surface of lower cover.
The invention also includes a kind of method for carrying out RT-PCR using portable infrared isothermal PCR platform as described above, packets
Include following steps:
Sample and RNA Extraction buffer is added into the extraction chamber RNA in step 1；
By stepper motor the lower section to the extraction chamber RNA, heating is arranged in infrared LED by step 2；
Solution in the extraction chamber RNA is cooled to room temperature by step 3；
Step 4, the RNA and waste obtained by externally-applied magnetic field separation and Extraction；
Step 5 removes waste, and washing buffer is added into the RNA that extraction obtains；
Detection buffer is added into the extraction chamber RNA for step 6, mixes, the mixture in the extraction chamber RNA is transferred to PCR
Sample room；
Infrared LED is arranged to the lower section of peroxidating hydrogen room by stepper motor for step 7, heats in hydrogen peroxide room
Hydrogen peroxide generates oxygen, and the mixture in the sample room PCR is pushed to enter the room PCR；
Step 8, PCR reaction and fluorescence signal detection；Wherein infrared LED is arranged under the room PCR by stepper motor
Side, to provide temperature condition for PCR reaction.
Further, step 4 and step 5 repeat 1-2 times.
Further, infrared LED is arranged to the lower section of peroxidating hydrogen room, heating specifically by stepper motor for step 7
Hydrogen peroxide in peroxidating hydrogen room generates oxygen, pushes the mixture in the sample room PCR to enter the room PCR, thus with enzyme and disease
The detection solution of substance mixes.The room PCR includes the first room PCR, the 2nd room PCR and the 3rd room PCR.Enzyme is that real-time fluorescence nucleic acid is permanent
Warm amplification enzyme (SAT enzyme)；The detection solution of pathogen is the inspection of the detection solution, Ureaplasma urealyticum (UU) of chlamydia trachomatis (CT)
Survey the detection solution of solution and genital tract mycoplasma (MG).First room PCR is both needed to be added in the 2nd room PCR and the 3rd room PCR
SAT enzyme, but only that detection one of the solution and the detection solution of MG of the detection solution of CT, UU is added.Preferably, CT
Detection solution, the detection solution of UU and the detection solution of MG be respectively distributed in 3 rooms PCR.
Further, step 7 further includes that parafilm is applied on the room PCR after mixture enters the room PCR, when use
The MWNT heating layer of micro-fluidic chip is heated with melted paraffin film.Wherein, the fusing point of parafilm is 40 DEG C.It is melted using 41 DEG C
Parafilm.Parafilm is used as protection cap, to prevent the degradation of primer and probe in detection solution, to play the micro-fluidic core of storage
Reaction system in the room piece PCR is for the subsequent effect used.
The present invention also provides above-mentioned micro-fluidic chips and portable infrared isothermal PCR platform to detect a variety of sexually transmitted diseases
The application of aspect.
The present invention provides a kind of infrared mediate rna isothermal RT-PCR (IR-MERIT PCR), detection of platform is a variety of spreads through sex intercourse
The mode of disease.
Present invention process is simple, cheap, is suitble to Large scale processes production.
Demand point is tested with microfluidic chip technology or the analytical equipment based on microfluid paper combines, and is substantially increased small
The function of type instrument.These instruments provide portable for consumer, and easy-to-use, the selection of cheap and quick molecular diagnosis can be in
Middle operation, convenient and privacy.
Below with reference to attached drawing, the invention will be further described, with absolutely prove the purpose of the present invention, technical characteristic and
Technical effect.
Detailed description of the invention
Fig. 1 is the explosive view of a specific embodiment of micro-fluidic chip according to the present invention；
Fig. 2 is the perspective view of a specific embodiment of micro-fluidic chip according to the present invention；
Fig. 3 is the schematic diagram of a specific embodiment of portable infrared isothermal PCR platform according to the present invention；
Fig. 4 is the partial exploded view of a specific embodiment of portable infrared isothermal PCR platform according to the present invention；
Fig. 5 is the explosive view of a specific embodiment of fluorescence detection modular assembly according to the present invention；
Fig. 6 is showing for a specific embodiment of fluorescence detection modular assembly and locating module component according to the present invention
It is intended to；
Fig. 7 is the front schematic view of a specific embodiment of micro-fluidic chip bracket according to the present invention；
Fig. 8 is the rear isometric view and transmission shaft of a specific embodiment of micro-fluidic chip bracket according to the present invention
Schematic diagram；
Fig. 9 is the schematic diagram of a specific embodiment of shading box according to the present invention；
Figure 10 is the schematic diagram of a specific embodiment of signal deteching circuit according to the present invention；
Figure 11 is a specific implementation of the method that portable infrared isothermal PCR platform according to the present invention carries out RT-PCR
The flow chart of example；
Figure 12 is fluorescence detection result curve figure.
Specific embodiment
Technical solution of the present invention is further described below by way of specific embodiment.Embodiment below is pair
Further explanation of the invention, is not intended to limit the scope of the invention.
Embodiment 1 designs and prepares micro-fluidic chip
Micro-fluidic chip (as illustrated in fig. 1 and 2) in a specific embodiment include the lower cover 18 being arranged from bottom to top,
MWNT heating layer, PCR room floor, upper cover 4, the extraction chamber RNA 7 and RNA extract chamber cap 6；In PCR room floor setting have family, room include according to
It is secondary by channel attached peroxidating hydrogen room 12, the sample room PCR 11 and the room PCR 9, further include temperature-controlled chamber 10.
MWNT heating layer includes matching respectively with peroxidating hydrogen room 12, temperature-controlled chamber 10, the room PCR 9 and the extraction chamber RNA 7
Corresponding peroxidating hydrogen room MWNT heating element 14, temperature-controlled chamber MWNT heating element 16,15 and of the room PCR MWNT heating element
The extraction chamber RNA MWNT heating element 13.The extraction chamber RNA 7 is arranged on the extraction chamber RNA MWNT heating element 13.
The upper surface of lower cover 18 is provided with the first double faced adhesive tape 17；It is provided on first double faced adhesive tape 17 and is mentioned respectively with RNA
Take room 7, peroxidating hydrogen room 12, the sample room PCR 11, temperature-controlled chamber 10, the corresponding hollow out in the room PCR 9；The upper surface of PCR room floor
It is provided with the second double faced adhesive tape 8；Be provided on second double faced adhesive tape 8 respectively with peroxidating hydrogen room 12, the sample room PCR 11, temperature
Control room 10, the corresponding hollow out in the room PCR 9 and channel；Upper cover 4 be provided with respectively with peroxidating hydrogen room 12 and the sample room PCR 11
Corresponding peroxidating hydrogen room through-hole and the sample room PCR through-hole；Micro-fluidic chip further include peroxidating hydrogen room through-hole double faced adhesive tape 5,
The sample room PCR through-hole double faced adhesive tape 3, peroxidating hydrogen room through-hole lid 1 and the sample room PCR through-hole lid 2.
MWNT heating layer is made of the mixture of MWNT and heat-conductive bonding agent, and wherein MWNT accounts for 4-10 mass parts, thermally conductive viscous
Mixture accounts for 100 mass parts.Preferably, MWNT accounts for 6.6 mass parts, and heat-conductive bonding agent accounts for 100 mass parts.The quantity of the room PCR 9 is 3
It is a, respectively the first room PCR, the 2nd room PCR and the 3rd room PCR.
The drawing of Autodesk CAD design micro-fluidic chip is used first.Then according to drawing, laser engraving machine is used
It is logical that the first double faced adhesive tape, the second double faced adhesive tape, peroxidating hydrogen room through-hole double faced adhesive tape, the sample room PCR are made on double faced adhesive tape
Hole double faced adhesive tape；Made on PMMA using mechanical sculpturing machine PCR layers, upper cover, lower cover, the extraction chamber RNA and RNA extract chamber cap.
Setting is had family on PCR layer, and it further includes temperature that room, which includes passing sequentially through channel attached peroxidating hydrogen room, the sample room PCR and the room PCR,
Spend control room.
The PCR layer of production, upper cover, lower cover, the extraction chamber RNA and RNA are extracted into chamber cap ethyl alcohol and deionized water is rinsed, and
It is dry with nitrogen before assembling.
After nitrogen is dry, apply in peroxidating hydrogen room, the room PCR, temperature-controlled chamber and the outer surface of the extraction chamber RNA bottom
The extraction chamber RNA and PCR room floor are placed on the first double faced adhesive tape after electroconductive binder is dry by MWNT heating layer；First is two-sided
The another side of adhesive tape is pasted onto the upper surface of lower cover.
Assembling process after nitrogen is dry specifically: after removing the lower part plastic lid of the first double faced adhesive tape, by first pair
Face tape-stripping is in the upper surface of lower cover；Then, the mixture of MWNT and heat-conductive bonding agent is applied with 6.6 to 100 mass ratio
Be added on design point, i.e., peroxidating hydrogen room, the room PCR, temperature-controlled chamber and the extraction chamber RNA bottom outer surface.In conductive adhesion
After agent is dry, remove the upper plastic lid of the first double faced adhesive tape, and by RNA extract room floor and PCR room floor to be placed on first two-sided
On adhesive tape layer.The SAT enzyme of 4 μ L is added to each room PCR, and the detection solution of CT, UU and the MG of 0.8 μ L are respectively added to
In first room PCR, the 2nd room PCR and the 3rd room PCR.By the way that the parafilm with 40 DEG C of fusing points is applied to after solution is dry
On PCR reaction chamber, micro-fluidic chip can be stored for using later.Parafilm is used as protection cap, to prevent in detection solution
The degradation of primer and probe.When using micro-fluidic chip, MWNT heating layer is heated to 41 DEG C with melted paraffin film.In paraffin
After film is completely melt, micro-fluidic chip is removed from hot plate and carries out PCR reaction and detection.
Embodiment 2, portable infrared isothermal PCR Platform Designing
In a specific embodiment (as shown in Figures 3 and 4), portable infrared isothermal PCR platform is by three modular assembly groups
At: temperature control modules component, fluorescence detection modular assembly 40 and locating module component.The portable infrared isothermal PCR platform with
Above-mentioned micro-fluidic chip matches.
Thermal module component includes infrared LED 22 (20W, 840nm), thermocouple 21 (TT-K-36-SLE, ETA), thermocouple
Cold junction compensation circuit and LED circuit；Infrared LED 22 is electrically connected with LED circuit；Thermocouple 21 and cold junction compensation circuit electricity
Connection.
Fluorescence detection modular assembly (as shown in Figure 5) include blue-ray LED 49 (1400mW, 460nm, SMB490-1100-01,
Epitex), bandpass filter 45 (532nm, HB-optical), the first camera lens 46 (JK-01-018, φ 6 × 3.5, double R4.5),
Lens bracket 48, the second camera lens 47 (JK-01-018, φ 6 × 3.5, double R4.5), Si-PIN photodiode 44 (BPW21,
Osram), radiator 42, substrate 50, screw 43 and printed circuit board；Fluorescence detection module is arranged to blue led 49 and issues light
By the first camera lens 46 to excite the fluorescence in PCR sample, fluorescence is by the second camera lens 47 by Si- after bandpass filter 45
PIN photodiode 44 captures light intensity signal；Light intensity signal is converted to voltage signal by printed circuit board.Radiator 42
It is fixed on the one side of substrate 50, blue led 49 is fixed on the another side of substrate 50.
Locating module component (as shown in Fig. 3,4,6 and 7) includes micro-fluidic chip bracket 64, transmission shaft 63, stepper motor
62 (28BYJ-48-5V) and shading box 61；Micro-fluidic chip bracket 64 is arranged in shading box 61, and stepper motor 62 passes through transmission
Axis 63 is connect with micro-fluidic chip bracket 64.Gear teeth track 65, the biography of transmission shaft 63 are provided on micro-fluidic chip bracket 64
Moving gear is configured to rotate to control rotation angle and the side of micro-fluidic chip bracket 64 on gear teeth track 65
To；Heat transfer hollow out 66 is provided on micro-fluidic chip bracket 64 so that the energy that infrared LED 22 generates can pass through heat transfer
Hollow out 66 reaches the micro-fluidic chip being placed on micro-fluidic chip bracket 64, to heat to micro-fluidic chip.
The lower section of micro-fluidic chip bracket 64 is arranged in infrared LED 22；Fluorescence detection modular assembly 40 is arranged in shading box
Top.
Micro-fluidic chip bracket, transmission shaft and shading box use black ink 3D printing, to reduce ambient light.It is micro-fluidic
Chip set is in centre with pin hole and in bottom with gear teeth track.Transmission gear will be rotated from stepper motor connection simultaneously
It is transmitted to micro-fluidic chip bracket.It is accurate to control PCR in micro-fluidic chip by adjusting the rotation angle and direction of stepper motor
The position of room.Fluorescence detection modular assembly is fixed by screws at the top of shading box, can be the above easy adjustment micro-fluidic chip
The height of one camera lens and the second camera lens.There are two tracks being pasted onto beside infrared LED on shading box.When shading box is infrared
When right above LED, infrared LED is used to heat the room PCR, or when shading box slides into one side, for adding during preparation step
The hot extraction chamber RNA.
The voltage signal of infrared LED is controlled by pid algorithm, and establishes PWM control to change voltage signal duty ratio.With
The increase of LED voltage signal dutyfactor, heat MWNT using more power.Specifically, PWM control follow P=100 ×
Log (| Tt-St |) %, wherein P is output power percentage, and Tt is target temperature, and St is sample temperature.P maximum value is 100%
(when difference between target and sample is 10 DEG C), and with sample temperature close to target temperature and slowly to reduce power defeated
Out.Blue-ray LED of triggering is per minute to excite the fluorescence in PCR sample, and photodiode capture light intensity signal is simultaneously
Voltage signal is transferred to by printed circuit board (PCB).
Embodiment 3, sample pretreatment and PCR program
As shown in figure 11, the diluted sample of 0.5mL buffer is saved with 0.5mL to mix.By 400 μ L mixing samples and
The extraction chamber RNA is added in the RNA Extraction buffer that 100 μ L contain magnetic bead.Then, micro-fluidic chip is placed on infrared LED, and will
Sample is heated to 60 DEG C and continues 5 minutes, then cools to room temperature 10 minutes.Magnet is placed in below the extraction chamber RNA.In magnetic bead quilt
It is absorbed into after bottom, pipettes waste liquid from extraction chamber.Then, it removes magnet and 1mL washing buffer is added in room with washing
Bead.Step 4 and step 5 are repeated twice.Then, 40 μ L detection buffer is added in the extraction chamber RNA, and sample is shifted
To the sample room PCR.After pasting PCR sample chamber cap, hydrogen peroxide room is heated to generate oxygen, oxygen again arrives sample driving
In three rooms PCR.Then, microfluid is sealed with ARseal adhesive tape (Adhesives Research, Inc., Glen Rock, PA)
The outlet in channel.Finally, micro-fluidic chip is put into chip set, shading box slides above infrared LED, at 42 DEG C into
The amplification of 40 minutes isothermals of row.
Testing result is shown in Figure 12, wherein Figure 12 A show using portable infrared isothermal PCR platform to different FITC concentration into
Row fluorescence detection.When the concentration is low, fluorescence intensity and FITC concentration are linear.It meets relationship F=φ Io (1-
10- ε lc), wherein F is fluorescence intensity, and φ is fluorescence quantum efficiency, and Io is excitating light strength, and ε is molar absorption coefficient, and l is liquid
The thickness in body pond.Figure 12 B is on a micro-fluidic chip while to detect the demonstration of CT, UU and MG.The concentration of each target is distinguished
For 5 × 105 copies/reaction, 500CFU/ reaction and 0 copy/reaction.CFU is Colony Forming Unit, is referred to thin in unit volume
Group's sum of the microorganisms such as bacterium, mould, yeast.When the fluorescence signal of MG remains zero, the fluorescence of CT and UU can detecte
Signal shows do not have cross contamination between chamber.Figure 12 C is the fluorescence signal of various concentration UU.For example, 50CFU/ reaction is this
The detection of the UU of assay kit limits, and presented IR-MERIT PCR (portable infrared isothermal PCR) platform can be used
Success detects, and verifies the ability of platform isothermal amplification detection.Figure 12 D is shown with Healthy Human Serum (rather than sample system
Deionized water during standby) dilution when various concentration UU fluorescence signal；The signal is similar to the signal of Figure 12 C, and display should
Stability and applicability of the platform for clinical application and its applicability to the measurement of other isothermal RNA amplifications.Figure 12 E is shown
The fluorescence signal of various concentration CT.Figure 12 F shows the fluorescence signal of various concentration MG.
Using infrared LED as heat source and MWNTs as a heat transfer medium, explore the feasibility of RNA isothermal duplication.Simultaneously
It also developed portable infrared isothermal PCR (IR MEdiated RNA Isothermal RT-PCR, IR-MERIT PCR) platform
The micro-fluidic chip of prototype and its compatibility is integrated with RNA extraction, Micropump and multi-target detection function integration.The platform can
The hypofluorescence signal (50CFU/UU reaction and 1 × 104 copy/CT reaction) for detecting multicell, for carrying out multiple pathogen simultaneously
Diagnosis；The platform provides measure comparable portability with standard real-time fluorescence nucleic acid constant-temperature amplification detection technique (SAT).
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be within the scope of protection determined by the claims.
Claims (10)
1. a kind of micro-fluidic chip, which is characterized in that including be arranged from bottom to top lower cover, MWNT heating layer, PCR room floor and on
Lid；Be arranged in the PCR room floor and have family, the room include pass sequentially through channel attached peroxidating hydrogen room, the sample room PCR and
The room PCR further includes temperature-controlled chamber.
2. micro-fluidic chip as described in claim 1, which is characterized in that the MWNT heating layer include respectively with the peroxide
Change hydrogen room, the temperature-controlled chamber and the room PCR and matches corresponding peroxidating hydrogen room MWNT heating element, temperature-controlled chamber
MWNT heating element and the room PCR MWNT heating element；
It further include that the extraction chamber RNA and RNA extract chamber cap；The MWNT heating layer further includes corresponding with the extraction chamber the RNA matching
The extraction chamber RNA MWNT heating element；The extraction chamber RNA is arranged on the extraction chamber the RNA MWNT heating element.
3. micro-fluidic chip as claimed in claim 2, which is characterized in that the upper surface of the lower cover is provided with the first double-sided adhesive
Band；Be provided on first double faced adhesive tape respectively with the extraction chamber RNA, the peroxidating hydrogen room, the sample room PCR,
The temperature-controlled chamber, the corresponding hollow out in the room PCR；The upper surface of the PCR room floor is provided with the second double faced adhesive tape；It is described
Be provided on second double faced adhesive tape respectively with the peroxidating hydrogen room, the sample room PCR, the temperature-controlled chamber, the PCR
The corresponding hollow out in room and the channel；The upper cover be provided with respectively with the peroxidating hydrogen room and the sample room pair PCR
The peroxidating hydrogen room through-hole and the sample room PCR through-hole answered；The micro-fluidic chip further include peroxidating hydrogen room through-hole double faced adhesive tape,
The sample room PCR through-hole double faced adhesive tape, peroxidating hydrogen room through-hole lid and the sample room PCR through-hole lid；
The MWNT heating layer is made of the mixture of MWNT and heat-conductive bonding agent, and wherein MWNT accounts for 4-10 mass parts, thermally conductive viscous
Mixture accounts for 100 mass parts；Preferably, MWNT accounts for 6.6 mass parts, and heat-conductive bonding agent accounts for 100 mass parts；Preferably, the room PCR
Quantity be 3.
4. a kind of portable infrared isothermal PCR platform, which is characterized in that with micro-fluidic chip as claimed in claim 4 is mating makes
With.
5. portable infrared isothermal PCR platform as claimed in claim 4, which is characterized in that examined including thermal module component, fluorescence
Survey modular assembly and locating module component；The thermal module component includes infrared LED, thermocouple, cold junction compensation electricity
Road and LED circuit；The infrared LED is electrically connected with the LED circuit；The thermocouple and the cold junction compensation circuit
Electrical connection；
The fluorescence detection modular assembly includes blue-ray LED, bandpass filter, the first camera lens, the second camera lens, Si-PIN photoelectricity two
Pole pipe and radiator；The fluorescence detection module is arranged to the blue led and issues light by first camera lens to excite
Fluorescence in PCR sample, the fluorescence is by second camera lens by the Si-PIN photoelectricity after the bandpass filter
Diode captures light intensity signal；
The locating module includes micro-fluidic chip bracket, transmission shaft, stepper motor and shading box；The micro-fluidic chip bracket
It is arranged in the shading box, the stepper motor is connect by the transmission shaft with the micro-fluidic chip bracket；
The lower section of the micro-fluidic chip bracket is arranged in the infrared LED；The fluorescence detection modular assembly is arranged described
The top of shading box.
6. portable infrared isothermal PCR platform as claimed in claim 5, which is characterized in that the fluorescence detection modular assembly is also
Including printed circuit board, the light intensity signal is converted to voltage signal by the printed circuit board；The micro-fluidic chip
Gear teeth track is provided on bracket, the transmission gear of the transmission shaft is configured to rotate on the gear teeth track
To control the rotation angle and direction of the micro-fluidic chip bracket；Heat transfer hollow out is provided on the micro-fluidic chip bracket
So that the energy that the infrared LED generates can be reached by the heat transfer hollow out and be placed on the micro-fluidic chip bracket
On the micro-fluidic chip.
7. a kind of production method of micro-fluidic chip as claimed in claim 3, which comprises the following steps:
Step A, first double faced adhesive tape, second double faced adhesive tape, the peroxidating hydrogen room through-hole double faced adhesive tape, institute are made
State the sample room PCR through-hole double faced adhesive tape；PCR layers, the upper cover, the lower cover, the extraction chamber RNA are made using PMMA
Chamber cap is extracted with the RNA；
Step B, by the step A production PCR layer, upper cover, lower cover, the extraction chamber RNA and RNA extraction chamber cap ethyl alcohol and go from
Sub- water rinses, and dry with nitrogen before assembling；
Step C, after the described step B, in the peroxidating hydrogen room, the room PCR, the temperature-controlled chamber and the RNA
The outer surface of extraction chamber bottom applies the MWNT heating layer, after the electroconductive binder is dry, by the extraction chamber RNA and
The PCR room floor is placed on first double faced adhesive tape；The another side of first double faced adhesive tape is pasted onto the upper of the lower cover
Surface.
8. a kind of method for carrying out RT-PCR using portable infrared isothermal PCR platform as claimed in claim 6, feature exist
In, comprising the following steps:
Sample and RNA Extraction buffer is added into the extraction chamber RNA in step 1；
By the stepper motor lower section to the extraction chamber RNA, heating is arranged in the infrared LED by step 2；
Solution in the extraction chamber RNA is cooled to room temperature by step 3；
Step 4, the RNA and waste obtained by externally-applied magnetic field separation and Extraction；
Step 5 removes waste, and washing buffer is added into the RNA that extraction obtains；
Detection buffer is added in step 6, the extraction chamber Xiang Suoshu RNA, mixes, the mixture in the extraction chamber RNA is shifted
To the sample room PCR；
The infrared LED is arranged to the lower section of the peroxidating hydrogen room by the stepper motor for step 7, heats the mistake
The hydrogen peroxide aoxidized in hydrogen room generates oxygen, and the mixture in the sample room PCR is pushed to enter the room PCR；
Step 8, PCR reaction and fluorescence signal detection；Wherein the infrared LED is arranged to described by the stepper motor
The lower section of the room PCR, to provide temperature condition for PCR reaction.
9. method according to claim 8, which is characterized in that
The step 7 further includes that parafilm is applied on the room PCR after mixture enters the room PCR, will when use
The MWNT heating layer of the micro-fluidic chip is heated to melt the parafilm.
10. micro-fluidic chip as described in claim 1 and portable infrared isothermal PCR platform as claimed in claim 4 are being examined
Survey the application in terms of a variety of sexually transmitted diseases.
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| CN201910247499.9A CN110004024A (en) | 2019-03-29 | 2019-03-29 | A kind of micro-fluidic chip and portable infrared isothermal PCR platform |
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Application publication date: 20190712 |