CN107930710A - Chemiluminescence testing microfluid control chip and chemiluminescence testing microfluid control chip system and their application - Google Patents

Abstract

The present invention relates to fluidic chip chemiluminescence technical field of immunoassay, more particularly to chemiluminescence testing microfluid control chip and chemiluminescence testing microfluid control chip system and their application.Chip includes：Sample injection unit (1), liquid storage unit (2), reaction member (3) and waste unit (4)；Further include and treat the quantitative dosing unit of sample sheet (5), including：The inlet (51) being connected with sample injection unit, the liquid outlet (52) being connected with waste unit, the temporary structure (54) for the temporary surplus liquid for carrying out quantitative D-M (Determiner-Measure) construction (53) and being connected with reaction member.Chip system is by upper, middle and lower up of three layers；Middle level is the chip；Levels are used to cover closing middle level；There are the sample holes being connected with sample injection unit, and the relief hole of corresponding liquid storage unit in upper strata.The chip detection process is simple, reaction sample can be quantified, high sensitivity, repeatability are strong.

Description

Chemiluminescence testing microfluid control chip and chemiluminescence testing microfluid control chip system withAnd their application

Technical field

The present invention relates to fluidic chip chemiluminescence technical field of immunoassay, more particularly to a kind of chemiluminescence detectionMicro-fluidic chip and a kind of chemiluminescence testing microfluid control chip system, and their applications in chemiluminescence detection.

Background technology

Microfluidic chip technology is otherwise known as Microfluid based Lab on a chip or chip lab, refers at one piece several squaresCentimetre chip on the chemistry or biology laboratory that build, it sample preparation involved in the fields such as chemistry and biology,Reaction, separates, detection, cell culture, sorting, and the basic operation unit such as cracking is integrated on the chip of one piece of very little, by micro- logicalRoad forms network, runs through whole system with controlled fluid, chemical to realize biology, the various work(in the field such as medical diagnosisEnergy.

The essential characteristic and sharpest edges of microfluidic chip technology be：A variety of cellular constructions can on small chip platformWith flexible combination so that chip design is upper flexible and changeable and multiple functional；The small required detection of chip internal structure unitSample size is considerably less, and the bigger serface of microstructure unit allows interior reagent quickly to spread to realize fast reaction and inspectionSurvey；Micro-fluidic chip is usually to be automatically performed internal-response by necessary instrument, so during actual test, micro-fluidic chipTechnology can reduce the technical requirements to medicine testing staff, reduce the human error of detection, and then reduce the medicine inspection of patientSurvey cost；Microfluidic chip technology using automation equipment due to being completed, so can be with so internal-response process is fully controllableIt is more accurate to obtain, and more sensitively detects data.

In medical science, in-vitro diagnosis is a very important branch, is referred in human vitronectin, to fromThe sample (body fluid such as such as blood, urine, saliva) of human body is detected so as to obtain clinical diagnosis information, is sentenced according to the informationWhether disconnected person under inspection suffers from certain disease or is subjected to the diagnostic method of certain germ infection.At present, with biotechnology, it is particularlyBiochemistry, immunology, the fast development of molecular biology, in-vitro diagnosis mainly have biochemistry according to testing principle and detection methodDiagnosis, immunodiagnosis, molecule diagnosis, microbe diagnosis, the diagnosis of blood coagulation class, fluidic cell diagnosis etc., its mesophytization, exempts fromEpidemic disease, molecule diagnosis are the major component fields of current China's in-vitro diagnosis.

In immunodiagnosis field, used detection method mainly has according to principle at present, and colloidal gold method, latex is than turbidMethod, fluorescent immune method, time-resolved fluorescence method, chemoluminescence method.Wherein colloidal gold method detection range is limited, and accuracy is not high,Although latex turbidimetry simple, intuitive, particular volume liquid eggs white matter can only be directed to, and detection range is narrow, fluorescence immunoassayMethod is simply in bacterium, virus, and the field such as skin activity is using more, and there are the problems such as unspecific staining, time resolutionFluorescence method is the further optimization to fluorescent immune method, but its operating procedure is more, complicated, it is necessary to professional skill skillfullyCan personnel operate.

Chemiluminescence immunoassay (CLIA) relies on high sensitivity, and specificity is good, and the degree of automation is high, and precision is good,Application of the advantages such as accuracy rate height in clinical diagnosis is more and more wider, the detection that applied chemistry luminescent detection techniques are carried out at presentProject mainly has：Infectious disease class, such as hepatitis B, hepatitis, AIDS, syphilis etc.；Tumor markers, such as alpha-fetoprotein, cancer embryoAntigen, cancer antigen, human chorionic gonadtropin etc.；Thyroid function index：Such as thyrotropic hormone, free thyroxine,Free triiodothyronine etc.；Steroids, such as sex hormone, growth hormone etc..

But it is especially few to carry out the patent that diagnoses in vitro of chemiluminescence using microfluidic chip technology, both at home and abroad by this twoKind of technology combine so that develop the chip product of maturation company it is less.Investigated by patent and find have in recent years both at home and abroadThe patent of chemiluminescence detection is carried out using microflow control technique on a small quantity, for example Chinese patent 201510696773 discloses one kindMagnetic microparticle chemiluminescence micro-fluidic chip, but there is no the dosing unit of reagent or sample in the chip, it is impossible to determine to participate in reactionSample volume, be easy to cause accuracy in detection decline, and microchannel structure completely encloses in chip, once introduce bubble,Whole chip will be caused to be difficult to complete subsequent detection；Disclosed for another example in Chinese patent 201611033217 a kind of micro-fluidicPapery chip and its chemical luminous immune detection method, but the chip completes sample feeding, different cores using capillarityPaper structure changes greatly inside piece, capillary force size heterogeneity and unmanageable, so the repeatability of chip chamber is relativelyDifference, can considerable decrease to the detection accuracy of single sample.

The content of the invention

The purpose of the invention is to overcome as above problem existing in the prior art, there is provided a kind of chemiluminescence detection miniflowChip is controlled, the micro-fluidic chip is simple with detection process, reaction sample can be quantified, each construction unit is interconnectedThe characteristics of, and detection sensitivity is high, repeatability is strong.

To achieve these goals, one aspect of the present invention provides a kind of chemiluminescence testing microfluid control chip, this is micro-fluidicChip includes：For receiving the sample injection unit of sample to be checked, the liquid storage unit for storing reaction reagent, being the sample to be checkedWaste unit with the reaction member of reaction reagent offer reacting environment and for collecting waste liquid；The micro-fluidic chip is alsoIncluding for carrying out quantitative dosing unit to the sample to be checked；

The dosing unit includes：The inlet that is connected with the sample injection unit, with what the waste unit was connected go out liquidMouthful, for treating sample sheet and/or reaction reagent carries out quantitative D-M (Determiner-Measure) construction and is used for what the reaction member was connectedThe temporary structure of temporary surplus liquid.

Preferably, which is additionally included in the unit that reports an error set between waste unit and liquid outlet, the reportWrong unit has chamber, which is used for the liquid for receiving the temporary structure from dosing unit.

Preferably, the unit that reports an error is connected with optical detection apparatus, and the optical detection apparatus is used for the chamberOptical detection is carried out, to judge the amount of liquid in chamber；Herein it should be noted that " connected " described herein can not be physicsIt is connected, and simply position alignment.

Preferably, which further includes the ventilation unit being connected with the waste unit, for for micro-fluidic corePiece system provides required external pressure.

Preferably, which further includes the tool interface system being connected with the ventilation unit, and the tool interface system is usedNecessary instrument outside connection fluidic chip system, the air pressure that the necessary instrument provides are provided to institute by the ventilation unitState micro-fluidic chip system.

Preferably, the necessary instrument include being used for providing the promotion pulsometer of air pressure, gas-guide tube and with the ventilationThe air-tightness interface of unit connection；The air-tightness interface is preferably tubaeform.

Preferably, ventilative but fluid-tight material is also filled with the ventilation unit；Preferably, it is described ventilative but impermeableThe material of water is aerosol.

Preferably, the reaction reagent is loaded into the liquid storage unit by reagent pouch；The reagent pouch includesFor the location hole being fixed in the liquid storage unit, seal and for opening and discharging described the sealThe crush-zone or needling structure of reaction reagent.

Preferably, filter element is additionally provided between the sample injection unit and the inlet originally to carry out to treat sampleFilter.

Preferably, mixing arrangement is additionally provided with the reaction member, for sample to be checked to be mixed with reaction reagentClose；The mixing arrangement, which is selected from, erects at least one of pillar, Z-shaped microchannel, W types mixer and triangle micro-structure.

Preferably, micro-valve door is also respectively provided with the inlet and liquid outlet of the dosing unit, so as to control liquidInflow and outflow.

The second aspect of the present invention also provides a kind of chemiluminescence testing microfluid control chip system, the micro-fluidic chip systemIt is made of the upper, middle and lower；

Wherein, media layer damage is chemiluminescence testing microfluid control chip as described above；

Wherein, superstructure and understructure, which are used to cover, closes the intermediate layer；It is provided with superstructure and sample introductionThe sample holes of unit connection, and with the relief hole corresponding to liquid storage unit, for providing external impetus to discharge liquid storage unitIn reaction reagent.

The third aspect of the present invention also provides chemiluminescence testing microfluid control chip as described above and/or as described aboveApplication of the chemiluminescence testing microfluid control chip system in chemiluminescence detection.

The present invention can obtain following beneficial effect：

1st, sample to be checked is detected using micro-fluidic chip provided by the invention and system combination chemoluminescence method, there is detectionThe advantages that optics background is low, and detection sensitivity is high, and the range of linearity is wide, and detection repeatability is strong, preferably coordinates particular detection instrument,It can realize full-automatic chip detection, accurate testing result can be quickly obtained without human interference.

2nd, the present invention uses microfluidic chip technology, treats sample and originally carries out volume quantitative first so that only particular volumeLong-pending sample reacts, and ensures the accuracy of testing result, simultaneously because chip structure is fixed, so for same sample itsDetection repeatability is strong, ensures the stability and reliability of testing result.

3rd, the present invention uses microfluidic chip technology, it would be desirable to which the liquid reagent for participating in chemiluminescence reaction is all pre-stored in storageIn liquid unit, it is entirely avoided the interference and pollution problem of flow path reagent in regular pipeline formula Chemiluminescence Apparatus, while full envelopeEnclosed liquid reagent preserves the shelf-life that can extend chip, it is ensured that chip remains able to obtain steady for a comparatively long period of timeFixed and accurate testing result.

4th, the present invention uses microfluidic chip technology, only carries out one-time detection for each detection sample, detection is completedChip is discarded immediately afterwards, it is entirely avoided the detection interference in hospital between different samples, while avoid completely between patientCross-infection.

5th, the present invention uses microfluidic chip technology, and the waste liquid and unnecessary sample after reaction is completed all are entirely sealed inChip internal, will not cause the leakage of waste liquid or sample, nuisanceless to detection environment, safe to hospital testing staff, noIt can cause the generation of nosocomial infection.

Brief description of the drawings

Fig. 1 is a kind of specific chemiluminescence testing microfluid control chip schematic diagram provided by the invention.

Fig. 2 shows a kind of specific necessary instrument and its connection mode with chemiluminescence testing microfluid control chip.

Fig. 3 is 4 kinds of (such as 3A, Fig. 3 B, Fig. 3 C and Fig. 3 D) different basis weights cellular construction schematic diagrams.

Fig. 4 A are a kind of schematic diagrames of reagent pouch provided by the invention；Fig. 4 B are shown by external pressure disrupting agentThe mode of pouch；Fig. 4 C are shown by way of acupuncture disrupting agent pouch.

Fig. 5 is the Rotating fields schematic diagram of the chemiluminescence testing microfluid control chip system of the present invention.

Fig. 6 is a kind of schematic diagram of superstructure provided by the invention.

Description of reference numerals

1 sample injection unit, 2 liquid storage unit, 3 reaction member

4 waste unit, 5 dosing unit 6 reports an error unit

7 ventilation unit, 8 tool interface system, 9 necessary instrument

10 filter element, 21 reagent pouch, 31 mixing arrangement

51 inlet, 52 liquid outlet, 53 D-M (Determiner-Measure) construction

54 55 micro-valve doors 91 of temporary structure promote pulsometer

92 gas-guide tube, 93 air-tightness interface, 211 location hole

212 seal, 213 crush-zone, 214 needling structure

Embodiment

The endpoint of disclosed scope and any value are not limited to the accurate scope or value herein, these scopes orValue should be understood to comprising the value close to these scopes or value.For number range, between the endpoint value of each scope, respectivelyIt can be combined with each other between the endpoint value of a scope and single point value, and individually between point value and obtain one or moreNew number range, these number ranges should be considered as specific open herein.

As shown in figures 1 and 3, first aspect present invention provides a kind of chemiluminescence testing microfluid control chip, this is micro-fluidicChip includes：For receiving the sample injection unit 1 of sample to be checked, the liquid storage unit 2 for storing reaction reagent, treating sample to be describedThis and the reaction reagent provide the reaction member 3 of reacting environment and the waste unit 4 for collecting waste liquid；The micro-fluidic corePiece is further included for carrying out quantitative dosing unit 5 to the sample to be checked；

The dosing unit 5 includes：The inlet 51 that is connected with the sample injection unit 1, be connected with the waste unit 4Liquid outlet 52, for treating sample sheet and/or reaction reagent carries out quantitative D-M (Determiner-Measure) construction 53 and connects with the reaction member 3What is connect is used to keep in the temporary structure 54 of surplus liquid.

According to the present invention, dosing unit 5 is used to the sample to be checked that participate in reaction is carried out quantitatively point to take, it can will be unnecessarySample transport to be checked at waste unit 4, ensure to only have the sample to be checked of designated volume to participate in follow-up chemical reaction so thatEnsure the accuracy of chip detection of the present invention.The dosing unit 5 can be it is multiple, its quantity can according to be actually needed comeIncrease or decrease.

Originally quantified it should be noted that, although merely defining and treating sample by dosing unit 5 herein, but this areaTechnical staff it should be appreciated that, dosing unit 5 can be by the setting of quantity and connection mode, to different samplesThis, for example, reaction reagent is quantified.For example, in subsequent reactions, can as needed to participating in the reagent of reaction, such asEnzymic-labelled antibody reagent is quantified, so as to ensure that the enzymic-labelled antibody reagent of only designated volume participates in subsequent reactions.TheseIt should be considered as within protection scope of the present invention.

Quantitative principle is carried out in conjunction with Fig. 3 to the dosing unit 5 to illustrate：Acted in the extracting of necessary instrument 9Under, sample to be checked is entered in D-M (Determiner-Measure) construction 53 by inlet 51, since the volume of sample to be checked is greater than D-M (Determiner-Measure) construction 53Volume, therefore unnecessary sample to be checked can be remained in temporarily in temporary structure 54, in the case of providing decompression in outside, closingLiquid at 52 both ends of inlet 51 and liquid outlet can be drawn into waste unit 4 by microchannel, so that remaining inSample to be checked at temporary structure 54 is extracted into waste unit 4, and the sample to be checked only in D-M (Determiner-Measure) construction 53 will not extractTo waste unit 4, so that the sample to be checked of the same volume of D-M (Determiner-Measure) construction 53 can only be retained.

Since chip structure is preferably prepared on a large scale mode using injection molding, so that each chip internalThe volume of D-M (Determiner-Measure) construction 53 is equal, to ensure that the volume that different chips are obtained for same sample to be checked point is also identical,It is so that repeated between improving batch interior repeatability of chip and criticizing.The D-M (Determiner-Measure) construction 53 can be different shape, such as Fig. 3 AIn triangle, the rectangle in Fig. 3 B, the semicircle in Fig. 3 C, the irregular shape in Fig. 3 D etc..

, according to the invention it is preferred to, the inlet 51 and liquid outlet 52 of the dosing unit 5 are in same horizontal lineOn, D-M (Determiner-Measure) construction 53 is in below the horizontal plane that inlet 51 and liquid outlet 52 are defined, and temporary structure 54 is in inlet 51Above the horizontal plane defined with liquid outlet 52, so that under external force extracting effect, the sample in D-M (Determiner-Measure) construction 53 will not be taken outWalk, the sample in only temporary structure 54 is pumped.But during under other conditions, such as in varying level line, it can also pass throughThe change of structure has the function that liquid quantitative, and those skilled in the art can specifically be set according to actual conditions.

According to the present invention, connected between the dosing unit 5 and sample injection unit 1 and waste unit 4 by microchannelConnect, in order to the flowing of liquid.Preferably, the connector between microchannel and waste unit 4 is located at the top of waste unit 4,Its main function is that the waste liquid after the completion of reaction or unnecessary sample to be checked are transported in waste unit 4, simultaneously because positionOn structural relation cause enter waste unit 4 inside waste liquid part before chip will not be back to by microchannel.

Wherein, the shape of the cross section of the microchannel can be rectangle, and circular, triangle is trapezoidal, or other various shapesShape.The width of microchannel can be 0.001mm-10mm, its depth can be 0.001mm-10mm.

According to the present invention, the waste unit 4 is preferably placed at the end of liquid flow path system in chip, to collect each stepThe waste liquid of biochemical reaction, prevents waste liquid outflow chip and pollutes external environment condition.Wherein, the waste unit 4 not office in structureIt is limited to the similarly suitable of the rectangle shown in Fig. 1, other shapes, such as cylinder, cone, or other various irregular shapesWith.Wherein, the volume of the waste unit 4 should be greater than the body of all 2 interior reagent volumes of liquid storage unit and sample to be checkedThe sum of product, so as to ensure that waste unit 4 can effectively collect issuable waste liquid in reaction process.

In the case of, according to the invention it is preferred to, also set respectively on the inlet 51 and liquid outlet 52 of the dosing unit 5It is equipped with micro-valve door 55.At the specific reaction moment, can be in an open state by controlling micro-valve door 55 to be changed by closed mode, orIt is in off state by open mode transformation, so as to control the flowing of liquid.When in off position, which leads to both sidesRoad is completely isolated, and liquid cannot pass through micro-valve door 55 and reach opposite side；In open mode, liquid can pass through the micro-valve door55 and smoothly reach opposite side.

Wherein, piston structure is preferably provided with micro-valve door 55, it is opened or closed can be completely by instrument according to predeterminedProgram is completed.Usually without under the intervention of instrument, micro-valve door 55 is in open mode or part micro-valve door 55 is in and opensState, and when needing to be turned off, the piston of micro-valve door is driven by the shaft of stepper motor in instrument, piston is rotated certainAngle, so that the fluid path of internal piston is closed so that the transformation of micro-valve door is in off state.It is it is preferred, therefore, that supportingInstrument 9 further includes stepper motor.

As a kind of alternative embodiment, the piston can be vertical slide and non-rotating, in general stateThe piston of lower micro-valve door is in open mode, and when needing to close, the piston at micro-valve door is promoted by the push-pull rod of instrument, willPiston is pushed into inside microchannels so as to block microchannel so that the micro-valve door is closed.

As another alternative embodiment, which can also be carried out by the cooperation of iron plate and electromagnetic fieldOperation is opened and closed, for example, a small iron plate can be pasted above micro-valve door 55, electromagnet is placed below micro-valve door,Under general state, electromagnet no power, iron plate is located at the top of micro-valve door 55, and micro-valve door 55 is in open mode, is needing to closeWhen closing, it is powered by instrument to electromagnet, the electromagnetic field that magnet produces can attract the iron plate of the top of micro-valve door 55, so that by ironPiece is moved to below micro-valve door so that micro-valve door is closed.

In the case of, according to the invention it is preferred to, in order to ensure that detection can be smoothed out, micro-fluidic chip of the invention also wrapsThe unit 6 that reports an error being arranged between liquid outlet 52 and waste unit 4 is included, the unit 6 that reports an error has chamber structure, which usesIn the liquid for receiving the temporary structure 54 from dosing unit 5.After liquid passes through dosing unit 5, unnecessary liquid can beExternal force effect is lower to be entered in the chamber structure for the unit 6 that reports an error by liquid outlet 52 by extracting, and according in the chamber structureThe filling degree of liquid judges to detect whether to be smoothed out.It is if hydraulically full in chamber structure, it is believed that quantitative singleLiquid is also filled with D-M (Determiner-Measure) construction 53 in member 5, at this time it is believed that chip operation is normally without reporting an error, if the chamber for the unit 6 that reports an errorIt is inadequate without liquid or amount of liquid inside cell structure, then it is assumed that the liquid volume of D-M (Determiner-Measure) construction 53 is also insufficient in dosing unit 5, thisWhen need to report an error and terminate the detection of chip.So chip internal self-detection mechanism can ensure only liquid quantitative entirely accurateIn the case of chip just may proceed to carry out follow-up detection, so as to ensure the accuracy of testing result.

Wherein, it is preferred that the unit 6 that reports an error is connected with optical detection apparatus, the optical detection apparatus can be used for pairThe chamber carries out optical detection, so as to judge the amount of liquid in chamber.

According to the present invention, the sample injection unit 1 is used for the sample to be checked for receiving user's injection.The sample to be checked can beIt is any to carry out the sample of chemiluminescence detection, for example, can be but be not limited to, whole blood, serum, blood plasma, urine, saliva,The various body body fluid such as sweat or by body various histoorgans secretion, can by the secretion intoLiquid materials after row dilution are detected as sample to be checked.

Wherein, the structure of the sample injection unit 1 can be cylinder, conical (for example, structure shown in Fig. 1), ladderShape, or other various irregular shapes.Its major function is to receive the sample to be checked of user's injection so that sample to be checked is being notedThe leakage of sample to be tested will not occur after entering to chip, sample can only be operated according to specific microchannel structure.

As an alternative embodiment, the sample injection unit 1 can be made into capillarity sampling structure, note at this timeThe sample to be checked entered can wick themselves into the construction unit in downstream, and capillarity can adsorb sample to be checkedIn chip, prevent the leakage of sample to be tested and pollute environment.

According to the present invention, it is described when the sample to be checked is needs sample to be checked (for example, the whole blood sample) filteredChemiluminescence testing microfluid control chip further includes the filter element being arranged between the sample injection unit 1 and the inlet 5110, that is, the filter element 10 is located at the downstream of sample injection unit 1, the upstream of dosing unit 5.The inside of filter element 10 can be withSample to be checked can be filtered by being preinstalled with, for example, in whole blood red blood cell hemofiltration film, it is single that the thickness of the hemofiltration film is less than filteringThe height of member 10, so that hemofiltration film can be fully seated inside filter element 10, while the shape and mistake of the hemofiltration filmThe interior wall construction for filtering unit 10 is consistent, so as to get the sample to be checked up to filter element 10 can not bypass hemofiltration film and be directly enteredDownstream passage.The hemofiltration film can be such that liquid is separated with red blood cell by physical pore size or biology, chemical reagent, and the biology, changeIt can be coagulant to learn reagent.The thickness of the hemofiltration film can be 0.1mm-10mm, and the height of the filter element 10 can be0.1mm-20mm。

According to the present invention, the liquid storage unit 2 is at least one, and Fig. 1 shows 8 liquid storage units 2, the liquid storage unit 2Main function be the various reaction reagents that will participate in chemiluminescence reaction, such as enzymic-labelled antibody reagent, micro- magnetic bead couplingAntibody reagent, cleaning solution, chemical luminous substrate reagent etc. are loaded into the chip internal of the present invention in advance, are carried out in chip of the present inventionDuring the test of sample to be checked, then by external force the reaction reagent inside the liquid storage unit 2 is discharged in sequence.Need hereinIt is noted that the size and shape of each liquid storage unit 2 of chip internal may be the same or different, its shape is also simultaneouslyThe circle shown in figure is not limited to, other shapes such as rectangle, diamond shape, polygon or irregular shape are equally applicable.TogetherWhen each liquid storage unit 2 size can be changed according to the volume size of the reaction reagent to be pre-installed, liquid storage unit 2 at this timeVolume and differ.

As shown in Figure 4 A, the present invention provides a kind of basic structure of liquid storage unit 2, prepackage reaction reagent to be filled in advanceFill out in reagent pouch 21, which is preferably flexible material, which includes but not limited to：Nitrocellulose is thinFilm, plastic film or metal aluminum foil, the plastic film can be but be not limited to polyester, polyethylene terephthalate(PET), at least one of makrolon (PC), polypropylene (PP) and polymethyl methacrylate (PMMA).Its common featureIt is the prepackage reaction reagent that inside can be discharged by way of extruding or acupuncture.Reagent pouch is loaded into prepackage reaction reagentAfter in 21, outlet is subjected to sealing forms seal 211 by way of thermoplastic envelope.The seal degree of seal 211 hereinThan shallower so that when external force pressurizes reagent pouch 21, seal 211 can rupture.Location hole 212 is used to aid in reagentPouch 21 is fixed at the liquid storage unit 2 in chip., can be with when needing to discharge 2 internal-response reagent of liquid storage unitBy way of in Fig. 4 B, by extruding the crush-zone 213 in reagent pouch 21, so that seal 211 is ruptured and releasedTapping body, can also puncture the bottom section of reagent pouch 21 such as needling structure 214 by the way of in Fig. 4 C by prickerPlace so that inside prepackage reaction reagent discharges.

According to the present invention, reaction member 3 is available to the main place that reaction reagent reacts with sample to be checked, at the same timeAnd chemical illuminating reagent participates in the main place of luminous detection after substrate reactions, therefore, reaction member 3 preferably uses translucencyPrepared by preferable material, for example, may be selected from polymethyl methacrylate (PMMA), makrolon (PC), polydimethylsiloxanesAlkane (PDMS) etc..The reaction member 3 is connected with each liquid storage unit 2 by microchannel, in order to the examination inside liquid storage unit 2It is directly entered after agent release in reaction member 3, participates in follow-up immune response and chemiluminescence reaction.

Herein it should be noted that, although present invention defines reaction member, but it is not that reaction is merely able to occur anti-Answer in unit.Since reaction member 3 is communicated with dosing unit 5, during reaction, reaction liquid can also enterReacted in dosing unit 5, dosing unit 5 can be considered as a kind of reaction member at this time.

Although the reaction member 2 has gathered immune response function and chemiluminescence detection function, optionally,This two parts function can be divided among in two differential responses units and carried out, while chemistry is carried out in another translucent constructionShine detection.Any combinations and/or deformation for so setting and carrying out on this basis should be considered as the present invention'sWithin protection domain.

A kind of preferred embodiment according to the present invention, the inside of the reaction member 3 are further prepared with mixing arrangement 31, itsMain function is that assisted reaction reagent and sample to be checked are mixed.As a preferred embodiment, the mixing arrangement31 are made of multiple setting pillars, which can be located at both sides inside reaction member, be extruded inside reaction member 3When, the liquid of inside can be promoted to be mixed, while liquid can form small whirlpool when by erectting pillar around pillarStream, the disturbance of the small vortex can accelerate the mixing between liquid, so that the immune response between accelerating each component, and then improve lifeChange reaction speed, reduce the total time of chip detection.Although mixing arrangement 31 shown here in the form of pillar is erect comeThe other structures shapes such as realization, but other micro-structures alternately, such as Z-type microchannel, W type mixers, triangle micro-structureFormula can also aid in and accelerate to mix between reagent.

A kind of preferred embodiment according to the present invention, the present invention between micro- path to chip system by pressurizeingOr decompression is so as to control the flowing of each liquid, to enter another construction unit from a construction unit.Therefore, it is described micro-Fluidic chip further includes the ventilation unit 7 being connected with the waste unit 4, for for micro-fluidic chip system provide needed forExternal pressure, so that internal liquid runs well under the auxiliary of external pressure.Ventilation unit 7 is also prevented from waste liquid list at the same timeWaste liquid in member 4 flows out to chip exterior and pollutes external environment condition.Although ventilation unit 7 as shown in Figure 1 is using W types microchannelOther various structures such as design, but other designs, such as circle, arc, Z-type, which can similarly play, ventilates and prevents outside waste liquidThe effect of stream, these designs should all be also treated as within protection scope of the present invention.

Preferably, predetermined substance can also be filled inside the ventilation unit 7, the work for playing ventilation but preventing waste liquid from outflowingWith, for example the material can be aerosol, or ventilative but fluid-tight loose cavernous structure material.

A kind of preferred embodiment according to the present invention, micro-fluidic chip of the invention preferably pass through exterior necessary instrumentAir pressure needed for 9 offer systems.Connect it is preferred, therefore, that the micro-fluidic chip further includes the instrument being connected with the ventilation unit 7Mouth 8, the tool interface system 8 are used to connect the necessary instrument 9 outside fluidic chip system, and the air pressure that the necessary instrument 9 provides is led toCross the ventilation unit 7 and be provided to the micro-fluidic chip system.Wherein, the size of the external pressure can compare atmospheric pressureGreatly, can also be smaller than atmospheric pressure, the size of the air pressure can be easily adjusted according to specific service condition difference.Meanwhile instituteIt is preferably air-tightness to state tool interface system 8, i.e., chip is when the air pressure regulator with necessary instrument 9 is docked, the positionIt is air tight.The function can be realized by plastic sealing ring or other assemblies.The air pressure regulator of necessary instrument 9 can beAir driven pump, gas storage vesica, pulsometer, extrusion pump etc..

A kind of preferred embodiment according to the present invention, as shown in Figure 2, the necessary instrument 9 includes being used to provide gasPromotion pulsometer 91, gas-guide tube 92 and the air-tightness interface 93 being connected with the ventilation unit 7 of pressure.When chip to be measured is putAfter entering into necessary instrument 9, the stepper motor (not shown) of instrument internal can promote pulsometer 91 so that air-tightness interface93 cling at the tool interface system 8 of chip to be measured, and it is air-tightness that this, which is close to mode, can be by the air-tightness interface 93It is upper to set sealing ring or O-ring to complete.Preferably, the air-tightness interface 93 is tubaeform, in the case of this is preferable, noNeed air-tightness interface 93 being accurately aligned with tool interface system 8, it is only necessary to which tool interface system 8 is located inside the air-tightness interface 93.Wait after the completion of being close to, the stepper motor being connected with pulsometer 91 is in the lock state, and the position of pulsometer 91 is consolidated at this timeIt is fixed.Rotation by another the stepper motor (not shown) being connected with 91 internal piston of pulsometer, can promote orPiston is extracted, so as to adjust the air pressure inside pulsometer 91, and then manipulates the air pressure of chip internal.Needing to add to chip internalDuring pressure, completed by stepper motor to promote piston, on the contrary, when needing to depressurize to chip internal, by stepper motor comePiston is extracted to realize.Chip internal fluid path network can drive fluid to shift due to increase or the reduction of air pressure.TreatingSurvey after the whole testing process completion of chip, by the rotation for the stepper motor being connected with pulsometer 91 come the air-tightnessInterface departs from chip.

As shown in Figure 5, according to the second aspect of the invention, there is provided a kind of chemiluminescence testing microfluid control chip system,The micro-fluidic chip system is made of the upper, middle and lower；

Wherein, media layer damage is chemiluminescence testing microfluid control chip as described above；

Wherein, superstructure and understructure, which are used to cover, closes the intermediate layer；It is provided with superstructure and sample introductionThe sample holes that unit 1 connects, and with the relief hole corresponding to liquid storage unit 2, for providing external impetus to discharge liquid storage listReaction reagent in member 2.

As shown in Figure 6, sample holes (aperture), the sample-adding for sample to be checked are provided with above superstructure.It is described intoSample hole is communicated with the sample injection unit 1 in media layer damage, to ensure that sample to be checked can enter the sample introduction list by sample holesIn member 1.In addition, being additionally provided with least one relief hole (macropore) in superstructure, the relief hole corresponds to media layer damageLiquid storage unit 2, for the entrance of the push rod of necessary instrument 9, so that external pressure is provided for liquid storage unit 2, by liquid storage unitThe reagent of the advance enclosed storage in 2 inside discharges, and therefore, necessary instrument 9 of the invention further includes at least one push-pull rod.InstituteRelief hole size and shape is stated by the structure size of the liquid storage pouch 21 of liquid storage unit in media layer damage and shape to determine, preferably, the diameter of the relief hole can be 0.5mm-50mm, its shape can be circular, rectangle, polygon, diamond shape, evenIrregular shape etc. is variously-shaped.

Preferably, the material of the upper, middle and lower is each independently selected from such as dimethyl silicone polymer (PDMS), poly- firstBase methyl acrylate (PMMA), makrolon (PC), polypropylene (PP), polyethylene terephthalate (PET), plastics are thinFilm, elastic emulsion, natural rubber, plastics and silica gel.

Preferably, the thickness of superstructure is 0.5-20mm, more preferably 0.5-10mm, more preferably 0.5mm-5mm；The thickness of media layer damage is 0.5-50mm；More preferably 2mm-20mm, more preferably 3mm-15mm；UnderstructureThickness is 0.5-20mm, more preferably 0.5-10mm, more preferably 0.5mm-5mm.

The preparation method of chemiluminescence testing microfluid control chip provided by the invention preferably includes following steps：

Reaction reagent needed for chemiluminescence reaction is preloaded onto in the reagent pouch 21 by step 1), and sealed by thermoplasticMode is sealed the seal 212 of the reagent pouch 21；And chip system middle level is placed on by location hole 211 and is tiedAt the specific liquid storage structure of structure, further the reagent pouch 21 of each liquid storage is fixed in chip system by way of thermoplastic envelopeOn Rotating fields.

Step 2) fits to understructure as above below media layer damage, its laminating type includes but are not limited to ultrasoundThe modes such as hot melt, gluing, ultraviolet light cure, thermoplastic envelope.

Step 3) fits to superstructure as above above media layer damage, its laminating type includes but are not limited to ultrasoundThe modes such as hot melt, gluing, ultraviolet light cure, thermoplastic envelope.

The flow that chemiluminescence detection is carried out using chemiluminescence testing microfluid control chip system provided by the invention is as follows(by taking whole blood as an example)：

Step 1) draws a certain amount of whole blood by pipettor, and whole blood is added to well, then by micro-fluidic chip bodySystem, which is placed in necessary instrument 9, to be started to test.

Pulsometer 91, air-tightness interface 93 are moved to tool interface system 8 by step 2) necessary instrument 9 by internal stepper motorIt is fixed behind place, then the piston in pulsometer 91 is stripped by stepper motor, under the action of draft, whole blood enter intoIn sample unit 1, then enter back into and red blood cell filtration is carried out in filter element 10, the blood plasma after filtering enters dosing unit 5, moreRemaining blood plasma enters in the unit 6 that reports an error, and it is (supporting to detect the optical signal for the unit 6 that reports an error by the optical detecting unit of necessary instrument 9Instrument 9 further includes optical detecting unit), if the chamber for the unit 6 that reports an error is not full of for empty or liquid, stop chip inspectionSurvey, and report an error, if liquid is full of herein, continue follow-up detecting step.

Step 3) discharges each reaction reagent in liquid storage unit 2 by the push-pull rod of necessary instrument 9 successively：Enzymic-labelled antibodyReagent, micro- magnetic bead coupled antibody reagent, the first cleaning solution, the second cleaning solution, the 3rd cleaning solution, the 4th cleaning solution.By the reagentReaction member 3 is discharged into successively, wherein needing repeatedly mixing cleaning complete in cleaning process, passes through the push-and-pull of necessary instrument 9Bar extrudes the reaction member 3 to be mixed in mixing arrangement 31.

Waste liquid after step 4) cleaning is completed is drawn into waste unit 4 by the pulsometer 91 on necessary instrument 9, mostThe first luminous substrate and the second luminous substrate in liquid storage unit 2 are discharged by the push-pull rod of necessary instrument 9 afterwards so that substrate intoEnter to reaction member 3, carry out chemiluminescence reaction, light is carried out by the light detection module on necessary instrument 9, such as CCD or PMTDetection, and provide testing result.

The sample volume of chemiluminescence testing microfluid control chip system detection provided by the invention is 10-200 μ L.

In the present invention, necessary instrument 9 is small portable apparatus, and necessary instrument 9 is except including pulsometer 91, gas-guide tube92 and air-tightness interface 93 outside, further preferably include push-and-pull bar unit, stepper motor module, light detection module.FurtherPreferably, the necessary instrument 9 further includes temperature control module, to carry out control on demand to the temperature in chip.

The third aspect of the present invention additionally provides chemiluminescence testing microfluid control chip as described above and/or the changeLearn application of the detection micro-fluidic chip system in chemiluminescence detection that shine.

The present invention will be described in detail by way of examples below.

Embodiment 1

The present embodiment is used to illustrate to detect the c reactive protein in whole blood (CRP) using magnetic granule chemoluminescence method

Enzymic-labelled antibody used in the present embodiment is horseradish peroxidase-labeled CRP (HRP-CRP), magnetic particle markerThe size of magnetic particle is 5 μm or so in CRP, and cleaning solution is containing 0.2 volume %BSA, the phosphoric acid of 0.5 volume %tween-20Salt buffer (pH 7.5), the first luminous substrate liquid are the acid solution containing luminol, and the second luminous substrate liquid is to spread out containing benzeneThe alkaline solution of biology.

(1) assembling of chip system

Above-mentioned each reaction reagent is mounted in reagent pouch 21 in advance, and (heating time 30s) will by way of thermoplastic envelopeThe seal 212 of the reagent pouch 21 is sealed；And the specific of chip system media layer damage is placed on by location hole 211At liquid storage structure, the reagent pouch 21 of each liquid storage is further fixed on chip system media layer damage by way of thermoplastic envelopeOn.

Understructure is fitted on media layer damage by the cured mode of light-sensitive emulsion (ultraviolet light hardening time 10min),Superstructure is fitted on media layer damage by same light-sensitive emulsion curing mode (ultraviolet light hardening time 10min), soPrepare complete available chip system.

(2) pattern detection

The method for drawing standard curve：Make dilution with normal person's whole blood, CRP standard items are diluted to following concentration：0pg/mL, 10pg/mL, 50pg/mL, 500pg/mL, 1000pg/mL, 5ng/mL, 10ng/mL.The CRP whole bloods of each concentration are equalCarry out following operation：Whole blood standard items of the 50 μ L containing CRP are taken with pipettor, chip system is injected into by the sample holes of chipMedia layer damage in, while the chip is put into necessary instrument 9 and starts to test.

Necessary instrument 9 adjusts the air pressure inside chip system media layer damage by internal pulsometer 91, will treat sampleThis sucks sample injection unit 1 and enters in filter element 10 successively carries out red blood cell filtration, and the blood plasma after then filtering enters fixedUnit 5 is measured, unnecessary sample to be checked enters the unit 6 that reports an error, and by detection of the instrument to the unit 6 that reports an error, judges quantitative at this timeWhether 53 internal liquid of D-M (Determiner-Measure) construction of unit 5 is full of, if being in normal full state, continues follow-up chip inspectionSurvey, otherwise instrument, which reports an error, exits testing process.Other unnecessary measuring samples are drawn into waste unit 4, then by withThe push-pull rod of set instrument 9 discharges the reaction reagent in liquid storage unit 2 successively：HRP marks CRP antibody reagents, and micro- magnetic bead coupling is anti-Body reagent, the first cleaning solution, the second cleaning solution, the 3rd cleaning solution, the 4th cleaning solution.It is single that these reagents are discharged into reaction successivelyIn member 3, after each reagent releases, necessary instrument 9 is passed through the reagent at reaction member 3 by the effect of push-pull rod mixedAttaching together and put 31 and be sufficiently mixed, the waste liquid after cleaning is completed is drawn into waste unit 4 by necessary instrument 9, finally byThe push-pull rod of necessary instrument 9 discharges the first luminous substrate liquid and the second luminous substrate liquid so that luminous substrate liquid enters reactionUnit 3 carries out chemiluminescence reaction, detects the intensity of the luminous signal by the light detection module on necessary instrument 9, and provideTesting result.Total detection time is 15min, and each standard items take its knot 5 times using 5 micro-fluidic chip system of determination respectivelyThe average value of fruit, draws standard curve.

Using same testing process, take 50 μ L whole blood samples to be detected to be detected, obtained according to the intensity of luminous signalGo out the CRP concentration in whole blood sample to be detected.

The result shows that the lowest detection of micro-fluidic chip of the present invention is limited to 10pg/mL, detection range 20-10000pg/mL.Pass through external chemiluminescence detector device (the Beckman Kurt UniCel that will be approved in testing result and commercially available industry800 Full-automatic chemiluminescence immunoassay analysis meters of DxIn) testing result contrasted, find this chip system detection it is linearRelated coefficient is R²>0.99, show that this detection chip system carries out CRP and standard in magnetic granule chemoluminescence method detection whole bloodThe accuracy of method is consistent.Meanwhile by the way that measure is carried out repeatedly and (used 10 times in the present embodiment) to same whole blood sample, obtainTesting result between the coefficient of variation be respectively less than 10%, show that this detection chip has good repeatability, can be furtherReference as CRP medical diagnosis.

Embodiment 2

The present embodiment detects the c reactive protein in whole blood (CRP) for explanation with coated antibody chemoluminescence method

Enzymic-labelled antibody used in the present embodiment is horseradish peroxidase-labeled CRP (HRP-CRP), and cleaning solution is bagPhosphate buffer (pH 7.5) containing 0.2 volume %BSA, 0.5 volume %tween-20, the first luminous substrate liquid be containingThe acid solution of luminol, the second luminous substrate liquid are the alkaline solution containing benzene derivative.Wherein coated antibody is C- reaction eggsWhite solution, is injected into by way of point sample instrument point sample in reaction member 3, and point sample is when 40 DEG C of bakings 3 are small so that point sample is anti-The CRP albumen at unit 3 is answered to be absorbed and fixed in reaction chamber.

Detection process is the same as embodiment 1.

The result shows that the lowest detection that CRP Concentration Testings are carried out using coated antibody method is limited to 30pg/mL, detection rangeFor 30-10000pg/mL.Pass through external chemiluminescence detector device (the Beckman storehouse that will be approved in testing result and commercially available industry800 Full-automatic chemiluminescence immunoassay analysis meters of your spy UniCel DxIn) testing result contrasted, find the detection of this chipLinearly dependent coefficient be R²>0.99, show that this detection chip carries out the CRP in coated antibody chemoluminescence method detection whole bloodIt is consistent with the accuracy of standard method.Meanwhile by carrying out repeatedly and (using 10 times in the present embodiment) to survey to same whole blood sampleFixed, the coefficient of variation between the testing result of acquisition is respectively less than 10%, shows that this detection chip has good repeatability, can be withIt is further used as the reference of CRP medical diagnosis.

The preferred embodiment of the present invention described in detail above, still, the present invention is not limited thereto.In the skill of the present inventionIn art concept, technical scheme can be carried out a variety of simple variants, including each technical characteristic with it is any itsIts suitable method is combined, these simple variants and combination should equally be considered as content disclosed in this invention, belong toProtection scope of the present invention.

Claims (10)

1. a kind of chemiluminescence testing microfluid control chip, it is characterised in that the micro-fluidic chip includes：For receiving sample to be checkedSample injection unit (1), the liquid storage unit (2) for storing reaction reagent, provide for the sample to be checked and the reaction reagentThe reaction member (3) of reacting environment and the waste unit (4) for collecting waste liquid；The micro-fluidic chip is further included for instituteState sample to be checked and carry out quantitative dosing unit (5)；

The dosing unit (5) includes：The inlet (51) and the waste unit (4) being connected with the sample injection unit (1) are evenThe liquid outlet (52) that connects, for treat sample sheet and/or reaction reagent carry out quantitative D-M (Determiner-Measure) construction (53) and with it is described anti-Answer the temporary structure (54) for being used to keep in surplus liquid that unit (3) connects.

2. chemiluminescence testing microfluid control chip according to claim 1, wherein, which is additionally included in waste liquidThe unit that reports an error (6) set between unit (4) and liquid outlet (52), the unit that reports an error (6) have chamber, which is used to connectReceive the liquid of the temporary structure (54) from dosing unit (5)；

Preferably, the unit that reports an error (6) is connected with optical detection apparatus, the optical detection apparatus be used for the chamber intoRow optical detection, to judge the amount of liquid in chamber.

3. chemiluminescence testing microfluid control chip according to claim 1 or 2, wherein, the micro-fluidic chip further include withThe ventilation unit (7) of waste unit (4) connection, for providing required external pressure for micro-fluidic chip system；

Preferably, which further includes the tool interface system (8) being connected with the ventilation unit (7), the tool interface system(8) it is used to connect the necessary instrument (9) outside fluidic chip system, the air pressure that the necessary instrument (9) provides passes through the ventilationUnit (7) is provided to the micro-fluidic chip system；

Preferably, the necessary instrument (9) is led to including the pulsometer (91) for providing air pressure, gas-guide tube (92) and with describedThe air-tightness interface (93) of gas unit (7) connection；The air-tightness interface (93) is preferably tubaeform；

Preferably, ventilative but fluid-tight material is also filled with the ventilation unit (7)；Preferably, it is described ventilative but impermeableThe material of water is aerosol.

4. according to the chemiluminescence testing microfluid control chip described in any one in claim 1-3, wherein, the reaction reagentIt is loaded into by reagent pouch (21) in the liquid storage unit (2)；The reagent pouch (21) includes being used to be fixed to instituteState the location hole (211) in liquid storage unit (2), seal (212) and for being opened the seal (212) and discharging instituteState the crush-zone (213) or needling structure (214) of reaction reagent；

Preferably, the material of the reagent pouch (21) in cellulose nitrate film, plastic film and metal aluminum foil extremelyFew one kind；

Preferably, the material of the plastic film is selected from polyester, polyethylene terephthalate, makrolon, polypropylene andAt least one of polymethyl methacrylate.

5. chemiluminescence testing microfluid control chip according to claim 1, wherein, the sample injection unit (1) and it is described intoFilter element (10) is additionally provided between liquid mouth (51) originally to be filtered to treat sample.

6. chemiluminescence testing microfluid control chip according to claim 1, wherein, also set up in the reaction member (3)There is mixing arrangement (31), for sample to be checked to be mixed with reaction reagent；

The mixing arrangement (31) selected from erect in pillar, Z-shaped microchannel, W types mixer and triangle micro-structure at least oneKind.

7. chemiluminescence testing microfluid control chip according to claim 1, wherein, the inlet of the dosing unit (5)(51) and on liquid outlet (52) micro-valve door (55) is also respectively provided with, so as to control the inflow and outflow of liquid.

8. according to the chemiluminescence testing microfluid control chip described in any one in claim 1-7, wherein, the sample to be checkedFor body body fluid or body secretes thing；

Preferably, the sample to be checked is selected from whole blood, serum, blood plasma, urine, saliva, sweat.

A kind of 9. chemiluminescence testing microfluid control chip system, it is characterised in that the micro-fluidic chip system by upper strata, middle level andLower floor forms；

Wherein, media layer damage is the chemiluminescence testing microfluid control chip described in any one in claim 1-8；

Wherein, superstructure and understructure, which are used to cover, closes the middle level；It is provided with superstructure and sample injection unit (1)The sample holes of connection, and with the relief hole corresponding to liquid storage unit (2), for providing external impetus to discharge liquid storage unit(2) reaction reagent in；

Preferably, the material of the upper, middle and lower is each independently selected from such as dimethyl silicone polymer, poly-methyl methacrylateEster, makrolon, polypropylene, polyethylene terephthalate, plastic film, elastic emulsion, natural rubber, plastics and siliconGlue；

Preferably, the thickness of superstructure is 0.5-20mm；The thickness of media layer damage is 0.5-50mm；The thickness of understructure is0.5-20mm。

10. described in the chemiluminescence testing microfluid control chip and/or claim 9 in claim 1-8 described in any oneApplication of the chemiluminescence testing microfluid control chip system in chemiluminescence detection.