CN102788780A - 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 the biochemistry detection field, relate in particular to a kind of micro-fluidic chip that is used for the biochemiluminescence detection and preparation method thereof.
Background technology
At present, in the research of chemistry and life science, fluorescence (Fluorescence) and chemiluminescence are to use one of high sensitivity quantitation detection method the most widely.Because the photon energy absorption efficiency of fluorophor is high, the excited fluorescence group in the molecule can discharge a large amount of luminous energy, produces the light signal of higher-strength, thereby, simple to The pretreatment, easy to detect.But, contain the complicated background fluorophor owing to be excited sample itself, can produce non-specific fluorescence excitation, the complicated spectrum of excitation source can disturb the photo-detector of fluorescence to detect, thereby, need the quality that various filtering apparatus could guarantee sample measurement.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 advantage such as detection threshold is low.Chemiluminescence (Chemiluminescence) be it mainly be according to determinand with have specific reagent and mix existence and the concentration that produces shiner and show determinand; Bioluminescence (bioluminescence) then is to rely on the enzyme be present in the biosomes such as firefly and photobacteria, and the energy of catalysis specificity chemical reaction generation is luminous and show the existence and the concentration of determinand.Biochemiluminescence detects the characteristic utilized testing concentration and luminous intensity in the above-mentioned reaction system to be quantitative relationship under certain condition just, confirms determinand content through the detection architecture luminous intensity.This has become high-sensitivity biological chemical analysis method commonly used.
Nearly microflow control technique that grew up in 20 years, advantage such as, traceization integrated with it, analysis speed are fast, accuracy height is widely used in fields such as biochemistry detection, medical research, environmental monitoring.Micro-fluidic chip can be accomplished a plurality of 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 a plurality of liquid flow path passages, carries out the high throughput testing of a plurality of samples.The micro-fluidic chip technology is combined with the biochemiluminescence detection technique, and the conduction of solution plurality of reagents, integrated the controlling of mixing and other steps are set up luminescence system; The micro-fluidic chip technology is combined with photoelectric detecting technology; Utilize detection means such as photodiode, photoelectrical coupler (CCD), photomultiplier to realize stable and high sensitivity original position Photoelectric Detection; In the hope of under the prerequisite that guarantees detection sensitivity; Improving versatility and convenience that biochemiluminescence detects, reduce reagent consumption and improve detection speed, is the research and the technological development direction of present micro-fluidic chip technology.
At present, the micro-fluidic chip relevant with luminous detection of bibliographical information exists following shortcomings: 1, rely on the complex optical path that microscope stage or lens and optical filter are formed: like application number is that 200910114403.8 the disclosed fluidic chip chemiluminescence of Chinese patent is measured the method for materials inside human single blood erythrocyte by mocro and Chinese patent disclosed fluorescence biosensor chip low-light spectrum detection device and the method for making that application number is 201110153526.X; 2, flow passage structure is single, thereby sample introduction causes reaction efficiency lower without fully mixing, and can't reach maximum emission intensity: like application number is 200910154432.7 the disclosed capillary electrophoresis separation of Chinese patent and the micro-fluidic chip of chemiluminescence detection; 3, the light-emitting zone full impregnated is bright, and the scattered light of different directions is not fully utilized, and the light detection efficiency is low.
Summary of the invention
An object of the present invention is provides a kind of micro-fluidic chip that biochemiluminescence detects that is used for for above-mentioned three technical matterss that solve at present relevant with luminous detection micro-fluidic chip existence; Micro-fluidic chip of the present invention improves the biochemical reaction luminous intensity through improving the solution mixability; Improve optical design and improve the light detection efficiency, thereby improve simple operation property and detection sensitivity that biochemiluminescence detects.
Another object of the present invention provides a kind of method for making that is used for the micro-fluidic chip of biochemiluminescence detection, and chip manufacture method of the present invention is simple, and is easy to operate.
The present invention realizes that the technical scheme that its first technical purpose adopts is: a kind of micro-fluidic chip that is used for the biochemiluminescence detection; Said chip comprises interface layer, photic zone, channel layer, reflector layer and the fixed bed that sets gradually from top to bottom; Connect up and down through bonding agent between described each layer and fix through securing member, described interface layer is provided with inlet opening, fluid hole and optical fiber and connects the hole, and described channel layer is provided with feed liquor flow channel, micro-mixer, detection cell, the waste liquid Buffer Pool that is communicated with successively; Described feed liquor flow channel is communicated with inlet opening; The waste liquid Buffer Pool is communicated with fluid hole, and described optical fiber connects the top that the hole is arranged on detection cell, and optical fiber connects between hole and the detection cell and is separated with photic zone; The top of detection cell and euphotic lower surface join, and the bottom of detection cell is connected with reflector layer.This micro-fluidic chip is through setting gradually interface layer, photic zone, channel layer, reflector layer and fixed bed, and inlet opening, feed liquor flow channel, micro-mixer are set on chip; Detection cell, waste liquid buffer zone, fluid hole; Optical fiber connects the hole; Said structure makes sample introduction get into the feed liquor flow channel from inlet opening, and through fully being mixed behind the micro-mixer, mixed sample introduction luminescence-producing reaction efficient improves greatly then.Optical fiber connects the top that the hole is arranged on detection cell; Optical fiber connects between hole and the 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, and 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 the detection cell causing and contaminated aqueous solution and stream unsmooth, guarantees the good light permeability between detection cell and the optical fiber simultaneously again.Inlet opening and fluid hole are used to insert feed liquor tubule and fluid tubule, and the optical fiber that faces toward the detection cell place connects the hole, is used to insert optical fiber.This micro-fluidic chip need not rely on the complex optical path of being made up of microscope stage or lens and optical filter; Do not need the external excitation light source; Need not apply external high pressure electric field driven stream, adopt micro-mixer, design specific flow passage structure; Solution just can reach maximum emission intensity behind sample introduction and mixer circulation passage.Light-emitting zone adopts reflective structure, and the scattered light that directive optical fiber connects different directions beyond the hole also can be fully utilized, and has improved the light detection efficiency.
As preferably, described bonding agent comprises dimethyl silicone polymer monomer and hardening agent, and the volume ratio of described dimethyl silicone polymer monomer and hardening agent is 3～20:1.Dimethyl silicone polymer monomer (PDMS) and hardening agent are commercial product, available from Dow Corning.
As preferably, described interface layer is provided with several inlet openings, and the aperture of described inlet opening is 1mm～2mm, and interface layer is provided with a fluid hole and an optical fiber connects the hole, and the aperture of described fluid hole is 1.5mm～2.5mm.The quantity of inlet opening can be set to a plurality of as required, to satisfy the needs of different testing goals.The size and the feed liquor aperture of the feed liquor flow channel tube of peristaltic pump are adaptive, and the size of fluid flow channel tube and fluid aperture are adaptive, and the aperture that described optical fiber connects the hole is 1.5mm～5.0mm.
As preferably, channel layer be provided with interface layer on several consistent feed liquor flow channels of feed liquor number of perforations, each feed liquor flow channel is communicated with micro-mixer respectively, the width of described feed liquor flow channel is 1.5mm～2.5mm, the degree of depth is 1mm～2mm.Channel layer be provided with interface layer on several consistent feed liquor flow channels of feed liquor number of perforations, such structure makes that the different sample introductions on a plurality of passages are fully mixed in micro-mixer, thereby improves reaction velocity and efficient.
As preferably, described micro-mixer is broken line shape raceway groove, establish in square to intersect and establish channel shape structure side by side in linear raceway groove or the rectangle, and the width and the degree of depth of micro-mixer raceway groove are respectively 0.5mm～1.5mm.Micro-mixer is broken line shape raceway groove, establish in square to intersect and establish channel shape structure side by side in linear raceway groove or the rectangle, can make multiple solution that fully mixing takes place when passing through fast.Certainly micro-mixer can also adopt other structures such as S shape, as long as the purpose that can satisfy the present technique scheme is all within the protection domain of this programme.
As preferably; Described detection cell is circle hole shape; The detection cell bottom is a reflector layer; The aperture of detection cell (d) and channel layer thickness (h), optical fiber core diameter () and numerical aperture (α) satisfy:
, the volume of described detection cell less than with the volume of waste liquid Buffer Pool.Be the abundant coupled into optical fibres of light that guarantees that detection cell sends; The detection cell bottom is a reflector layer; The diameter of detection cell (d) should be complementary with channel layer thickness (h), light core diameter () and numerical aperture (α); Satisfy:
; Described numerical aperture (α) is by fiber optic materials and the decision of optical fiber core diameter; The detection cell front end connects the waste liquid Buffer Pool, and the volume of detection cell is to influence the detection effect in order to prevent waste liquid from blowing back into detection cell less than waste liquid Buffer Pool volume.
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, polymethylmethacrylate (PMMA) the material substrate that can select thickness 2mm～7.5mm for use is as interface layer; Selection is convenient; The 2nd, the degree of depth that such structure makes inlet opening, fluid hole and optical fiber connect the hole is set to the best, makes things convenient for the inflow and the outflow of sample introduction, and optical fiber insertion back connection length reaches best.Euphotic thickness is preferably 0.2mm～0.8mm; Polyethylene terephthalate (PET) film that can select thickness 0.2mm～0.8mm for use is as photic zone; 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 guarantees the good light permeability between detection cell and the optical fiber again.The thickness of channel layer is preferably 2.5mm～3.5mm; The PMMA material substrate that can adopt thickness 2.5mm～3.5mm is as channel layer; The feed liquor flow channel, micro-mixer, detection cell, the waste liquid Buffer Pool that are communicated with successively that are fit to the width and the degree of depth can be set on channel layer like this; Each channel width and the degree of depth are provided with 1.5mm～2.5mm and 1mm～2mm respectively; Purpose is to keep a suitable flow path resistance, guarantees feed liquor unobstructed operation under peristaltic pump drives, and 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, and the minute surface of selecting 0.2mm～1.5mm for use is as reflector layer, because biochemiluminescence is unoriented, minute surface can reflex to fiber port with a big chunk scattered light, has improved the light detection efficiency.Of is preferably 2mm～7.5mm, can select for use like this thickness be 2mm～7.5mm PMMA material substrate as fixed bed, be arranged on below the reflector layer, be used for reinforcing chip.
The present invention realizes that the technical scheme that its another technical purpose adopts is: a kind of method for making that is used for the micro-fluidic chip of biochemiluminescence detection, and described method for making is following steps,
1) uses laser marking machine on interface layer, to carve inlet opening, fluid hole, optical fiber and connect the 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 fastener hole respectively; 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 the hole is 1.5mm～5.0mm; The width of feed liquor flow channel is 1.5mm～2.5mm, and the degree of depth is 1mm～2mm, and the width and the degree of depth of micro-mixer raceway groove are respectively 0.5mm～1mm; Described detection cell circle hole shape; The aperture of detection cell (d) and channel layer thickness (h), optical fiber core diameter () and numerical aperture (α) satisfy:
, described numerical aperture (α) determines by fiber optic materials and optical fiber core diameter, the volume of described detection cell less than with the volume of waste liquid Buffer Pool;
2) preparation of bonding agent: is that 3～20:1 is mixed and made into by dimethyl silicone polymer monomer and hardening agent according to volume ratio, leaves standstill till the bubble collapse, and described dimethyl silicone polymer monomer and hardening agent are commercial product;
3) use the bonding agent of preparation in the step 2 to apply the surface of contact between each layer; And interface layer, photic zone, channel layer, luminescent layer and fixed bed stacked from top to bottom; Fastening at corner location with securing member, place 70～90 ℃ of baking ovens to solidify then and processed in 60～120 minutes to be used for the micro-fluidic chip that biochemiluminescence detects.
Compared with prior art, the invention has the beneficial effects as follows:
(1) detects solution and take place to mix fast and fully, improved the reaction luminous intensity through micro-mixer;
(2) the reflector layer minute surface of luminescence detecting pool below can reduce the scattered light loss, has improved the light detection efficiency;
(3) the luminous detection pool size reaches more excellent coupling efficiency according to optical fiber core diameter and numerical aperture optimal design, has removed light path devices such as complicated lens simultaneously from;
(4) this facture of microchip method is simple, easy to operate, and detection speed is fast, and detection sensitivity is high.
Description of drawings
Fig. 1 is used for a kind of structural representation of the micro-fluidic chip of biochemiluminescence detection for the present invention;
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 that the present invention is used for the micro-fluidic chip of biochemiluminescence detection;
Fig. 5 is the third structural representation that the present invention is used for the micro-fluidic chip of biochemiluminescence detection;
Among the figure: 1, interface layer, 11, inlet opening, 12, fluid hole, 13, optical fiber connects the hole, 2, photic zone, 3, channel layer, 31, the feed liquor flow channel, 32, micro-mixer, 33, detection cell, 34, the waste liquid Buffer Pool, 4, reflector layer, 5, fixed bed, 6, securing member.
Embodiment
Below through specific embodiment and combine accompanying drawing that technical scheme of the present invention is further specified, needed raw material is all commercially available or adopt known method synthetic among the embodiment.
Embodiment 1:
As shown in Figure 1; A kind of micro-fluidic chip that is used for the biochemiluminescence detection; Chip comprises interface layer 1, photic zone 2, channel layer 3, reflector layer 4 and the fixed bed 5 that sets gradually from top to bottom, bonds together up and down through bonding agent between each layer and fixing through quadruplet securing member 6, and interface layer 1 is a PMMA material substrate; The thickness of interface layer 1 is 4mm; Interface layer 1 is provided with the fluid hole 12 of the inlet opening 11 of 2 diameter 1mm, a diameter 1.5mm, the optical fiber of a diameter 2.25mm connects hole 13, and channel layer 3 adopts the PMMA material substrate of thickness 3mm, and channel layer 3 is provided with two feed liquor flow channels being communicated with successively 31, micro-mixer 32, 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 a broken line shape, 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, micro-mixer 32 front end joint detection ponds 33, and connecting length between micro-mixer 32 and the detection cell 33 is 1.5mm; (aperture d calculates according to formula
detection cell 33 for the circular hole of diameter 4mm; Channel layer thickness 3mm in the present embodiment; The optical fiber core diameter of being selected for use is that 1mm and numerical aperture are 0.37); The bottom is a reflector layer 4, and it is 1mm that reflector layer 4 adopts thickness, and 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 connection 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, and optical fiber connects the top that hole 13 is arranged on detection cell 33, and optical fiber connects between hole 13 and the 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.Fixed bed 5 is the PMMA material substrate of 7mm, and length and width are of a size of 50mm * 50mm, and the fastener hole of diameter 5mm is arranged at corner location.Securing member 6 is tightened to one with interface layer 1, photic zone 2, channel layer 3, reflector layer 4 and fixed bed 5, is configured for the micro-fluidic chip (see figure 2) that biochemiluminescence detects.
The making flow process of micro-fluidic chip is following in the present embodiment:
1) uses Chinese workers' board laser marking machine on the interface layer 1 of thickness, to carve inlet opening 11, fluid hole 12, optical fiber and connect hole 13 as 4mm PMMA material; Be to carve inlet opening 11, fluid hole 12 on the PET film photic zone 2 of 0.2mm at thickness; Be to carve micro-mixer 32, detection cell 33, waste liquid Buffer Pool 34 on the channel layer 3 of 3mmPMMA material at thickness; At interface layer 1, photic zone 2, channel layer 3, thickness is that the fixed bed 5 of the PMMA material of 2mm carves fastener hole respectively, conveniently fixes with securing member; The aperture of inlet opening 11 is 1mm; The aperture of fluid hole 12 is 1.5mm; The width of feed liquor flow channel 31 is 2mm; The degree of depth is 1.5mm; The width and the degree of depth of micro-mixer 32 raceway grooves are respectively 0.5mm and 0.6mm, and (aperture d calculates according to formula
detection cell 33, channel layer thickness 3mm in the present embodiment for the circular hole of diameter 4mm; The optical fiber core diameter of being selected for use is that 1mm and numerical aperture are 0.37), the volume of detection cell 33 less than with the volume of waste liquid Buffer Pool 34;
2) preparation of bonding agent: is that 10:1 is mixed and made into by dimethyl silicone polymer monomer and hardening agent according to volume ratio, leaves standstill till the bubble collapse, and described dimethyl silicone polymer monomer and hardening agent are commercial product; Sylgard 184 silicone elastomer PDMS matrix and hardening agent are the mixed of 10:1 by volume and stir, in fuming cupboard, leave standstill till the bubble collapse, as bonding agent;
3) use the bonding agent of preparation in the step 2 to apply the surface of contact between each layer; And interface layer 1, photic zone 2, channel layer 3, luminescent layer 4 and fixed bed 5 stacked from top to bottom; Fastening at corner location with securing member 6, place 80 ℃ of baking ovens to solidify then and processed in 90 minutes to be used for the micro-fluidic chip that biochemiluminescence detects.
Embodiment 2:
In the embodiment shown in fig. 4; A kind of micro-fluidic chip that is used for the biochemiluminescence detection; Chip comprises interface layer 1, photic zone 2, channel layer 3, reflector layer 4 and the fixed bed 5 that sets gradually from top to bottom, bonds together up and down through bonding agent between each layer and fixing through quadruplet securing member 6, and interface layer 1 is a PMMA material substrate; The thickness of interface layer 1 is 3.5mm; Interface layer 1 is provided with the fluid hole 12 of the inlet opening 11 of 3 diameter 1.5mm, a diameter 2mm, the optical fiber of a diameter 1.5mm connects hole 13, and channel layer 3 adopts the PMMA material substrate of thickness 2.5mm, and channel layer 3 is provided with 3 feed liquor flow channels being communicated with successively 31, micro-mixer 32, 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 the square, the raceway groove wall scroll length of intersecting is 10mm, and channel width is 1mm, and the degree of depth is 0.5mm, micro-mixer 32 front end joint detection ponds 33, and connecting length between micro-mixer 32 and the detection cell 33 is 0.5mm; Detection cell 33 is the circular hole of diameter 3mm (the optical fiber core diameter of being selected for use in the present embodiment is that 0.8mm and numerical aperture are 0.37); The bottom is a 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, and the front end of detection cell 33 connects waste liquid Buffer Pool 34, and connection 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 the top that hole 13 is arranged on detection cell 33, and optical fiber connects between hole 13 and the detection cell 33 and is separated with photic zone 2, and 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.Fixed bed 5 is the PMMA material substrate of 3mm, and length and width are of a size of 50mm * 50mm, and the fastener hole of diameter 5mm is arranged at corner location.Securing member 6 is tightened to one with interface layer 1, photic zone 2, channel layer 3, reflector layer 4 and fixed bed 5, constitutes micro-fluidic chip.
The making flow process of micro-fluidic chip is following in the present embodiment:
1) uses Chinese workers' board laser marking machine on the interface layer 1 of thickness, to carve inlet opening 11, fluid hole 12, optical fiber and connect hole 13 as 3.5mm PMMA material; Be to carve inlet opening 11, fluid hole 12 on the PET film photic zone 2 of 0.3mm at thickness; 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; At interface layer 1, photic zone 2, channel layer 3, thickness is that the fixed bed 5 of the PMMA material of 3mm carves fastener hole respectively, conveniently fixes with securing member; The aperture of inlet opening 11 is 1.5mm; The aperture of fluid hole 12 is 2mm; The width of feed liquor flow channel 31 is 1mm; The degree of depth is 1mm; The width and the degree of depth of micro-mixer 32 raceway grooves are respectively 1mm and 0.5mm, and (aperture d calculates according to formula
detection cell 33, channel layer thickness 2.5mm in the present embodiment for the circular hole of diameter 3mm; The optical fiber core diameter of being selected for use is that 0.8mm and numerical aperture are 0.37), the volume of detection cell 33 less than with the volume of waste liquid Buffer Pool 34;
2) preparation of bonding agent: is that 3:1 is mixed and made into by dimethyl silicone polymer monomer and hardening agent according to volume ratio, leaves standstill till the bubble collapse, and described dimethyl silicone polymer monomer and hardening agent are commercial product; Sylgard 184 silicone elastomer PDMS matrix and hardening agent are the mixed of 3:1 by volume and stir, in fuming cupboard, leave standstill till the bubble collapse, as bonding agent;
3) use the bonding agent of preparation in the step 2 to apply the surface of contact between each layer; And interface layer 1, photic zone 2, channel layer 3, luminescent layer 4 and fixed bed 5 stacked from top to bottom; Fastening at corner location with securing member 6, place 70 ℃ of baking ovens to solidify then and processed in 60 minutes to be used for the micro-fluidic chip that biochemiluminescence detects.
Embodiment 3:
In Fig. 5 embodiment; A kind of micro-fluidic chip that is used for the biochemiluminescence detection; Chip comprises interface layer 1, photic zone 2, channel layer 3, reflector layer 4 and the fixed bed 5 that sets gradually from top to bottom, bonds together up and down through bonding agent between each layer and fixing through quadruplet securing member 6, and interface layer 1 is a PMMA material substrate; The thickness of interface layer 1 is 4.5mm; Interface layer 1 is provided with the fluid hole 12 of the inlet opening 11 of 4 diameter 2mm, a diameter 2.5mm, the optical fiber of a diameter 5mm connects hole 13, and channel layer 3 adopts the PMMA material substrate of thickness 3.5mm, and channel layer 3 is provided with 4 feed liquor flow channels being communicated with successively 31, micro-mixer 32, 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 the 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, micro-mixer 32 front end joint detection ponds 33, and connecting length between micro-mixer 32 and the detection cell 33 is 2mm; Detection cell 33 is the circular hole of diameter 5.5mm (the optical fiber core diameter of being selected for use in the present embodiment is that 1mm and numerical aperture are 0.5); The bottom is a 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, and the front end of detection cell 33 connects waste liquid Buffer Pool 34, and connection 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 the top that hole 13 is arranged on detection cell 33, and optical fiber connects between hole 13 and the detection cell 33 and is separated with photic zone 2, and 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.Fixed bed 5 is the PMMA material substrate of 4mm, and length and width are of a size of 50mm * 50mm, and the fastener hole of diameter 5mm is arranged at corner location.Securing member 6 is tightened to one with interface layer 1, photic zone 2, channel layer 3, reflector layer 4 and fixed bed 5, constitutes micro-fluidic chip.
The making flow process of micro-fluidic chip is following in the present embodiment:
1) uses Chinese workers' board laser marking machine on the interface layer 1 of thickness, to carve inlet opening 11, fluid hole 12, optical fiber and connect hole 13 as 4.5mm PMMA material; Be to carve inlet opening 11, fluid hole 12 on the PET film photic zone 2 of 0.4mm at thickness; 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; At interface layer 1, photic zone 2, channel layer 3, thickness is that the fixed bed 5 of the PMMA material of 4mm carves fastener hole respectively, conveniently fixes 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, and the degree of depth is 2mm, and the width and the degree of depth of micro-mixer 32 raceway grooves are respectively 0.8mm and 1mm; Detection cell 33 is the circular hole of diameter 5.5mm (the optical fiber core diameter of being selected for use in the present embodiment is that 1mm and numerical aperture are 0.5), the volume of detection cell 33 less than with the volume of waste liquid Buffer Pool 34;
2) preparation of bonding agent: is that 20:1 is mixed and made into by dimethyl silicone polymer monomer and hardening agent according to volume ratio, leaves standstill till the bubble collapse, and described dimethyl silicone polymer monomer and hardening agent are commercial product; Sylgard 184 silicone elastomer PDMS matrix and hardening agent are the mixed of 20:1 by volume and stir, in fuming cupboard, leave standstill till the bubble collapse, as bonding agent;
3) use the bonding agent of preparation in the step 2 to apply the surface of contact between each layer; And interface layer 1, photic zone 2, channel layer 3, luminescent layer 4 and fixed bed 5 stacked from top to bottom; Fastening at corner location with securing member 6, place 90 ℃ of baking ovens to solidify then and processed in 120 minutes to be used for the micro-fluidic chip that biochemiluminescence detects.
The flow process that is used for the ATP bioluminescent detection to arbitrary micro-fluidic chip that is used for the biochemiluminescence detection that makes among embodiment 1, embodiment 2 or the embodiment 3 is following:
The inlet opening 11 that 1) will be used on the micro-fluidic chip interface layer 1 that biochemiluminescence detects is connected with peristaltic pump (model LongerPump BT100-2J) through the feed liquor flow channel tube; Fluid hole 12 is connected with the fluid flow channel tube; Detection fiber one end inserts optical fiber and connects hole 13; The other end is connected to photon counter (IDQ id100-MMF50-ULN), and photon counter is connected on the computing machine; The number of peristaltic pump is consistent with the number of inlet opening; Inlet opening 11 is connected with the fluid flow channel tube with feed liquor flow channel tube, fluid hole 12, detection fiber and optical fiber connect hole 13 and uses bonding agent to solidify respectively, and bonding agent is selected from the ethene-vinyl acetate hot melt adhesive.
2) wash down stream, use the aqueous slkali (can select for use NaOH solution, KOH solution) of 0.1～1mol/L successively, 0.1～1mol/L acid solution (can be selected HCl solution, H for use
2SO
4Solution), the aqueous slkali of 0.1～1mol/L, with the liquid flow path passage and the solution pool of the flow velocity of 1uL/s～10uL/s flushing micro-fluidic chip,, use ultrapure water again, with the liquid flow path passage and the solution pool of the flow velocity flushing micro-fluidic chip of 1uL/s～10uL/s;
3) in feed liquor flow channel 11, feed the sample liquid that 15uL～100uL luciferase luminous detection liquid and 15uL～100uL contain ATP respectively with the flow velocity of 0.1uL/s～3uL/s, can record the response curve (Fig. 3-1) of bioluminescence reaction.
Comparative example 1
With reference to the micro-fluidic chip that is used for the biochemiluminescence detection that embodiment 1 ~ 3 makes, difference is not contain reflector layer.With reference to being used for the flow process that micro-fluidic chip that biochemiluminescence detects is used for the ATP bioluminescent detection, record response curve (Fig. 3-2).
Comparative example 2
With reference to the micro-fluidic chip that is used for the biochemiluminescence detection that embodiment 1 ~ 3 makes, difference is not have micro-mixer on the channel layer, and the feed liquor flow channel directly imports detection cell.With reference to being used for the flow process that micro-fluidic chip that biochemiluminescence detects is used for the ATP bioluminescent detection, record response curve (Fig. 3-3).
Comparative example 3
With reference to the micro-fluidic chip that is used for the biochemiluminescence detection that embodiment 1 ~ 3 makes, difference is not contain reflector layer, and does not have micro-mixer on the channel layer, and the feed liquor flow channel directly imports detection cell.With reference to being used for the flow process that micro-fluidic chip that biochemiluminescence detects is used for the ATP bioluminescent detection, record response curve (Fig. 3-4).
Just to the explanation rather than the restriction of technical scheme of the present invention, those of ordinary skill is within any alternative all protection domains in technical scheme of the present invention of being done on the basis of present technique scheme in the art for the foregoing description.
Claims (8)
1. one kind is used for the micro-fluidic chip that biochemiluminescence detects; It is characterized in that: said chip comprises interface layer (1), photic zone (2), channel layer (3), reflector layer (4) and the fixed bed (5) that sets gradually from top to bottom; Connect up and down through bonding agent between described each layer and fixing through securing member (6); Described interface layer (1) is provided with inlet opening (11), fluid hole (12) and optical fiber and connects hole (13); Described channel layer (3) is provided with feed liquor flow channel (31), micro-mixer (32), detection cell (33), the waste liquid Buffer Pool (34) that is communicated with successively; Described feed liquor flow channel (31) is communicated with inlet opening (11); Waste liquid Buffer Pool (34) is communicated with fluid hole (12), and described optical fiber connects the top that hole (13) is arranged on detection cell (33), and optical fiber connects between hole (13) and the 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, and the bottom of detection cell (33) is connected with reflector layer (4).
2. a kind of micro-fluidic chip that biochemiluminescence detects that is used for according to claim 1; It is characterized in that: described bonding agent comprises dimethyl silicone polymer monomer and hardening agent, and the volume ratio of described dimethyl silicone polymer monomer and hardening agent is 3～20:1.
3. a kind of micro-fluidic chip that biochemiluminescence detects that is used for 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; Interface layer (1) is provided with a fluid hole (12) and an optical fiber connects hole (13), and 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 claim 1 or 3 described a kind of micro-fluidic chips that biochemiluminescence detects that are used for; It is characterized in that: channel layer (3) is provided with interface layer (1) and goes up several consistent feed liquor flow channels (31) of inlet opening (11) number; 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. a kind of micro-fluidic chip that biochemiluminescence detects that is used for according to claim 1; It is characterized in that: described micro-mixer (32) is broken line shape raceway groove, establish in square to intersect and establish channel shape structure side by side in linear raceway groove or the rectangle, and the width and the degree of depth of micro-mixer (32) raceway groove are respectively 0.5mm～1.5mm.
6. a kind of micro-fluidic chip that biochemiluminescence detects that is used for according to claim 1; It is characterized in that: described detection cell (33) is circle hole shape; Detection cell (33) bottom is reflector layer (4); Satisfy with channel layer thickness (h), optical fiber core diameter () and numerical aperture (α) in the aperture (d) of detection cell (33):
, the volume of described detection cell (33) is less than the volume of waste liquid Buffer Pool (34).
7. according to claim 1 or 3 or 5 or 6 described a kind of micro-fluidic chips that biochemiluminescence detects that are used for; 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, and the thickness of reflector layer (4) is 0.2mm～3.5mm, and the thickness of fixed bed (5) is 2mm～7.5mm.
8. method for making that is used for the micro-fluidic chip that biochemiluminescence detects as claimed in claim 1, it is characterized in that: described method for making is following steps,
1) uses 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 fastener hole respectively; 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; The width and the degree of depth of micro-mixer (32) raceway groove are respectively 0.5mm～1.5mm; Described detection cell (33) circle hole shape; The aperture (d) of detection cell (33) and channel layer thickness (h), optical fiber core diameter () and numerical aperture (α) satisfy:
, described numerical aperture (α) determines by fiber optic materials and optical fiber core diameter, the volume of described detection cell (33) less than with the volume of waste liquid Buffer Pool (34);
2) preparation of bonding agent: is that 3～20:1 is mixed and made into by dimethyl silicone polymer monomer and hardening agent according to volume ratio, leaves standstill till the bubble collapse, and described dimethyl silicone polymer monomer and hardening agent are commercial product;
3) use the bonding agent of preparation in the step 2 to apply the surface of contact between each layer; And interface layer (1), photic zone (2), channel layer (3), luminescent layer (4) and fixed bed (5) stacked from top to bottom; Fastening at corner location with securing member (6), place 70～90 ℃ of baking ovens to solidify then and processed in 60～120 minutes to be used for the micro-fluidic chip that biochemiluminescence detects.
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