CN207533259U - ELISA detects micro-fluidic chip and ELISA detection micro-fluidic chip systems - Google Patents

Abstract

The utility model is related to micro-fluidic chip ELISA detection technique fields, more particularly to ELISA detection micro-fluidic chips and ELISA detection micro-fluidic chip systems.Chip includes：Sample injection unit (1), liquid storage unit (2), reaction member (3) and waste unit (4)；The micro-fluidic chip further includes to treat quantitative the first dosing unit (5) of sample sheet, the first sample chamber (52) and the second sample chamber (53) that the first dosing unit includes the first slit (51) and separated by the first slit；First sample chamber is communicated with the sample injection unit, and the second sample chamber is communicated with the waste unit.Chip system is by upper, middle and lower up of three layers；Middle level is the chip；Levels close middle level for covering；There is the relief hole of the sample holes being connect with sample injection unit and corresponding liquid storage unit on upper strata.The chip detection process is simple, reaction sample can be quantified, high sensitivity, repeatability are strong.

Description

ELISA detects micro-fluidic chip and ELISA detection micro-fluidic chip systems

Technical field

The utility model is related to micro-fluidic chip ELISA detection technique fields, more particularly to a kind of ELISA detections are micro-fluidic Chip and a kind of ELISA detection micro-fluidic chip systems.

Background technology

Microfluidic chip technology (microfluidics) is otherwise known as Microfluid based Lab on a chip or chip lab (lab-on-a-chip), the chemistry or biology laboratory that are built on one piece several square centimeters of chip are referred to, it is chemistry It with sample preparation involved in the fields such as biology, reacts, detaches, detection, cell culture, sorting, the basic operations such as cracking Unit is integrated on the chip of one piece of very little, and network is formed by microchannel, with controlled fluid through whole system, to realize life Various functions in the fields such as object, chemistry, medical diagnosis.

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

In medical science, in-vitro diagnosis (In-Vitro-Diagnostics, IVD) is one very important point Branch, refers in human vitronectin, the sample (body fluid such as such as blood, urine, saliva) for deriving from human body is detected to obtain Clinical diagnosis information, according to the information come judge subject whether with certain disease or be subjected to certain germ infection diagnosis side Method.At present, with biotechnology, particularly biochemistry, immunology, the fast development of molecular biology, in-vitro diagnosis according to Testing principle and detection method mainly have biochemical diagnosis, immunodiagnosis, molecule diagnosis, microbe diagnosis, the diagnosis of blood coagulation class, streaming Cyto-diagnosis etc., mesophytization are immunized, and molecule diagnosis is the major component field 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 turbid Method, fluorescent immune method, time-resolved fluorescence method, enzyme-linked immunosorbent assay (ELISA), chemoluminescence method.Wherein colloidal gold Method detection range is limited, and accuracy is not high, although latex turbidimetry simple, intuitive, can only be directed to particular volume liquid eggs white matter, And detection range is narrow, and fluorescent immune method is only in bacterium, virus, and the fields such as skin activity exist using more The problems such as unspecific staining, time-resolved fluorescence method are that fluorescent immune method is advanced optimized, but its operating procedure compared with It is more, it is complicated for operation, skilled efficient staff is needed to operate.

Enzyme-linked immunosorbent assay (ELISA) is a kind of detection technique to grow up the 1970s, but due to The advantages such as this method has high sensitivity, and high specificity, accuracy rate is high, and detectable target molecule species are more, diagnose in vitro In application it is also more and more extensive.At present, ELISA method can be used for the various protein in body fluid in clinical examination, including The few protein of content, the steroid hormone of steroids such as small-molecular-weight etc., antibiotic and drug, pathogen antigen, The detection of HBsAg, HBeAg etc..

But ELISA method is typically to be operated in laboratory or clinical laboratory by professional technician, should be grasped by hand in the process Make step complexity, and time-consuming for whole operation process, detection easily causes operation error etc., domestic at present to use micro-fluidic chip The patent that technology carries out ELISA method detection is especially few, both at home and abroad combines both technologies and then develops ripe chip production The company of product is less.It is investigated by patent and finds have in recent years carry out ELISA method inspection using microflow control technique on a small quantity both at home and abroad The patent of survey, such as Chinese patent 201510648955 disclose a kind of ELISA detection devices for microspironema pallidum detection And chip, but the chip is driven using platinum electrode, needs to apply platinum electrode liquid inside high voltage ability driving chip Body moves, and chip interior channel design is simple, it is difficult to carry out accurate quantification to sample to be tested；Chinese patent for another example A kind of integrated ELISA chips and its detection method based on distance detection target are disclosed in 201611159534, but is used The gas volume that immune response generates carrys out driving signal and generates distance, and quantitative detection is carried out according to distance, and detection mode is opposite Low in optical method detection sensitivity, accuracy is difficult to ensure that, while internal immune response is difficult to accurately control.

Utility model content

The purpose of this utility model is to overcome as above problems of the existing technology, and it is micro- to provide a kind of ELISA detections Fluidic chip, the micro-fluidic chip is simple with detection process, reaction sample can be quantified, each structural unit mutually interconnects The characteristics of logical, detection sensitivity is high, repeatability is strong.

To achieve these goals, the utility model provides a kind of ELISA detections micro-fluidic chip, the micro-fluidic chip packet It includes：For receive sample to be checked sample injection unit, for the liquid storage unit of storage reaction reagent, for the sample to be checked and described Reaction reagent provides the reaction member of reacting environment and the waste unit for collecting waste liquid；The micro-fluidic chip further includes use In carrying out the first quantitative dosing unit to the sample to be checked；

Wherein, first dosing unit includes the first slit and the first sample chamber separated by first slit Room and the second sample chamber；The first sample chamber is communicated with the sample injection unit, and second sample chamber is given up with described Liquid unit communicates.

Preferably, which is additionally included in first set between waste unit and the second sample chamber and reports an error list Member, described first reports an error unit with chamber, which is used to receive the liquid of the second sample chamber from the first dosing unit Body.

Wherein, described first unit that reports an error is connected with optical detection apparatus, and the optical detection apparatus is used for the chamber Room carries out optical detection, to judge the amount of liquid in chamber；Herein it should be noted that " connected " described herein can not be object Reason is connected, and only position alignment.

Preferably, which further includes to carry out the reaction reagent the second quantitative dosing unit；

Wherein, second dosing unit includes the second slit and the first reagent chamber separated by second slit Room and the second reagent chamber；First reagent chamber is communicated with the liquid storage unit, and second reagent chamber is given up with described Liquid unit communicates.

Preferably, which is additionally included in second set between waste unit and the second reagent chamber and reports an error list Member, described second reports an error unit with chamber, which is used to receive the liquid of the second reagent chamber from the second dosing unit Body；

Wherein, described second unit that reports an error is connected with optical detection apparatus, and the optical detection apparatus is used for the chamber Room carries out optical detection, to judge the amount of liquid in chamber；Herein it should be noted that " connected " described herein can not be object Reason is connected, and only position alignment.

Preferably, mixing arrangement is provided in the reaction member, for sample to be checked and reaction reagent are mixed；

Wherein, the mixing arrangement is selected from erectting in pillar, Z-shaped microchannel, W types mixer and triangle micro-structure extremely Few one kind.

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

Preferably, which further includes the tool interface system being connect with the ventilation unit, and the tool interface system is used Necessary instrument outside connection flow control chip system, the air pressure that the necessary instrument provides are provided to institute by the ventilation unit State micro-fluidic chip system.

Preferably, the necessary instrument include for provide the pulsometer of air pressure, gas-guide tube and with the ventilation unit The air-tightness interface of connection；The air-tightness interface is tubaeform.

Preferably, filter element is additionally provided between the sample injection unit and first dosing unit to treat sample sheet It is filtered.

The second aspect of the utility model also provides a kind of ELISA detections micro-fluidic chip system, the micro-fluidic chip body System is made of the upper, middle and lower；

Wherein, media layer damage detects micro-fluidic chip for ELISA as described above；

Wherein, superstructure and understructure close the middle layer for covering；It is provided in superstructure and sample introduction The sample holes of unit connection and the relief hole corresponding to liquid storage unit, for providing external impetus to discharge in liquid storage unit Reaction reagent.

The utility model can obtain following advantageous effect：

1st, the utility model detects sample to be checked using microfluidic chip technology combination ELISA, has detection optics background The advantages that low, detection sensitivity is high, and the range of linearity is wide, and detection is repeatable strong, while coordinate particular detection instrument, it can realize Full-automatic chip detection, accurate testing result can be quickly obtained without human interference.

2nd, the utility model uses microfluidic chip technology, treats sample and originally carries out volume quantitative first so that only spy The sample for determining volume reacts, and ensures the accuracy of testing result, simultaneously because chip structure is fixed, so for same This its detection repeatability is strong, ensures the stability and reliability of testing result.

3rd, the utility model uses microfluidic chip technology, it would be desirable to which the liquid reagent for participating in enzyme linked immunoassay all prestores In liquid storage unit, it is entirely avoided the interference and pollution problem of flow path reagent in routine experiment detection, while totally enclosed type liquid Body reagent, which preserves, can extend shelf-life of chip, it is ensured that chip remains able to be stablized and accurate for a comparatively long period of time True testing result.

4th, the utility model uses microfluidic chip technology, only carries out one-time detection for each detection sample, detects Chip is discarded immediately after completion, it is entirely avoided the detection interference in hospital between different samples, while patient is avoided completely Between cross-infection.

5th, the utility model uses microfluidic chip technology, and the waste liquid and extra sample after reaction is completed are all completely close Chip interior is enclosed in, the leakage of waste liquid or sample will not be caused, it is nuisanceless to detection environment, to hospital's testing staff's safety Height will not lead to the generation of nosocomial infection.

Description of the drawings

Fig. 1 is a kind of specific ELISA detections micro-fluidic chip schematic diagram provided by the utility model.

Fig. 2 shows a kind of specific necessary instrument and its connection mode with ELISA detection micro-fluidic chips.

Fig. 3 is the structure of the first dosing unit of chip provided by the utility model and its Operational Mechanisms figure.

Fig. 4 is the structure of the second dosing unit of chip provided by the utility model and its Operational Mechanisms figure.

Fig. 5 A are a kind of schematic diagrames of reagent pouch provided by the utility model；Fig. 5 B are shown to be ruptured by external pressure The mode of reagent pouch；Fig. 5 C are shown in a manner that needle pierces disrupting agent pouch.

Fig. 6 is that the ELISA of the utility model detects the layer structure diagram of micro-fluidic chip system.

Fig. 7 is a kind of schematic diagram of superstructure provided by the utility model.

Reference sign

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

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

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

10 second dosing units 11 second report an error 12 filter element of unit

21 reagent pouch, 31 mixing arrangement, 51 first slit

52 53 second sample chamber of first sample chamber, 54 first baffle

91 push 92 gas-guide tube of pulsometer, 93 air-tightness interface

101 second slit, 102 first reagent chamber, 103 second reagent chamber

104 second baffle, 211 location hole, 212 seal

213 crush-zone, 214 needling structure

Specific embodiment

The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or Value should be understood to comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively It between the endpoint value of a range and individual point value and can be individually combined with each other between point value and obtain one or more New numberical range, these numberical ranges should be considered as specific open herein.

As shown in figures 1 and 3, the utility model first aspect provides a kind of ELISA detections micro-fluidic chip, and feature exists In the micro-fluidic chip includes：For receive sample to be checked sample injection unit 1, for storage reaction reagent liquid storage unit 2, The reaction member 3 of reacting environment and waste unit for collecting waste liquid are provided for the sample to be checked and the reaction reagent 4；The micro-fluidic chip further includes to carry out the sample to be checked the first quantitative dosing unit 5；

Wherein, first dosing unit 5 includes the first slit 51 and the first sample separated by first slit 51 52 and second sample chamber 53 of this chamber；The first sample chamber 52 is communicated with the sample injection unit 1, second sample chamber Room 53 is communicated with the waste unit 4.

According to the utility model, the first dosing unit 5 is used to that the sample to be checked that participate in reaction quantitatively divide to take, It by extra sample transport to be checked at waste unit 4, can ensure to only have the sample to be checked of designated volume to participate in subsequent ELISA Reaction, so as to ensure the accuracy of the utility model chip detection.

Quantitative principle is carried out in conjunction with Fig. 3 to first dosing unit 5 to illustrate：As shown in Fig. 3 A, the The primary structure of a certain amount of unit 5 has a first baffle 54, first sample chamber 52, the second sample chamber 53, the first slit 51, and And micro-valve door is provided on the microchannel of 52 side of first sample chamber, the first baffle 54 is opposite with first slit 51 But do not connect, 52 and second sample chamber 53 of first sample chamber passes through the first baffle 54 and first slit 51 The space defined communicates.First dosing unit 5 carries out quantitative main operational principle to sample, before quantitative, first leads to The push-pull rod crossed in necessary instrument 9 closes micro-valve door (as shown in Fig. 3 B), then by sample entrance port to first sample Sample to be quantified is added in chamber 52, sample to be quantified can be first filled in first sample chamber 52, and extra liquid can be got over It crosses the first slit 51 and enters the second sample chamber 53, and then micro- access by being connected with waste unit 4 flows into downstream knot Structure unit (Fig. 3 C).Then as shown in fig.3d, micro-valve door is opened by necessary instrument 9, the sample after quantifying can be flowed into In downstream configurations unit.

Since chip structure is prepared on a large scale mode using injection molding, so the first gear of each chip interior Plate 54, first sample chamber 52, the second sample chamber 53, the position all same of the first slit 51, so as to ensure that each chip should After the volume of the quantitative chamber of first dosing unit 5 is identical, and then the different chips of guarantee are quantitative to same sample to be tested Volume is also identical, repeatability between which improves batch interior repeatability of chip and criticize.Optionally, should Quantitative chamber can also use other shapes, such as round, trapezoidal, square or other various irregular shapes, quantitative baffle Other shapes can also be used, for example other are variously-shaped for vertical baffle, arc-shaped baffle etc..

According to the utility model, by micro- logical between first dosing unit 5 and sample injection unit 1 and waste unit 4 Road is attached, in order to the flowing of liquid.Preferably, the connector between microchannel and waste unit 4 is located at waste unit 4 Top, main function is that the waste liquid after the completion of reaction or extra sample to be checked are transported in waste unit 4, at the same by The waste liquid entered inside waste unit 4 is caused not flow back into chip forepart by microchannel in the structural relation on position Point.

Wherein, the shape of the cross section of the microchannel can be rectangle, round, triangle, trapezoidal or other various shapes Shape.The width of microchannel 215 can be 0.001mm-10mm, and depth can be 0.001mm-10mm.

Wherein, piston structure is preferably provided in the micro-valve door, opening or closing can be completely by instrument according to pre- Program is determined to complete.Usually that under the intervention of instrument, micro-valve door is not in opening state or part micro-valve door is in opening state, 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 by a certain angle, So that the fluid path of internal piston is closed so that the transformation of micro-valve door is in off state.It is preferred, therefore, that necessary instrument 9 Further include stepper motor.

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

As another alternative embodiment, micro-valve door can also be opened by the cooperation of iron plate and electromagnetic field And shutoff operation, for example, a small iron plate can be pasted above micro-valve door, electromagnet is placed below micro-valve door, general Under state, electromagnet no power, iron plate is located above micro-valve door, and micro-valve door is in opening state, when needing to close, passes through instrument Device to be powered to electromagnet, and the electromagnetic field that magnet generates can attract the iron plate above micro-valve door, so as to move iron plate to micro-valve door Lower section so that micro-valve door is closed.

Herein it should be noted that, although being as above only quantitative to the micro-valve door in the first dosing unit 5 and first single The introduction that member 5 is carried out with waste unit 4 by the connection mode of micro- access and position, but it is equal to the micro-valve door referred to below It is applicable in.In the case of no explanation on the contrary, the effect and selection of the micro-valve door hereinafter referred to and other structures unit lead to It crosses mode that micro- access connect with waste unit 4 and position can refer to above.

According to the utility model, under preferable case, in order to ensure that detection can be smoothed out, the utility model it is micro-fluidic Chip further includes first be arranged between the first dosing unit 5 and waste unit 4 and reports an error unit 6, and described first reports an error unit 6 With chamber structure, which is used to receive the liquid of the second sample chamber 53 from the first dosing unit 5.Pass through in liquid After first dosing unit 5, extra liquid can enter the first chamber structure for reporting an error unit 6 by extracting under external force In, and judge to detect whether to be smoothed out according to the filling degree of liquid in the chamber structure.If in chamber structure It is hydraulically full, it is believed that liquid to be also filled at first sample chamber 52 in the first dosing unit 5, at this time it is believed that chip operation Normally without reporting an error, if first report an error unit 6 chamber structure inside it is inadequate without liquid or amount of liquid, then it is assumed that first is fixed The liquid volume for measuring first sample chamber 52 in unit 5 is also insufficient, needs to report an error and terminate the detection of chip at this time.Such chip Chip just will continue to carry out subsequent detection in the case that built in self testing mechanism can ensure only liquid quantitative entirely accurate, from And ensure the accuracy of testing result.

Wherein, it is preferred that described first unit 6 that reports an error is connected with optical detection apparatus, and the optical detection apparatus can be used In carrying out optical detection to the chamber, so as to judge the amount of liquid in chamber.

According to the utility model, the chip of the utility model includes at least one liquid storage unit 2, and Fig. 1 illustrates 9 liquid storages Unit 2, the main function of the liquid storage unit 2 are the various reagents that will participate in enzyme linked immunoassay, such as enzymic-labelled antibody examination Agent, sample diluting liquid, cleaning solution, enzyme linked immunological substrate reagent etc. are loaded into the utility model chip interior in advance, in this practicality When novel chip carries out the test of sample to be checked, then the reagent inside the liquid storage unit 2 is released in sequence by external force Come.Herein it should be noted that the size and shape of each liquid storage unit 2 of chip interior may be the same or different, Shape is also not limited to circle shown in figure, and other shapes such as rectangle, diamond shape, polygon or irregular shape are also the same It is applicable in.The size of each liquid storage unit 2 can change according to the volume size of the reagent to be pre-installed simultaneously, at this time liquid storage list The volume of member 2 simultaneously differs.

As shown in Figure 5A, the utility model provides a kind of basic structure of liquid storage unit 2, and prepackage reaction reagent is pre- It is first loaded into reagent pouch 21, which is preferably flexible material, which includes but not limited to：Nitrocellulose Film, 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 feature It is that can discharge internal prepackage reaction reagent by way of squeezing or needle pierces.Reagent pouch is loaded into prepackage reaction reagent After in 21, outlet is sealed to form seal 211 by way of thermoplastic envelope.The seal degree of seal 211 herein Than shallower so that when external force pressurizes reagent pouch 21, seal 211 can rupture.Location hole 212 is used to assist reagent capsule Bag 21 is fixed at the liquid storage unit 2 in chip.When needing to discharge 2 internal-response reagent of liquid storage unit, Ke Yitong The mode in Fig. 5 B is crossed, by squeezing the crush-zone 213 in reagent pouch 21, so that seal 211 is ruptured and discharged Liquid can also puncture the bottom section of reagent pouch 21 such as needling structure 214 by the way of in Fig. 5 C by pricker Place so that inside prepackage reaction reagent releases.

According to a kind of preferred embodiment of the utility model, the chip, which further includes, quantifies the reaction reagent The second dosing unit 10, for the precisely quantitative of reaction reagent.For example, when need to treat sample be originally diluted when, The diluted sample of predetermined extension rate can be obtained.It is as shown in Figure 4, second dosing unit 10 include second baffle 104, Second slit 101 and the first reagent chamber 102 and the second reagent chamber 103 separated by the slit 101；Described first Reagent chamber 102 is communicated with the liquid storage unit 2, and second reagent chamber 103 is communicated with the waste unit 4.Described Two baffles 104 are opposite with second slit 101 but do not connect, and 102 and second reagent chamber 103 of the first reagent chamber is logical It crosses the second baffle 104 and is communicated with the space that second slit 101 defines.Wherein, second dosing unit 10 is determined Amount principle is identical with the quantitative principle of the first dosing unit 5, and it is no longer repeated herein.

It is further preferred that in order to further ensure that the reaction reagent that can obtain predetermined amount, which also wraps It includes second set between 4 and second reagent chamber 103 of waste unit to report an error unit 11, described second unit 11 that reports an error has Chamber, the chamber are used to receive the liquid of the second reagent chamber 103 from the second dosing unit 10.Wherein, second report Wrong unit 11 report an error principle with first report an error unit 6 the principle that reports an error it is identical, it is no longer repeated herein.

Wherein, more preferably, described second unit 11 that reports an error is connected with optical detection apparatus, the optical detection apparatus For carrying out optical detection to the chamber, to judge the amount of liquid in chamber.

It is herein it should be noted that, although as shown in Figure 1, merely define the second dosing unit of a liquid storage unit 2 10.But those skilled in the art it should be appreciated that, the second dosing unit 10 can pass through quantity and connection mode Setting, quantifies different reaction reagents.These, which should be considered as, is within the protection scope of the utility model.

According to the utility model, the waste unit 4 is preferably placed at the end of liquid flow path system in chip, to collect The waste liquid of each step ELISA reactions prevents waste liquid outflow chip and pollutes external environment.Wherein, the waste unit 4 in structure simultaneously The rectangle shown in Fig. 1, other shapes, such as cylinder are not limited to, it is conical or other various irregular shapes also same Sample is applicable in.Wherein, the volume of the waste unit 4 should be greater than all liquid storage units 2 (in the presence of) interior reagent The sum of volume of volume and sample to be checked, so as to ensure waste unit 4 can to waste liquid issuable in reaction process into Row is effective to be collected.

According to the utility model, reaction member 3 is available to the main place that immune response occurs for antigen-antibody reagent, together When be also ELISA reagent participate in substrate reactions after optical detection main place, therefore herein will using translucency it is relatively good Material is prepared, such as polymetylmethacrylate or polycarbonate or polydimethylsiloxane etc..Reaction Unit 3 is connect with each liquid storage unit 2 by microchannel, anti-convenient for being directly entered this after the reagent release inside liquid storage unit 2 Unit 3 is answered, participates in subsequent enzyme linked immunoassay.

Although this reaction member 3 has gathered immune response function and optical detection function, optionally, can incite somebody to action This two parts function is divided among in two different structure units and carries out.Any group for so setting and carrying out on this basis It closes and/or deforms be considered as and be within the protection scope of the utility model.

According to a kind of preferred embodiment of the utility model, the inside of the reaction member 3 is further prepared with mixing arrangement 31, main function is that assisted reaction reagent and sample to be checked are mixed.As a preferred embodiment, the mixing Device 31 is made of multiple setting pillars, which can be located at both sides inside reaction member, in 3 inside quilt of reaction member During extruding, internal liquid can be promoted to be mixed, while liquid can be formed small when by erectting pillar around pillar Vortex, the disturbance of the small vortex can accelerate the mixing between liquid, so as to accelerate the immune response between each component, and then improve Biochemical reaction speed reduces the total time of chip detection.Although mixing arrangement 31 shown here is using the form for erectting pillar It realizes, but other micro-structures alternately, such as Z-type microchannel, W type mixers, the other structures such as triangle micro-structure Form can also assist and accelerate to mix between reagent.

According to the utility model, the sample injection unit 1 is used to receive the sample to be checked of user's injection.The sample to be checked can Think one or more in the body fluid such as the whole blood of human or animal, blood plasma, urine, saliva, sperm, spinal cord, amniotic fluid.In addition, this Utility model chip can be also used for field of detection of food safety, and detection sample can be food leachate, and extracting solution elutes Liquid or other liquid, to remains of pesticide in food, the bacterium in food, virus or other pathogens, nutrition contained by food product The content of substance is detected.Likewise, the utility model chip can also be applied to environmental testing, sample is detected at this time Can be river water, seawater or any liquid being derived from environment to be detected, it can be organic to the poisonous and harmful substance in environment Or inorganic pollution is used for quickly detecting.

Wherein, the structure of the sample injection unit 1 can be cylindrical, conical (for example, structure shown in Fig. 1), ladder Shape 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 noted The 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 time The sample to be checked entered can wick themselves into the structural unit in downstream, and capillarity can adsorb sample to be checked In chip, prevent the leakage of sample to be tested and pollute environment.

According to the utility model, when the sample to be checked is sample to be checked (for example, the whole blood sample) for needing to filter, institute It states micro-fluidic chip and further includes the filter element 12 being arranged between the sample injection unit 1 and first dosing unit 5, That is, the filter element 12 is located at the downstream of sample injection unit 1, the upstream of the first dosing unit 5.It can be pre- inside filter element 12 Equipped with can filter sample to be checked, for example, in whole blood red blood cell hemofiltration film, the thickness of the hemofiltration film is less than filter element 10 height, so that hemofiltration film can be fully seated inside filter element 10, while the shape of the hemofiltration film and filtering The interior wall construction of unit 12 is consistent, so as to get the sample to be checked up to filter element 10 can not bypass hemofiltration film and be directly entered down Swim channel.The hemofiltration film can be such that liquid is detached with red blood cell by physical pore size or biology, chemical reagent, the biology, chemistry Reagent can be coagulant.The thickness of the hemofiltration film can be 0.1mm-10mm, and the height of the filter element 10 can be 0.1mm-20mm。

According to a kind of preferred embodiment of the utility model, the utility model passes through between micro- access to chip system Pressure or decompression are carried out so as to control the flowing of each liquid, to enter another structural unit from a structural unit.Cause This, the micro-fluidic chip further includes the ventilation unit 7 being connect with the waste unit 4, for being carried for micro-fluidic chip system For required external pressure, so that internal liquid runs well under the auxiliary of external pressure.Ventilation unit 7 can also be prevented simultaneously Only the waste liquid in waste unit 4 flows out to chip exterior and pollutes external environment.Although ventilation unit 7 as shown in Figure 1 uses W Type microchannel is designed, but other are designed, such as round, arc, other various structures such as Z-type, which can similarly play, ventilates and prevent Only the effect of waste liquid outflow, these designs should all also be treated as being within the protection scope of the utility model.

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

According to a kind of preferred embodiment of the utility model, the micro-fluidic chip of the utility model preferably passes through outside Air pressure needed for 9 offer system of necessary instrument.It is connect it is preferred, therefore, that the micro-fluidic chip is further included with the ventilation unit 7 Tool interface system 8, the tool interface system 8 is used to connect necessary instrument 9 outside flow control chip system, and the necessary instrument 9 provides Air pressure the micro-fluidic chip system is provided to by the ventilation unit 7.Wherein, the size of the external pressure can compare Atmospheric pressure is big, can also be smaller than atmospheric pressure, and the size of the air pressure can be easily adjusted according to specific service condition difference.Together When, the tool interface system 8 is preferably air-tightness, i.e. chip, should when the air pressure regulator with necessary instrument 9 is docked Position is air tight.The function can be realized by plastic sealing ring or other assemblies.The air pressure regulator of necessary instrument 9 can To be air driven pump, gas storage vesica, pulsometer, extrusion pump etc..

According to a kind of preferred embodiment of the utility model, as shown in Figure 2, the necessary instrument 9 includes carrying For promotion pulsometer 91, gas-guide tube 92 and the air-tightness interface 93 being connect with the ventilation unit 7 of air pressure.When chip to be measured After being put into necessary instrument 9, the stepper motor (not shown) of instrument internal can push pulsometer 91 so that air-tightness Interface 93 is clung 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 being connect in the air-tightness Sealing ring or O-ring is set to complete on mouth 93.Preferably, the air-tightness interface 93 is tubaeform, in the preferred situation Under, it does not need to precisely align air-tightness interface 93 and tool interface system 8, it is only necessary to which tool interface system 8 is located at the air-tightness interface 93 Inside.It waits after the completion of being close to, the stepper motor being connect with pulsometer 91 is in the lock state, at this time the position of pulsometer 91 It is fixed.By the rotation of another stepper motor (not shown) being connected with 91 internal piston of pulsometer, can push Or extracting piston, so as to adjust the air pressure inside pulsometer 91, and then manipulate the air pressure of chip interior.It is needing to chip interior During pressurization, completed by stepper motor to push piston, on the contrary, when needing to depressurize to chip interior, pass through stepper motor It is realized to extract piston.Chip interior fluid path network can drive fluid to shift due to increase or the reduction of air pressure. It is airtight come this by the rotation of stepper motor being connect with pulsometer 91 after the entire testing process of chip to be measured is completed Property interface be detached from chip.

As shown in Figure 6, second aspect according to the present utility model provides a kind of ELISA detections micro-fluidic chip system, The micro-fluidic chip system is made of the upper, middle and lower；

Wherein, media layer damage detects micro-fluidic chip for ELISA as described above；

Wherein, superstructure and understructure close the middle layer for covering；It is provided in superstructure and sample introduction The sample holes and the relief hole corresponding to liquid storage unit 2 that unit 1 connects, for providing external impetus to discharge liquid storage unit 2 In reaction reagent.

As shown in Figure 7, sample holes (aperture) are provided with above superstructure, for the sample-adding of sample to be checked.It is described into Sample 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 holes In member 1.In addition, being further preferably provided at least one relief hole (macropore) in superstructure, the relief hole is tied corresponding to middle level The liquid storage unit 2 of structure, for the entrance of the push rod of necessary instrument 9, so as to provide external pressure for liquid storage unit 2, by liquid storage The reagent of the inside of unit 2 enclosed storage in advance releases, and therefore, the necessary instrument 9 of the utility model further includes at least one Push-pull rod.The relief hole size and shape by the structure size of the liquid storage pouch 21 of liquid storage unit in media layer damage and shape Lai It determining, it is preferred that the diameter of the relief hole can be 0.5mm-50mm, and shape can be round, rectangle, polygon, Diamond shape or even irregular shape etc. are variously-shaped.

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

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

The preparation method of ELISA detections micro-fluidic chip provided by the utility model preferably includes following steps：

Reaction reagent needed for enzyme linked immunoassay is preloaded onto in the reagent pouch 21 by step 1), and sealed by thermoplastic The seal 212 of the reagent pouch 21 is sealed by mode；And pass through location hole 211 and be placed on chip system media layer damage Specific liquid storage structure at, the reagent pouch 21 of each liquid storage is further fixed on chip system middle level by way of thermoplastic envelope In structure.

Step 2) carries out point sample fixed trapped antibody at the reaction member 3 of chip, and then adding in closed protein reagent will It is closed on the surface of the unlocked capture antibody in 3 inside of reaction member.Understructure is fitted into media layer damage again after the completion In the following, its laminating type includes but are not limited to the modes such as ultrasonic hot melt, gluing, ultraviolet light curing, thermoplastic envelope.

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

Use ELISA provided by the utility model detection micro-fluidic chip system carry out ELISA detections flow it is following (with For whole blood, and the second dosing unit 10 is arranged on the lower section of dilution to be quantified to dilution)：

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

For step 2) necessary instrument 9 by internal stepper motor by pulsometer 91, air-tightness interface 93 is moved to tool interface system 8 It is fixed behind place.The micro-valve door between the first dosing unit 5 and reaction member is closed, then by stepper motor in pulsometer 91 Piston be stripped, under the action of draft, whole blood enters in sample injection unit 1, then enters back into filter element 12 and carries out Red blood cell filtration, the blood plasma after filtering enter the first dosing unit 5, and extra blood plasma reports an error into first in unit 6, passes through The optical detecting unit of necessary instrument 9 detects the first optical signal (necessary instrument 9 further includes optical detecting unit) for reporting an error unit 6, If the first chamber for reporting an error unit 6 is not full of for empty or liquid, stop chip detection, and report an error, if this Place's liquid is full of, then continues subsequent detecting step.Same mode discharges the dilution in liquid storage unit 2, and carries out It is quantitative.

Step 3) opens the micro-valve door between the first dosing unit 5 and the second dosing unit 10 and reaction member 3, closes anti- The micro-valve door between unit and waste unit is answered, discharges each reaction in liquid storage unit 2 successively by the push-pull rod of necessary instrument 9 Reagent：First cleaning solution, the second cleaning solution, third cleaning solution, enzymic-labelled antibody, the 4th cleaning solution, the 5th cleaning solution.This is tried Agent is discharged into reaction member 3 successively, wherein needing repeatedly to mix cleaning in cleaning process completely, passes through pushing away for necessary instrument 9 Pull rod squeezes the reaction member 3 to be mixed in mixing arrangement 31.

Waste liquid after step 4) cleaning is completed is drawn by the pulsometer 91 on necessary instrument 9 in waste unit 4, most Afterwards pass through the push-pull rod of necessary instrument 9 discharge liquid storage unit 2 in the first chromogenic substrate and the second chromogenic substrate so that substrate into Enter to reaction member 3, carry out enzyme linked immunoassay, light is carried out by the light detection module on necessary instrument 9, such as CCD or PMT Detection, and provide testing result.

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

In the utility model, necessary instrument 9 is small portable apparatus, and necessary instrument 9 is in addition to including pulsometer 91, air guide Except pipe 92 and air-tightness interface 93, further preferably include push-and-pull bar unit, stepper motor module, light detection module.Into one Step is preferred, and 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 utility model additionally provides ELISA as described above detection micro-fluidic chip and/or described ELISA detects application of the micro-fluidic chip system in ELISA detections.

The utility model will be described in detail by embodiment below.

Embodiment 1

The present embodiment detects the c reactive protein (CRP) in people's whole blood for explanation using Double antibody sandwich ELISA

Enzymic-labelled antibody used in the present embodiment is horseradish peroxidase-labeled CRP (HRP-CRP), and cleaning solution contains The phosphate buffer (pH 7.5) of 0.5%tween-20, dilution are the phosphate buffers (pH 7.2) of 0.01mol/L, Coated antibody is anti-human CRP antibody, and closed reagent is the phosphate buffer (pH 7.5) containing 0.2%BSA inert proteins.Enzyme The reaction substrate joined needed for immune response is TMB chromogenic substrates.

(1) assembling of chip system

By above-mentioned each reaction reagent in advance in reagent pouch 21, and (heating time 30s) will by way of thermoplastic envelope The seal 212 of the reagent pouch 21 is sealed；And pass through location hole 211 and be placed on the specific of chip system media layer damage At liquid storage structure, the reagent pouch 21 of each liquid storage is further fixed on chip system media layer damage by way of thermoplastic envelope On.Point sample instrument is reused by inside coated antibody reagent point sample to reaction member 3, point sample is dried 3 hours after 40 DEG C so that point Coated antibody of the sample at reaction member 3 is sufficiently secured in reaction member 3.100 μ L closed reagents are added in pipettor to arrive In reaction member 3, dried in 40 DEG C 3 hours and Seal treatment is carried out to antibody.

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), so Prepare 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 equal Carry out following operation：Whole blood standard items of the 50 μ L containing CRP are taken with pipettor, chip system is injected by the sample holes of chip In media layer damage, 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 sample This 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 fixed Unit 5 is measured, extra 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 time Whether 53 internal liquid of D-M (Determiner-Measure) construction of unit 5 is full of, if in normal full state, continues follow-up chip inspection It surveys, otherwise instrument, which reports an error, exits testing process.Other extra measuring samples are drawn into waste unit 4, then by matching The push-pull rod of set instrument 9 discharges the reaction reagent in liquid storage unit 2 successively：Dilution, the first cleaning solution, the second cleaning solution, the Three cleaning solutions, enzymic-labelled antibody reagent, the 4th cleaning solution, the 5th cleaning solution.These reagents are discharged into reaction member 3 successively In, after each reagent releases, the reagent at reaction member 3 is passed through mixing by necessary instrument 9 by the effect of push-pull rod Device 31 is sufficiently mixed, cleaning complete after waste liquid be drawn into waste unit 4 by necessary instrument 9, finally by with Cover the push-pull rod release TMB chromogenic substrate reagents of instrument 9 so that substrate reagent, which enters, carries out enzyme linked immunological in reaction member 3 Reaction detects the intensity of optical signalling by the light detection module on necessary instrument 9, (its Detection wavelength is 450nm), and provides Testing result.Total detection time is 30min, and each standard items take its knot 5 times using 5 micro-fluidic chip system of determination respectively The average value of fruit draws standard curve.

Using same testing process, 50 μ L whole blood samples to be detected is taken to be detected, are obtained according to the intensity of optical signalling Go out the CRP concentration in whole blood sample to be detected.

The result shows that the lowest detection that CRP Concentration Testings are carried out using double antibody sandwich method is limited to 40pg/mL, model is detected It encloses for 40-8000pg/mL.It (is provided by the way that the result of testing result and laboratory routine enzyme-linked immunoassay is compared One specific bibliography), find the laboratory of the testing result and normal process obtained using the utility model chip ELISA testing results have high consistency, show there is accuracy using the testing result that the utility model chip obtains.Together When, it is measured, the change between the testing result of acquisition by carrying out repeatedly and (being used 10 times in the present embodiment) to same whole blood sample Different coefficient is respectively less than 10%, shows that this detection chip has good repeatability, can be further used as the ginseng of CRP medical diagnosis It examines.

The preferred embodiment of the utility model described in detail above, still, the utility model is not limited to this.At this In the range of the technology design of utility model, a variety of simple variants can be carried out to the technical solution of the utility model, including each Technical characteristic is combined with any other suitable method, these simple variants and combination should equally be considered as the utility model Disclosure of that belongs to the scope of protection of the utility model.

Claims (10)

1. a kind of ELISA detects micro-fluidic chip, which is characterized in that the micro-fluidic chip includes：For receiving sample to be checked Sample injection unit (1) provides instead for the liquid storage unit (2) of storage reaction reagent, for the sample to be checked and the reaction reagent Answer the reaction member (3) in place and for collecting the waste unit of waste liquid (4)；The micro-fluidic chip is further included for described Sample to be checked carries out quantitative the first dosing unit (5)；

Wherein, first dosing unit (5) separated including the first slit (51) and by first slit (51) first Sample chamber (52) and the second sample chamber (53)；The first sample chamber (52) communicates with the sample injection unit (1), described Second sample chamber (53) is communicated with the waste unit (4).

2. ELISA according to claim 1 detects micro-fluidic chip, which is characterized in that the micro-fluidic chip is additionally included in First set between waste unit (4) and the second sample chamber (53) reports an error unit (6), and described first reports an error unit (6) tool There is chamber, which comes from the liquid of the second sample chamber (53) of the first dosing unit (5) for reception；

Wherein, described first unit (6) that reports an error is connected with optical detection apparatus, and the optical detection apparatus is used for the chamber Optical detection is carried out, to judge the amount of liquid in chamber.

3. ELISA according to claim 1 or 2 detects micro-fluidic chip, which is characterized in that the micro-fluidic chip further includes For carrying out quantitative the second dosing unit (10) to the reaction reagent；

Wherein, second dosing unit (10) separates including the second slit (101) and by second slit (101) First reagent chamber (102) and the second reagent chamber (103)；First reagent chamber (102) and the liquid storage unit (2) phase Logical, second reagent chamber (103) communicates with the waste unit (4).

4. ELISA according to claim 1 or 2 detects micro-fluidic chip, which is characterized in that the micro-fluidic chip further includes Second set between waste unit (4) and the second reagent chamber (103) reports an error unit (11), and described second reports an error unit (11) there is chamber, which comes from the liquid of the second reagent chamber (103) of the second dosing unit (10) for reception；

Wherein, described second unit (11) that reports an error is connected with optical detection apparatus, and the optical detection apparatus is used for the chamber Room carries out optical detection, to judge the amount of liquid in chamber.

5. ELISA according to claim 1 detects micro-fluidic chip, which is characterized in that is set in the reaction member (3) There is mixing arrangement (31), for sample to be checked to be mixed with reaction reagent；

Wherein, the mixing arrangement (31) is selected from erectting in pillar, Z-shaped microchannel, W types mixer and triangle micro-structure extremely Few one kind.

6. ELISA according to claim 1 or 2 detects micro-fluidic chip, which is characterized in that the micro-fluidic chip further includes The ventilation unit (7) being connect with the waste unit (4), for providing required external pressure for micro-fluidic chip system.

7. ELISA according to claim 6 detects micro-fluidic chip, which is characterized in that the micro-fluidic chip further include with The tool interface system (8) of the ventilation unit (7) connection, the tool interface system (8) is for connecting matching outside micro-fluidic chip system Instrument (9) is covered, the air pressure that the necessary instrument (9) provides is provided to the micro-fluidic chip body by the ventilation unit (7) System.

8. ELISA according to claim 7 detects micro-fluidic chip, which is characterized in that the necessary instrument (9) is including use In the pulsometer (91), gas-guide tube (92) and the air-tightness interface (93) being connect with the ventilation unit (7) that provide air pressure；Institute It is tubaeform to state air-tightness interface (93).

9. ELISA according to claim 1 detects micro-fluidic chip, which is characterized in that the sample injection unit (1) and described Filter element (12) is additionally provided between first dosing unit (5) to be originally filtered to treat sample.

10. a kind of ELISA detects micro-fluidic chip system, which is characterized in that the micro-fluidic chip system by upper strata, middle level and under Layer composition；

Wherein, media layer damage detects micro-fluidic chip for the ELISA described in any one in claim 1-9；

Wherein, superstructure and understructure close the middle level for covering；It is provided in superstructure and sample injection unit (1) The sample holes of connection and the relief hole corresponding to liquid storage unit (2), for providing external impetus to discharge liquid storage unit (2) In reaction reagent.