CN102788780B - Microfluidic chip for biological chemiluminescence detection and manufacturing method thereof - Google Patents
Microfluidic chip for biological chemiluminescence detection and manufacturing method thereof Download PDFInfo
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Abstract
The invention discloses a microfluidic chip for biological chemiluminescence detection. The chip comprises an interface layer, a transparent layer, a channel layer, a reflecting layer and a fixed layer, which are sequentially arranged from top to bottom. The layers are connected by adhesives and fixed by fasteners; the interface layer is provided with a liquid inlet hole, a liquid outlet hole and an optical fiber interface; the channel layer is provided with a liquid inlet flow channel, a micro mixer, a detecting pool and a waste liquid buffer pool, which are successively communicate with each other; the transparent layer is arranged between the optical fiber interface and the detecting pool; the detecting pool is opened to the lower surface of the channel layer; and the bottom of the detecting pool is connected with the reflecting layer. The invention also discloses a manufacturing method of the microfluidic chip. The microfluidic chip improves reaction luminescence intensity of the detection solution and optical detection efficiency, and has advantages of simple preparation method, convenient operation, fast detection, high sensitivity, and accurate detection result.
Description
Technical field
The present invention relates to biochemistry detection field, relate in particular to a kind of micro-fluidic chip detecting for biochemiluminescence and preparation method thereof.
Background technology
At present, in the research of chemistry and life science, fluorescence (Fluorescence) and chemiluminescence are one of high sensitivity quantitation detection methods being most widely used.Because the photon energy absorption efficiency of fluorophor is high, the excited fluorescence group in molecule can discharge a large amount of luminous energy, produces the light signal of higher-strength, thereby, simple to the pre-service of sample, easy to detect.But, contain complicated background fluorescence group owing to being excited sample itself, can produce non-specific fluorescence excitation, the spectrum of excitation source complexity can disturb the photo-detector of fluorescence to detect, thereby, need to there is the quality of various filtering apparatus guarantee sample measurements.Be different from fluorescence, biochemiluminescence is the autoluminescence that a species specificity biochemical reaction inspires, and does not need exciting of external light source, thereby, have that ground unrest is little, response rapidly and the advantage such as detection threshold is low.Chemiluminescence (Chemiluminescence) be it be mainly according to determinand with there is specific reagent and mix and produce shiner and show existence and the concentration of determinand, bioluminescence (bioluminescence) is to rely on to be present in enzyme in the biosome such as firefly and photobacteria, and energy that catalysis specificity chemical reaction produces is luminous and show existence and the concentration of determinand.Biochemiluminescence detects the characteristic of having utilized just testing concentration and luminous intensity in above-mentioned reaction system to be quantitative relationship under certain condition, determines determinand content by detection system luminous intensity.This has become more conventional high-sensitivity biological chemical analysis method.
Nearly microflow control technique growing up for 20 years,, traceization integrated with it, the advantage such as analysis speed is fast, accuracy is high, be widely used in the fields such as biochemistry detection, medical research, environmental monitoring.Micro-fluidic chip can complete multiple solution operating steps such as sample introduction, mixing, reaction, detection on a slice chip, also can on a slice chip, realize the spatial structure of multiple liquid flow path passages, and the high flux that carries out multiple samples detects.Micro-fluidic chip technology is combined with biochemiluminescence detection technique, and luminescence system is set up in the integrated manipulation that solves the step such as conduction, mixing of plurality of reagents; Micro-fluidic chip technology is combined with photoelectric detecting technology, utilize the detection means such as photodiode, photoelectrical coupler (CCD), photomultiplier to realize stable and high sensitivity original position Photoelectric Detection, to ensureing under the prerequisite of detection sensitivity, improve versatility and convenience that biochemiluminescence detects, reducing reagent consumption and improve detection speed, is important research and the technological development direction of current micro-fluidic chip technology.
At present, the micro-fluidic chip relevant with luminous detection of bibliographical information exists following shortcomings: the complex optical path that 1, relies on microscope stage or lens and optical filter composition: as the disclosed fluidic chip chemiluminescence of Chinese patent that application number is 200910114403.8 is measured the method for materials inside human single blood erythrocyte by mocro and the disclosed fluorescence micro-spectrum detection device of biological chip of Chinese patent and the method for making that application number is 201110153526.X; 2, flow passage structure is single, thereby sample introduction is without fully mixing and cause reaction efficiency lower, cannot reach maximum emission intensity: as the application number disclosed capillary electrophoresis separation of Chinese patent that is 200910154432.7 and the micro-fluidic chip of chemiluminescence detection; 3, light-emitting zone all-transparent, the scattered light of different directions is not fully utilized, and light detection efficiency is low.
Summary of the invention
An object of the present invention is above-mentioned three technical matterss in order to solve at present relevant with luminous detection micro-fluidic chip existence and a kind of micro-fluidic chip detecting for biochemiluminescence is provided, micro-fluidic chip of the present invention improves biochemical reaction luminous intensity by improving solution mixability, improve optical design and improve light detection efficiency, thereby improve simple operation and detection sensitivity that biochemiluminescence detects.
Another object of the present invention is to provide a kind of method for making of the micro-fluidic chip detecting for biochemiluminescence, and chip manufacture method of the present invention is simple, easy to operate.
The present invention realizes the technical scheme that its first technical purpose adopts: a kind of micro-fluidic chip detecting for biochemiluminescence, described chip comprises the interface layer setting gradually from top to bottom, photic zone, channel layer, reflector layer and fixed bed, between described each layer, connect up and down and fix by securing member by bonding agent, described interface layer is provided with inlet opening, fluid hole and optical fiber connect hole, described channel layer is provided with the feed liquor flow channel being communicated with successively, micro-mixer, detection cell, waste liquid Buffer Pool, described feed liquor flow channel is communicated with inlet opening, waste liquid Buffer Pool is communicated with fluid hole, described optical fiber connects hole and is arranged on the top of detection cell, optical fiber connects between hole and detection cell and is separated with photic zone, the top of detection cell and euphotic lower surface join, the bottom of detection cell is connected with reflector layer.This micro-fluidic chip is by setting gradually interface layer, photic zone, channel layer, reflector layer and fixed bed, and inlet opening, feed liquor flow channel be set on chip, micro-mixer, detection cell, waste liquid buffer zone, fluid hole, optical fiber connects hole, said structure makes sample introduction enter feed liquor flow channel from inlet opening, and then by fully being mixed after micro-mixer, mixed sample introduction luminescence-producing reaction efficiency improves greatly.Optical fiber connects hole and is arranged on the top of detection cell, optical fiber connects between hole and detection cell and is separated with photic zone, the top of detection cell and euphotic lower surface join, the bottom of detection cell is connected with reflector layer, photic zone is arranged on optical fiber, and to connect the optical fiber that can avoid optical fiber and solution directly to contact between hole and detection cell causing and contaminated aqueous solution and stream unsmooth, ensures again the good light permeability between detection cell and optical fiber simultaneously.Inlet opening and fluid hole are used for inserting feed liquor tubule and fluid tubule, connect hole, for inserting optical fiber facing to the optical fiber at detection cell place.This micro-fluidic chip does not need to rely on the complex optical path being made up of microscope stage or lens and optical filter, do not need external excitation light source, do not need to apply external high pressure electric field driven stream, adopt micro-mixer, design specific flow passage structure, solution, after sample introduction and mixer circulation passage, just can reach maximum emission intensity.Light-emitting zone adopts reflective structure, and directive optical fiber connects the scattered light of different directions beyond hole and also can be fully utilized, and has improved light detection efficiency.
As preferably, described bonding agent comprises dimethyl silicone polymer monomer and hardening agent, and described dimethyl silicone polymer monomer and the volume ratio of hardening agent are 3～20:1.Dimethyl silicone polymer monomer (PDMS) and hardening agent are commercial product, purchased from Dow Corning.
As preferably, described interface layer is provided with several inlet openings, and the aperture of described inlet opening is 1mm～2mm, is provided with a fluid hole and an optical fiber connects hole on interface layer, and the aperture of described fluid hole is 1.5mm～2.5mm.The quantity of inlet opening can be set to multiple as required, to meet the needs of different testing goals.The size of the feed liquor flow channel tube of peristaltic pump and feed liquor aperture adaptation, the size of fluid flow channel tube and fluid aperture adaptation, the aperture that described optical fiber connects hole is 1.5mm～5.0mm.
As preferably, channel layer is provided with several feed liquor flow channels consistent with feed liquor number of perforations on interface layer, and each feed liquor flow channel is communicated with micro-mixer respectively, and the width of described feed liquor flow channel is 1.5mm～2.5mm, and the degree of depth is 1mm～2mm.On channel layer, be provided with several feed liquor flow channels consistent with feed liquor number of perforations on interface layer, such structure is fully mixed the different sample introductions on multiple passages in micro-mixer, thereby improves reaction velocity and efficiency.
As preferably, described micro-mixer is fold-line-shaped raceway groove, establish in square to intersect in linear raceway groove or rectangle and establish channel shape structure side by side, and width and the degree of depth of micro-mixer raceway groove are respectively 0.5mm～1.5mm.Micro-mixer is fold-line-shaped raceway groove, establish in square to intersect in linear raceway groove or rectangle and establish channel shape structure side by side, can make multiple solution that fully mixing occurs in passing through fast.Certainly micro-mixer can also adopt other structures such as S shape, as long as the object that can meet the technical program is all within the protection domain of this programme.
As preferably, described detection cell is circle hole shape, and detection cell bottom be reflector layer, and the aperture (d) of detection cell is satisfied with channel layer thickness (h), optical fiber core diameter () and numerical aperture (α):
, the volume of described detection cell is less than the volume with waste liquid Buffer Pool.For the abundant coupled into optical fibres of light that ensures that detection cell sends, detection cell bottom is reflector layer, the diameter (d) of detection cell should match with channel layer thickness (h), light core diameter () and numerical aperture (α), meets:
, described numerical aperture (α) is determined by fiber optic materials and optical fiber core diameter, detection cell front end connection waste liquid Buffer Pool, and the volume of detection cell is less than waste liquid Buffer Pool volume, is to affect detection effect in order to prevent waste liquid from blowing back into detection cell.
As preferably, the thickness of interface layer is 2mm～7.5mm, and euphotic thickness is 0.2mm～0.8mm, and the thickness of channel layer is 2.5mm～3.5mm, and the thickness of reflector layer is 0.8mm～1.5mm, and the thickness of fixed bed is 2mm～7.5mm.The thickness of interface layer is preferably 2mm～7.5mm, the one, can select polymethylmethacrylate (PMMA) the material substrate of thickness 2mm～7.5mm as interface layer, selection is convenient, the 2nd, the degree of depth that such structure makes inlet opening, fluid hole and optical fiber connect hole is set to the best, facilitate inflow and the outflow of sample introduction, and after optical fiber insertion, connecting length reaches best.Euphotic thickness is preferably 0.2mm～0.8mm, can select polyethylene terephthalate (PET) film of thickness 0.2mm～0.8mm as photic zone, the optical fiber that the photic zone that 0.2mm～0.8mm is set in the middle of interface layer and channel layer can be avoided optical fiber and solution directly to contact causing and contaminated aqueous solution and stream are unsmooth, and the while ensures again the good light permeability between detection cell and optical fiber.The thickness of channel layer is preferably 2.5mm～3.5mm, can adopt the PMMA material substrate of thickness 2.5mm～3.5mm as channel layer, the feed liquor flow channel, micro-mixer, detection cell, the waste liquid Buffer Pool that are communicated with successively that are applicable to width and the degree of depth can be set like this on channel layer, each channel width and the degree of depth arrange respectively 1.5mm～2.5mm and 1mm～2mm, object is to maintain a suitable flow path resistance, ensure feed liquor unobstructed operation under peristaltic pump drives, solution can not be detained of a specified duration in passage simultaneously; Feed liquor flow channel length is 5mm～10mm, and front end connects micro-mixer.The thickness of reflector layer is preferably 0.2mm～3.5mm, selects the minute surface of 0.2mm～1.5mm as reflector layer, and because biochemiluminescence is unoriented, minute surface can reflex to fiber port by a big chunk scattered light, has improved light detection efficiency.Fixed bed thickness is preferably 2mm～7.5mm, can select like this thickness be 2mm～7.5mm PMMA material substrate as fixed bed, be arranged on below reflector layer, be used for reinforcing chip.
The present invention realizes the technical scheme that its another technical purpose adopts: a kind of method for making of the micro-fluidic chip detecting for biochemiluminescence, and described method for making is following steps,
1) use laser marking machine on interface layer, to carve inlet opening, fluid hole, optical fiber and connect hole, on photic zone, carve inlet opening, fluid hole, on channel layer, carve feed liquor flow channel, micro-mixer, detection cell, waste liquid Buffer Pool, on interface layer, photic zone, channel layer and fixed bed, carve respectively fastener hole; The aperture of described inlet opening is 1mm～2mm, the aperture of fluid hole is 1.5mm～2.5mm, the aperture that described optical fiber connects hole is 1.5mm～5.0mm, the width of feed liquor flow channel is 1.5mm～2.5mm, the degree of depth is 1mm～2mm, width and the degree of depth of micro-mixer raceway groove are respectively 0.5mm～1mm, described detection cell circle hole shape, and the aperture (d) of detection cell meets with channel layer thickness (h), optical fiber core diameter () and numerical aperture (α):
, described numerical aperture (α) is by fiber optic materials and the decision of optical fiber core diameter, and the volume of described detection cell is less than the volume with waste liquid Buffer Pool;
2) preparation of bonding agent: be that 3～20:1 is mixed by dimethyl silicone polymer monomer and hardening agent according to volume ratio, till leaving standstill bubble collapse, described dimethyl silicone polymer monomer and hardening agent are commercial product;
3) use the bonding agent of preparation in step 2 surface of contact between applying each layer, and interface layer, photic zone, channel layer, luminescent layer and fixed bed are stacked from top to bottom, fastening with securing member at corner location, be then placed in 70～90 DEG C of baking ovens and solidify the micro-fluidic chip of making for 60～120 minutes for biochemiluminescence detection.
Compared with prior art, the invention has the beneficial effects as follows:
(1) detect solution and occur to mix fast and fully through micro-mixer, improved reaction luminous intensity;
(2) the reflector layer minute surface of luminescence detecting pool below can reduce scattered light loss, has improved light detection efficiency;
(3) luminescence detecting pool size, according to optical fiber core diameter and numerical aperture optimal design, reaches preferably coupling efficiency, has removed the light path devices such as complicated lens from simultaneously;
(4) this facture of microchip method is simple, easy to operate, and detection speed is fast, and detection sensitivity is high.
Brief description of the drawings
Fig. 1 is a kind of structural representation of the present invention for the micro-fluidic chip of biochemiluminescence detection;
Fig. 2 is the A-A diagrammatic cross-section of Fig. 1;
Fig. 3 is the photon counting response curve figure of the test result of embodiment;
Fig. 4 is the another kind of structural representation of the present invention for the micro-fluidic chip of biochemiluminescence detection;
Fig. 5 is the present invention's the third structural representation for the micro-fluidic chip of biochemiluminescence detection;
In figure: 1, interface layer, 11, inlet opening, 12, fluid hole, 13, optical fiber connects hole, 2, photic zone, 3, channel layer, 31, feed liquor flow channel, 32, micro-mixer, 33, detection cell, 34, waste liquid Buffer Pool, 4, reflector layer, 5, fixed bed, 6, securing member.
Embodiment
Below by specific embodiment by reference to the accompanying drawings technical scheme of the present invention being further described in detail, in embodiment, needed raw material is all commercially available or adopt known method synthetic.
Embodiment 1:
As shown in Figure 1, a kind of micro-fluidic chip detecting for biochemiluminescence, chip comprises the interface layer 1 setting gradually from top to bottom, photic zone 2, channel layer 3, reflector layer 4 and fixed bed 5, between each layer, bond together up and down by bonding agent and pass through quadruplet securing member 6 and fix, interface layer 1 is PMMA material substrate, the thickness of interface layer 1 is 4mm, on interface layer 1, be provided with the inlet opening 11 of 2 diameter 1mm, the fluid hole 12 of a diameter 1.5mm, the optical fiber of a diameter 2.25mm connects hole 13, channel layer 3 adopts the PMMA material substrate of thickness 3mm, channel layer 3 is provided with two feed liquor flow channels 31 that are communicated with successively, a micro-mixer 32, a detection cell 33, a waste liquid Buffer Pool 34, each channel width is 2mm, degree of depth 1.5mm, the length of feed liquor flow channel 31 is 5mm, and front end connects micro-mixer 32, micro-mixer 32 is fold-line-shaped, and single folding length is 10mm, and channel width is 0.5mm, and the degree of depth is 0.6mm, and channel pitch is 1mm, and micro-mixer 32 front ends connect detection cell 33, and between micro-mixer 32 and detection cell 33, connecting length is 1.5mm, detection cell 33 is that (aperture d is according to formula for the circular hole of diameter 4mm
calculate, channel layer thickness 3mm in the present embodiment, selected optical fiber core diameter is that 1mm and numerical aperture are 0.37), bottom is reflector layer 4, it is 1mm that reflector layer 4 adopts thickness, length and width are of a size of the minute surface of 24mmx24mm, and the front end of detection cell 33 connects waste liquid Buffer Pool 34, and connecting length is 2mm, the diameter of waste liquid Buffer Pool 34 is 6mm, and the degree of depth is 2.7mm.
Feed liquor flow channel 31 is communicated with inlet opening 11, waste liquid Buffer Pool 34 is communicated with fluid hole 12, optical fiber connects hole 13 and is arranged on the top of detection cell 33, optical fiber connects between hole 13 and detection cell 33 and is separated with photic zone 2, it is the PET film of 0.2mm that photic zone 2 adopts thickness,, the lower surface of the top of detection cell 33 and photic zone 2 joins, and the bottom of detection cell 33 is connected with reflector layer 4.The PMMA material substrate that fixed bed 5 is 7mm, length and width are of a size of 50mm × 50mm, have the fastener hole of diameter 5mm at corner location.Interface layer 1, photic zone 2, channel layer 3, reflector layer 4 and fixed bed 5 are tightened to one by securing member 6, is configured for the micro-fluidic chip (see figure 2) that biochemiluminescence detects.
In the present embodiment, the making flow process of micro-fluidic chip is as follows:
1) use Chinese workers board laser marking machine on the interface layer 1 for 4mm PMMA material, to carve inlet opening 11, fluid hole 12, optical fiber at thickness and connect hole 13, on the PET film photic zone 2 that is 0.2mm at thickness, carve inlet opening 11, fluid hole 12, be to carve micro-mixer 32, detection cell 33, waste liquid Buffer Pool 34 on the channel layer 3 of 3mmPMMA material at thickness, the fixed bed 5 of the PMMA material that is 2mm at interface layer 1, photic zone 2, channel layer 3, thickness carves respectively fastener hole, is conveniently fixed with securing member; The aperture of inlet opening 11 is 1mm, the aperture of fluid hole 12 is 1.5mm, and the width of feed liquor flow channel 31 is 2mm, and the degree of depth is 1.5mm, width and the degree of depth of micro-mixer 32 raceway grooves are respectively 0.5mm and 0.6mm, and detection cell 33 is that (aperture d is according to formula for the circular hole of diameter 4mm
calculate, channel layer thickness 3mm in the present embodiment, selected optical fiber core diameter is that 1mm and numerical aperture are 0.37), the volume of detection cell 33 is less than the volume with waste liquid Buffer Pool 34;
2) preparation of bonding agent: be that 10:1 is mixed by dimethyl silicone polymer monomer and hardening agent according to volume ratio, till leaving standstill bubble collapse, described dimethyl silicone polymer monomer and hardening agent are commercial product; Sylgard 184 silicone elastomer PDMS matrix and hardening agent are mixed and stirred for the ratio of 10:1 by volume, in fuming cupboard, leave standstill to till bubble collapse, as bonding agent;
3) use the bonding agent of preparation in step 2 surface of contact between applying each layer, and interface layer 1, photic zone 2, channel layer 3, luminescent layer 4 and fixed bed 5 are stacked from top to bottom, fastening with securing member 6 at corner location, be then placed in 80 DEG C of baking ovens and solidify the micro-fluidic chip of making for 90 minutes for biochemiluminescence detection.
Embodiment 2:
In the embodiment shown in fig. 4, a kind of micro-fluidic chip detecting for biochemiluminescence, chip comprises the interface layer 1 setting gradually from top to bottom, photic zone 2, channel layer 3, reflector layer 4 and fixed bed 5, between each layer, bond together up and down by bonding agent and pass through quadruplet securing member 6 and fix, interface layer 1 is PMMA material substrate, the thickness of interface layer 1 is 3.5mm, on interface layer 1, be provided with the inlet opening 11 of 3 diameter 1.5mm, the fluid hole 12 of a diameter 2mm, the optical fiber of a diameter 1.5mm connects hole 13, channel layer 3 adopts the PMMA material substrate of thickness 2.5mm, channel layer 3 is provided with 3 feed liquor flow channels 31 that are communicated with successively, a micro-mixer 32, a detection cell 33, a waste liquid Buffer Pool 34, each channel width is 1mm, degree of depth 1mm, the length of feed liquor flow channel 31 is 7.5mm, and front end connects micro-mixer 32, micro-mixer 32 is for establishing the raceway groove of intersection linear structure in square, the raceway groove wall scroll length of intersecting is 10mm, and channel width is 1mm, and the degree of depth is 0.5mm, and micro-mixer 32 front ends connect detection cells 33, and between micro-mixer 32 and detection cell 33, connecting length is 0.5mm, the circular hole (in the present embodiment, selected optical fiber core diameter is that 0.8mm and numerical aperture are 0.37) that detection cell 33 is diameter 3mm, bottom is reflector layer 4, it is 0.8mm that reflector layer 4 adopts thickness, length and width are of a size of the minute surface of 24mmx24mm, the front end of detection cell 33 connects waste liquid Buffer Pool 34, and connecting length is 2mm, the diameter of waste liquid Buffer Pool 34 is 5mm, and the degree of depth is 2.5mm.
Feed liquor flow channel 31 is communicated with inlet opening 11, waste liquid Buffer Pool 34 is communicated with fluid hole 12, optical fiber connects hole 13 and is arranged on the top of detection cell 33, optical fiber connects between hole 13 and detection cell 33 and is separated with photic zone 2, it is the PET film of 0.3mm that photic zone 2 adopts thickness, detection cell 33 is offered to the lower surface of channel layer 3, and the bottom of detection cell 33 is connected with reflector layer 4.The PMMA material substrate that fixed bed 5 is 3mm, length and width are of a size of 50mm × 50mm, have the fastener hole of diameter 5mm at corner location.Interface layer 1, photic zone 2, channel layer 3, reflector layer 4 and fixed bed 5 are tightened to one by securing member 6, forms micro-fluidic chip.
In the present embodiment, the making flow process of micro-fluidic chip is as follows:
1) use Chinese workers board laser marking machine on the interface layer 1 for 3.5mm PMMA material, to carve inlet opening 11, fluid hole 12, optical fiber at thickness and connect hole 13, on the PET film photic zone 2 that is 0.3mm at thickness, carve inlet opening 11, fluid hole 12, be to carve micro-mixer 32, detection cell 33, waste liquid Buffer Pool 34 on the channel layer 3 of 2.5mmPMMA material at thickness, the fixed bed 5 of the PMMA material that is 3mm at interface layer 1, photic zone 2, channel layer 3, thickness carves respectively fastener hole, is conveniently fixed with securing member; The aperture of inlet opening 11 is 1.5mm, and the aperture of fluid hole 12 is 2mm, and the width of feed liquor flow channel 31 is 1mm, and the degree of depth is 1mm, and width and the degree of depth of micro-mixer 32 raceway grooves are respectively 1mm and 0.5mm, and detection cell 33 is that (aperture d is according to formula for the circular hole of diameter 3mm
calculate, channel layer thickness 2.5mm in the present embodiment, selected optical fiber core diameter is that 0.8mm and numerical aperture are 0.37), the volume of detection cell 33 is less than the volume with waste liquid Buffer Pool 34;
2) preparation of bonding agent: be that 3:1 is mixed by dimethyl silicone polymer monomer and hardening agent according to volume ratio, till leaving standstill bubble collapse, described dimethyl silicone polymer monomer and hardening agent are commercial product; Sylgard 184 silicone elastomer PDMS matrix and hardening agent are mixed and stirred for the ratio of 3:1 by volume, in fuming cupboard, leave standstill to till bubble collapse, as bonding agent;
3) use the bonding agent of preparation in step 2 surface of contact between applying each layer, and interface layer 1, photic zone 2, channel layer 3, luminescent layer 4 and fixed bed 5 are stacked from top to bottom, fastening with securing member 6 at corner location, be then placed in 70 DEG C of baking ovens and solidify the micro-fluidic chip of making for 60 minutes for biochemiluminescence detection.
Embodiment 3:
In Fig. 5 embodiment, a kind of micro-fluidic chip detecting for biochemiluminescence, chip comprises the interface layer 1 setting gradually from top to bottom, photic zone 2, channel layer 3, reflector layer 4 and fixed bed 5, between each layer, bond together up and down by bonding agent and pass through quadruplet securing member 6 and fix, interface layer 1 is PMMA material substrate, the thickness of interface layer 1 is 4.5mm, on interface layer 1, be provided with the inlet opening 11 of 4 diameter 2mm, the fluid hole 12 of a diameter 2.5mm, the optical fiber of a diameter 5mm connects hole 13, channel layer 3 adopts the PMMA material substrate of thickness 3.5mm, channel layer 3 is provided with 4 feed liquor flow channels 31 that are communicated with successively, a micro-mixer 32, a detection cell 33, a waste liquid Buffer Pool 34, each channel width is 1.5mm, degree of depth 2mm, the length of feed liquor flow channel 31 is 10mm, and front end connects micro-mixer 32, micro-mixer 32 is established channel shape structure side by side in rectangle, and raceway groove wall scroll length is 10mm side by side, and channel width is 0.8mm, the degree of depth is 1mm, channel pitch is 1mm, and micro-mixer 32 front ends connect detection cell 33, and between micro-mixer 32 and detection cell 33, connecting length is 2mm, the circular hole (in the present embodiment, selected optical fiber core diameter is that 1mm and numerical aperture are 0.5) that detection cell 33 is diameter 5.5mm, bottom is reflector layer 4, it is 1.5mm that reflector layer 4 adopts thickness, length and width are of a size of the minute surface of 24mmx24mm, the front end of detection cell 33 connects waste liquid Buffer Pool 34, and connecting length is 2mm, the diameter of waste liquid Buffer Pool 34 is 6.5mm, and the degree of depth is 3mm.
Feed liquor flow channel 31 is communicated with inlet opening 11, waste liquid Buffer Pool 34 is communicated with fluid hole 12, optical fiber connects hole 13 and is arranged on the top of detection cell 33, optical fiber connects between hole 13 and detection cell 33 and is separated with photic zone 2, it is the PET film of 0.4mm that photic zone 2 adopts thickness, detection cell 33 is offered to the lower surface of channel layer 3, and the bottom of detection cell 33 is connected with reflector layer 4.The PMMA material substrate that fixed bed 5 is 4mm, length and width are of a size of 50mm × 50mm, have the fastener hole of diameter 5mm at corner location.Interface layer 1, photic zone 2, channel layer 3, reflector layer 4 and fixed bed 5 are tightened to one by securing member 6, forms micro-fluidic chip.
In the present embodiment, the making flow process of micro-fluidic chip is as follows:
1) use Chinese workers board laser marking machine on the interface layer 1 for 4.5mm PMMA material, to carve inlet opening 11, fluid hole 12, optical fiber at thickness and connect hole 13, on the PET film photic zone 2 that is 0.4mm at thickness, carve inlet opening 11, fluid hole 12, be to carve micro-mixer 32, detection cell 33, waste liquid Buffer Pool 34 on the channel layer 3 of 3.5mmPMMA material at thickness, the fixed bed 5 of the PMMA material that is 4mm at interface layer 1, photic zone 2, channel layer 3, thickness carves respectively fastener hole, is conveniently fixed with securing member; The aperture of inlet opening 11 is 2mm, the aperture of fluid hole 12 is 2.5mm, the width of feed liquor flow channel 31 is 1.5mm, the degree of depth is 2mm, width and the degree of depth of micro-mixer 32 raceway grooves are respectively 0.8mm and 1mm, the circular hole (in the present embodiment, selected optical fiber core diameter is that 1mm and numerical aperture are 0.5) that detection cell 33 is diameter 5.5mm, the volume of detection cell 33 is less than the volume with waste liquid Buffer Pool 34;
2) preparation of bonding agent: be that 20:1 is mixed by dimethyl silicone polymer monomer and hardening agent according to volume ratio, till leaving standstill bubble collapse, described dimethyl silicone polymer monomer and hardening agent are commercial product; Sylgard 184 silicone elastomer PDMS matrix and hardening agent are mixed and stirred for the ratio of 20:1 by volume, in fuming cupboard, leave standstill to till bubble collapse, as bonding agent;
3) use the bonding agent of preparation in step 2 surface of contact between applying each layer, and interface layer 1, photic zone 2, channel layer 3, luminescent layer 4 and fixed bed 5 are stacked from top to bottom, fastening with securing member 6 at corner location, be then placed in 90 DEG C of baking ovens and solidify the micro-fluidic chip of making for 120 minutes for biochemiluminescence detection.
As follows for the flow process of ATP bioluminescent detection arbitrary micro-fluidic chip detecting for biochemiluminescence making in embodiment 1, embodiment 2 or embodiment 3:
1) inlet opening on the micro-fluidic chip interface layer 1 detecting for biochemiluminescence 11 is connected with peristaltic pump (model LongerPump BT100-2J) by feed liquor flow channel tube, fluid hole 12 is connected with fluid flow channel tube, detection fiber one end is inserted optical fiber and is connect hole 13, the other end is connected to photon counter (IDQ id100-MMF50-ULN), and photon counter is connected on computing machine; The number of peristaltic pump is consistent with the number of inlet opening; Inlet opening 11 is connected with fluid flow channel tube with feed liquor flow channel tube, fluid hole 12, detection fiber and optical fiber connect hole 13 and uses respectively bonding agent to solidify, and bonding agent is selected from ethene-vinyl acetate hot melt adhesive.
2) wash down stream, use successively the aqueous slkali (can select NaOH solution, KOH solution) of 0.1～1mol/L, 0.1～1mol/L acid solution (can be selected HCl solution, H
2sO
4solution), the aqueous slkali of 0.1～1mol/L, rinses liquid flow path passage and the solution pool of micro-fluidic chip with the flow velocity of 1uL/s～10uL/s,, then use ultrapure water, rinse liquid flow path passage and the solution pool of micro-fluidic chip with the flow velocity of 1uL/s～10uL/s;
3) with the flow velocity of 0.1uL/s～3uL/s in the interior luminous detection liquid of 15uL～100uL luciferase and the 15uL～100uL sample liquid containing ATP that passes into respectively of feed liquor flow channel 11, can record the response curve (Fig. 3-1) of bioluminescence reaction.
Comparative example 1
The micro-fluidic chip detecting for biochemiluminescence made from reference to embodiment 1 ~ 3, difference is not containing reflector layer.Flow process with reference to the micro-fluidic chip detecting for biochemiluminescence for ATP bioluminescent detection, records response curve (Fig. 3-2).
Comparative example 2
The micro-fluidic chip detecting for biochemiluminescence made from reference to embodiment 1 ~ 3, difference is on channel layer that, without micro-mixer, feed liquor flow channel directly imports detection cell.Flow process with reference to the micro-fluidic chip detecting for biochemiluminescence for ATP bioluminescent detection, records response curve (Fig. 3-3).
Comparative example 3
The micro-fluidic chip detecting for biochemiluminescence made from reference to embodiment 1 ~ 3, difference is not containing reflector layer, and on channel layer without micro-mixer, feed liquor flow channel directly imports detection cell.Flow process with reference to the micro-fluidic chip detecting for biochemiluminescence for ATP bioluminescent detection, records response curve (Fig. 3-4).
Above-described embodiment is explanation instead of the restriction to technical solution of the present invention just, any substituting all within the protection domain of technical solution of the present invention that those of ordinary skill is done on the basis of the technical program in the art.
Claims (6)
1. the micro-fluidic chip detecting for biochemiluminescence, it is characterized in that: described chip comprises the interface layer (1) setting gradually from top to bottom, photic zone (2), channel layer (3), reflector layer (4) and fixed bed (5), between described each layer, connect up and down by bonding agent and pass through securing member (6) and fix, described interface layer (1) is provided with inlet opening (11), fluid hole (12) and optical fiber connect hole (13), described channel layer (3) is provided with the feed liquor flow channel (31) being communicated with successively, micro-mixer (32), detection cell (33), waste liquid Buffer Pool (34), described feed liquor flow channel (31) is communicated with inlet opening (11), waste liquid Buffer Pool (34) is communicated with fluid hole (12), described optical fiber connects hole (13) and is arranged on the top of detection cell (33), optical fiber connects between hole (13) and detection cell (33) and is separated with photic zone (2), the lower surface of the top of detection cell (33) and photic zone (2) joins, the bottom of detection cell (33) is connected with reflector layer (4), described micro-mixer (32) is fold-line-shaped raceway groove, in square, establish in the linear raceway groove of intersection or rectangle and establish channel shape structure side by side, width and the degree of depth of micro-mixer (32) raceway groove are respectively 0.5mm～1.5mm, described detection cell (33) is circle hole shape, detection cell (33) bottom is reflector layer (4), the aperture d of detection cell (33) and channel layer thickness h, optical fiber core diameter and numerical aperture α meet:
, the volume of described detection cell (33) is less than the volume of waste liquid Buffer Pool (34).
2. a kind of micro-fluidic chip detecting for biochemiluminescence according to claim 1, it is characterized in that: described bonding agent comprises dimethyl silicone polymer monomer and hardening agent, described dimethyl silicone polymer monomer and the volume ratio of hardening agent are 3～20:1.
3. a kind of micro-fluidic chip detecting for biochemiluminescence according to claim 1, it is characterized in that: described interface layer (1) is provided with several inlet openings (11), the aperture of described inlet opening (11) is 1mm～2mm, on interface layer (1), be provided with a fluid hole (12) and an optical fiber connects hole (13), the aperture of described fluid hole (12) is 1.5mm～2.5mm, and the aperture that described optical fiber connects hole (13) is 1.5mm～5.0mm.
4. according to a kind of micro-fluidic chip detecting for biochemiluminescence described in claim 1 or 3, it is characterized in that: channel layer (3) is provided with several feed liquor flow channels (31) consistent with upper inlet opening (11) number of interface layer (1), each feed liquor flow channel (31) is communicated with micro-mixer (32) respectively, the width of described feed liquor flow channel (31) is 1.5mm～2.5mm, and the degree of depth is 1mm～2mm.
5. according to a kind of micro-fluidic chip detecting for biochemiluminescence described in claim 1 or 3, it is characterized in that: the thickness of interface layer (1) is 2mm～7.5mm, the thickness of photic zone (2) is 0.2mm～0.8mm, the thickness of channel layer (3) is 2.5mm～3.5mm, the thickness of reflector layer (4) is 0.2mm～3.5mm, and the thickness of fixed bed (5) is 2mm～7.5mm.
6. a method for making for the micro-fluidic chip detecting for biochemiluminescence as claimed in claim 1, is characterized in that: described method for making is following steps,
1) use laser marking machine on interface layer (1), to carve inlet opening (11), fluid hole (12), optical fiber and connect hole (13), on photic zone (2), carve inlet opening (11), fluid hole (12), on channel layer (3), carve feed liquor flow channel (31), micro-mixer (32), detection cell (33), waste liquid Buffer Pool (34), on interface layer (1), photic zone (2), channel layer (3) and fixed bed (5), carve respectively fastener hole; The aperture of described inlet opening (11) is 1mm～2mm, the aperture of fluid hole (12) is 1.5mm～2.5mm, the width of feed liquor flow channel (31) is 1.5mm～2.5mm, the degree of depth is 1mm～2mm, width and the degree of depth of micro-mixer (32) raceway groove are respectively 0.5mm～1.5mm, described detection cell (33) is circle hole shape, and the aperture d of detection cell (33) and channel layer thickness h, optical fiber core diameter and numerical aperture α meet:
, described numerical aperture α is by fiber optic materials and the decision of optical fiber core diameter, and the volume of described detection cell (33) is less than the volume of waste liquid Buffer Pool (34);
2) preparation of bonding agent: be that 3～20:1 is mixed by dimethyl silicone polymer monomer and hardening agent according to volume ratio, till leaving standstill bubble collapse, described dimethyl silicone polymer monomer and hardening agent are commercial product;
3) use the bonding agent of preparation in step 2 surface of contact between applying each layer, and interface layer (1), photic zone (2), channel layer (3), reflector layer (4) and fixed bed (5) are stacked from top to bottom, fastening at securing member for corner location (6), be then placed in 70～90 DEG C of baking ovens and solidify the micro-fluidic chip of making for 60～120 minutes for biochemiluminescence detection.
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| CN105665042B (en) * | 2015-01-20 | 2017-06-27 | 天津农学院 | The application method of micro-fluidic chip |
| CN104668003B (en) * | 2015-01-20 | 2016-06-29 | 天津农学院 | Micro-fluidic chip |
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