CN102788781A - Microfluidic chip for biological chemiluminescence detection and detection method thereof - Google Patents
Microfluidic chip for biological chemiluminescence detection and detection 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 and the transparent layer are respectively provided with a liquid inlet hole and a liquid outlet hole; the interface layer is also provided with a 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 optical fiber interface is arranged above the detecting pool; the bottom of the optical fiber interface is connected with a light penetration layer; the detecting pool is opened to the lower surface of the channel layer; the bottom of the detecting pool is connected with the reflecting layer; and the bottom of the waste liquid buffer pool is connected with the fixed layer. The invention also discloses a detection 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 and detection method thereof that biochemiluminescence detects that be used for.
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
The objective of the 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 detection method that is used for the micro-fluidic chip of biochemiluminescence detection, and this detection method is simple to operate, response speed is fast, and detection sensitivity is high.
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; Described 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, be respectively equipped with inlet opening and fluid hole on described interface layer, the photic zone, also be provided with optical fiber on the interface layer and connect the hole through securing member; 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, and 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; The bottom that optical fiber connects the hole is connected with photic zone; Detection cell is offered the lower surface to channel layer, and the bottom of detection cell is connected with reflector layer, and the bottom of described waste liquid Buffer Pool is connected with fixed bed.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 reaction rate 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 lower surface of detection cell and channel layer connects; 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.
1. as preferred, described interface layer is provided with several inlet openings, and the aperture of described inlet opening is 0.8mm～2.2mm, and the aperture of the fluid hole that is provided with on the interface layer is 1.2mm～2.8mm.The quantity of inlet opening can be set to a plurality of as required, to satisfy the needs of different testing goals.The feed liquor aperture is adaptive with the size of the feed liquor flow channel tube that is attached thereto, and the size of fluid aperture and fluid flow channel tube is adaptive, and the aperture that optical fiber connects the hole is 1 mm～5.5mm.
As preferably, channel layer be provided with interface layer on the consistent feed liquor flow channel 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 1.2mm～2.8mm, the degree of depth is 1.2mm～2.2mm.Channel layer be provided with interface layer on the consistent feed liquor flow channel 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.
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 0.45mm～1.05mm.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 two kinds of 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; Satisfy with channel layer thickness (h), optical fiber core diameter () and numerical aperture (α) in the aperture of detection cell (d):
, the volume of described detection cell is less than 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 3.4mm～4.6mm, and euphotic thickness is 0.25mm～0.45mm, and the thickness of channel layer is 2.4mm～3.6mm, and the thickness of reflector layer is 0.7mm～1.6mm, and the thickness of fixed bed is 1.8mm～4.2mm.The thickness of interface layer is preferably 3.4mm～4.6mm; The one, polymethylmethacrylate (PMMA) the material substrate that can select thickness 3.4mm～4.6mm 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 0.25mm～0.45mm; Polyethylene terephthalate (PET) film that can select thickness 0.25mm～0.45mm for use is as photic zone; Optical fiber that the photic zone that 0.25mm～0.45mm 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 2.4mm～3.6mm; The PMMA material substrate that can adopt thickness 2.4mm～3.6mm 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 set to 1.2mm～2.2mm 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 0.7mm～1.6mm, and the minute surface of selecting 0.7mm～1.6mm 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 1.8mm～4.2mm, can select for use like this thickness be the PMMA material substrate of 1.8mm～4.2mm as fixed bed, be arranged on below the reflector layer, be used for reinforcing chip.
The present invention realizes that its second technical scheme that goal of the invention adopted is: a kind of detection method that is used for the micro-fluidic chip of biochemiluminescence detection, and described detection method is following steps,
The inlet opening that 1) will be used on the micro-fluidic chip interface layer that biochemiluminescence detects is connected with peristaltic pump through the feed liquor flow channel tube; Fluid hole is connected with the fluid flow channel tube, detection fiber one end inserts optical fiber and connects the hole; The other end is connected to photon counter, and photon counter is connected on the computing machine; The number of peristaltic pump is consistent with the number of inlet opening;
2) wash down stream; Use the aqueous slkali of 0.1～1mol/L successively; 0.1～1mol/L acid solution, the aqueous slkali of 0.1～1mol/L is 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, wash the liquid flow path passage and the solution pool of micro-fluidic chip with the flow velocity of 1uL/s～10uL/s;
3) in the feed liquor flow channel, feed the analyte sample fluid 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 photon counting response curve of bioluminescence reaction.
As preferably, inlet opening is connected with the fluid flow channel tube with feed liquor flow channel tube, fluid hole in the step 1), detection fiber and optical fiber connects the hole and use bonding agent to solidify respectively, and bonding agent is selected from the ethene-vinyl acetate hot melt adhesive.
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:
In the embodiment shown in fig. 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 1.5mm, a diameter 2mm, 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; Optical fiber connects the top that hole 13 is arranged on detection cell 33, and the bottom that optical fiber connects hole 13 is connected with photic zone 2, and detection cell 33 is offered to the lower surface of channel layer 3; The bottom of detection cell 33 is connected with reflector layer 4, and the bottom of waste liquid Buffer Pool 34 is connected with fixed bed 5.Fixed bed 5 is the PMMA material substrate of 2mm, 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 the micro-fluidic chip (see figure 2).
A kind of method for making that is used for the micro-fluidic chip of biochemiluminescence detection 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 width and the degree of depth of micro-mixer 32 raceway grooves are respectively 0.5mm; Described detection cell 33 is circle hole shape; Satisfy with channel layer thickness (h), optical fiber core diameter () and numerical aperture (α) in the aperture of detection cell 33 (d):
; Described numerical aperture (α) is by fiber optic materials and the decision of optical fiber core diameter; The optical fiber core diameter is 1mm, and numerical aperture is 0.37, 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 10:1 be mixed and made into hardening agent according to volume ratio by dimethyl silicone polymer monomer (PDMS), leaves standstill till the bubble collapse, and PDMS and hardening agent are commercial product; PDMS can select the Sylgard 184 silicone elastomer base of Dow Corning for use, and hardening agent can be selected the Sylgard 184 silicone elastomer curing agent of Dow Corning for use.
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 60～120 minutes (present embodiment places 80 ℃ of baking ovens to solidify 120 minutes) then and process the micro-fluidic chip that is used for the biochemiluminescence detection.
A kind of detection method that is used for the micro-fluidic chip of biochemiluminescence detection, described detection method is following steps,
Two inlet openings 11 that 1) will be used on the micro-fluidic chip interface layer 1 that biochemiluminescence detects are connected with two peristaltic pumps (model LongerPump BT100-2J) through two feed liquor flow channel tubes; Fluid hole 12 is connected with a fluid flow channel tube, the detection fiber of core diameter 1000um, numerical aperture 0.37; 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; 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 1000uL 0.5mol/L NaOH solution successively, 1000uL 0.5mol/L HCl solution; 1000uL 0.5mol/L NaOH solution through micro-fluidic chip, is used the 3000ul ultrapure water with the flow velocity of 1uL/s again; Flow velocity with 1uL/s passes through micro-fluidic chip, cleans stream;
3) flow velocity with 1uL/s feeds 60uL luciferase luminous detection liquid and 60uL ATP sample liquid respectively at two feed liquor flow channels 11, can record the photon counting response curve (Fig. 3-1) of bioluminescence reaction.
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 1mm, a diameter 1.5mm, 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 linear raceway groove of intersection 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, 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, and length and width are of a size of 24mmx24mm, minute surface; 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 the bottom that optical fiber connects hole 13 is connected with photic zone 2, and detection cell 33 is offered to the lower surface of channel layer 3; The bottom of detection cell 33 is connected with reflector layer 4, and the bottom of waste liquid Buffer Pool 34 is connected with fixed bed 5.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.
A kind of method for making that is used for the micro-fluidic chip of biochemiluminescence detection 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 width and the degree of depth of micro-mixer 32 raceway grooves are respectively 0.8mm; Described detection cell 33 is circle hole shape; Satisfy with channel layer thickness (h), optical fiber core diameter () and numerical aperture (α) in the aperture of detection cell 33 (d):
; Described numerical aperture (α) is by fiber optic materials and the decision of optical fiber core diameter; The desirable 0.8mm of optical fiber core diameter; Numerical aperture is desirable 0.37, 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 3:1 be mixed and made into hardening agent according to volume ratio by dimethyl silicone polymer monomer (PDMS), leaves standstill till the bubble collapse, and PDMS and hardening agent are commercial product; PDMS can select the Sylgard 184 silicone elastomer base of Dow Corning for use, and hardening agent can be selected the Sylgard 184 silicone elastomer curing agent of Dow Corning for use.
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.
A kind of detection method that is used for the micro-fluidic chip of biochemiluminescence detection, described detection method is following steps,
Three inlet openings 11 that 1) will be used on the micro-fluidic chip interface layer 1 that biochemiluminescence detects are connected with three peristaltic pumps (model LongerPump BT100-2J) through three feed liquor flow channel tubes; Fluid hole 12 is connected with a fluid flow channel tube, the detection fiber of core diameter 1000um, numerical aperture 0.37; 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; 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 1000uL 0.1mol/L KOH solution successively, 1000uL 0.1mol/L H
2SO
4Solution, 1000uL 0.1mol/L KOH solution with the liquid flow path passage and the solution pool of the flow velocity of 5uL/s flushing micro-fluidic chip, is used the 3000ul ultrapure water again, with the liquid flow path passage and the solution pool of the flow velocity flushing micro-fluidic chip of 10uL/s;
3) flow velocity with 0.1uL/s feeds 15uL luciferase luminous detection liquid and 15uL ATP sample liquid respectively to two feed liquor flow channels 11, can record the photon counting response curve of bioluminescence reaction.
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, and it is 1.5mm that reflector layer 4 adopts thickness, and length and width are of a size of the minute surface of 24mm*24mm; 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 the bottom that optical fiber connects hole 13 is connected with photic zone 2, and detection cell 33 is offered to the lower surface of channel layer 3; The bottom of detection cell 33 is connected with reflector layer 4, and the bottom of waste liquid Buffer Pool 34 is connected with fixed bed 5.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.
A kind of method for making that is used for the micro-fluidic chip of biochemiluminescence detection 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 width and the degree of depth of micro-mixer 32 raceway grooves are respectively 1mm; Described detection cell 33 is circle hole shape; Satisfy with channel layer thickness (h), optical fiber core diameter () and numerical aperture (α) in the aperture of detection cell 33 (d):
; Described numerical aperture (α) is by fiber optic materials and the decision of optical fiber core diameter; The desirable 1mm of optical fiber core diameter, numerical aperture is desirable 0.5, 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 20:1 be mixed and made into hardening agent according to volume ratio by dimethyl silicone polymer monomer (PDMS), leaves standstill till the bubble collapse, and PDMS and hardening agent are commercial product; PDMS can select the Sylgard 184 silicone elastomer base of Dow Corning for use, and hardening agent can be selected the Sylgard 184 silicone elastomer curing agent of Dow Corning for use.
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 100 minutes to be used for the micro-fluidic chip that biochemiluminescence detects.
A kind of detection method that is used for the micro-fluidic chip of biochemiluminescence detection, described detection method is following steps,
Four inlet openings 11 that 1) will be used on the micro-fluidic chip interface layer 1 that biochemiluminescence detects are connected with four peristaltic pumps (model LongerPump BT100-2J) through four feed liquor flow channel tubes; Fluid hole 12 is connected with a fluid flow channel tube, the detection fiber of core diameter 1000um, numerical aperture 0.5; 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; 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 1000uL 1mol/L NaOH solution successively; 1000uL 1mol/L HCl solution, 1000uL 1mol/L NaOH solution is with the liquid flow path passage and the solution pool of the flow velocity of 10uL/s flushing micro-fluidic chip; Use the 3000ul ultrapure water again, wash the liquid flow path passage and the solution pool of micro-fluidic chip with the flow velocity of 10uL/s;
3) flow velocity with 3uL/s feeds 100uL luciferase luminous detection liquid and 100uL ATP sample liquid respectively to four feed liquor flow channels 11, can record the photon counting response curve (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.Be used for the ATP bioluminescent detection with reference to embodiment 1 ~ 3, 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.Be used for the ATP bioluminescent detection with reference to embodiment 1 ~ 3, 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.Be used for the ATP bioluminescent detection with reference to embodiment 1 ~ 3, 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: described 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); Be respectively equipped with inlet opening (11) and fluid hole (12) on described interface layer (1), the photic zone (2); Also be provided with optical fiber on the interface layer (1) and connect 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, and 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 the top that hole (13) is arranged on detection cell (33), and the bottom that optical fiber connects hole (13) is connected with photic zone (2), and 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), and the bottom of described waste liquid Buffer Pool (34) is connected with fixed bed (5).
2. 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 0.8mm～2.2mm; The aperture that interface layer (1) is gone up the fluid hole (12) that is provided with is 1.2mm～2.8mm, and the aperture that described optical fiber connects hole (13) is 1 mm～5.5mm.
3. a kind of micro-fluidic chip that biochemiluminescence detects that is used for according to claim 1 and 2; It is characterized in that: channel layer (3) is provided with interface layer (1) and goes up the consistent feed liquor flow channel (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 1.2mm～2.8mm, and the degree of depth is 1.2mm～2.2mm.
4. 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 0.45mm～1.05mm.
5. 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; 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).
6. according to claim 1 or 2 or 4 or 5 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 3.4mm～4.6mm; The thickness of photic zone (2) is 0.25mm～0.45mm; The thickness of channel layer (3) is 2.4mm～3.6mm, and the thickness of reflector layer (4) is 0.7mm～1.6mm, and the thickness of fixed bed (5) is 1.8mm～4.2mm.
7. one kind with the described detection method that is used for the micro-fluidic chip that biochemiluminescence detects of claim 1, and it is characterized in that: described detection method is following steps,
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 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, and photon counter is connected on the computing machine; The number of peristaltic pump is consistent with the number of inlet opening;
2) wash down stream; Use the aqueous slkali of 0.1～1mol/L successively; 0.1～1mol/L acid solution, the aqueous slkali of 0.1～1mol/L is 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, wash the liquid flow path passage and the solution pool of micro-fluidic chip with the flow velocity of 1uL/s～10uL/s;
3) in feed liquor flow channel (11), feed the analyte sample fluid 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 photon counting response curve of bioluminescence reaction.
8. a kind of detection method that is used for the micro-fluidic chip of biochemiluminescence detection according to claim 7; It is characterized in that: inlet opening in the step 1) (11) connects hole (13) with feed liquor flow channel tube, fluid hole (12) with fluid flow channel tube, detection fiber and optical fiber and uses bonding agent to solidify respectively, and bonding agent is selected from the ethene-vinyl acetate hot melt adhesive.
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