CN103529195B - Detection method applied to measurement of trace target materials - Google Patents
Detection method applied to measurement of trace target materials Download PDFInfo
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- CN103529195B CN103529195B CN201310507020.3A CN201310507020A CN103529195B CN 103529195 B CN103529195 B CN 103529195B CN 201310507020 A CN201310507020 A CN 201310507020A CN 103529195 B CN103529195 B CN 103529195B
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Abstract
The invention discloses a detection method applied to measurement of trace target materials. The detection method comprises the following steps: (1) taking a sample feeding device and fixing a biological chip in a detection area; (2) closing a sample feeding channel and an auxiliary channel, and pumping buffer solution into a detection channel; (3) closing the detection channel and pumping samples to be detected, wherein the samples to be detected in the section form a detection section; (4) closing the sample feeding channel and the auxiliary channel, pumping the buffer solution into the detection channel, and pushing the detection section and covering the detection area; (5) performing a first conjugation reaction; (6) pushing the detection section out of the detection area by using the buffer solution after the first conjugation reaction is ended, and closing the detection channel; (7) replacing the samples to be detected through the same manners of the steps (3) and (4) by using signal material liquid so as to generate a signal section and cover the detection area; (8) performing a second conjugation reaction to generate samples; (9) closing the sample feeding channel and the auxiliary channel, pushing residual material liquid out of the detection channel by using the buffer solution; and (10) collecting and analyzing the samples in the detection area.
Description
Technical field
The present invention relates to a kind of detection method for blood sample trace target substance, belong to field of biomedicine technology.
Background technology
At present, the generation of human body diseases often produces the variation of kind or the quantity of biomolecule in vivo, and people can be carried out the early diagnosis of disease or be understood the differentiation of disease by the variation of the generation of some significant biomolecule in detection bodies or quantity.For example, myoglobins Myoglobin when heart injury in blood, Mb, creatine kinase isozyme (Creatine KinaseMB, CK-MB) and cardiac muscle troponin I (Cardiac Troponin I, cTnI) protein molecular will produce, Enrichment.Clinician comes Diagnosing Cardiac degree of injury or result for the treatment of by the content of these biochemical markers in body.But the specificity marker molecule content of most diseases is all very low, even detects these significant target materials in ultratrace level at trace and remain a huge challenge.
About trace, chemically refer to minimum amount, few only vestige a little.Trace analysis trace analysis, refer to content in material 1,000,000/analytical approach of following combination.The implication of trace one word changes to some extent along with the development of Analytical Methods of Trace.Therefore, trace is the value of confining spectrum gradual change.
Trace analysis comprises to be measured the total concentration of trace element in sample and measures trace element in sample or the distribution situation of specimen surface by probe technique.Generally be divided into 3 basic steps: sampling, sample pretreatment and mensuration, lay particular emphasis on mensuration link herein, but understand for contributing to, the first two link is only made a brief description.
Because tested element content in sample is very low, distribute very inhomogeneously, particularly environmental sample, often in time, spatial variations fluctuation is very large, give one's full attention to samples' representativeness and ensure certain sample size.In order to strengthen the Detection capability of trace constituent and to remove basic interference, separation and the enrichment of trace components are usually absolutely necessary, and have two schemes: one is that key component is separated from sample, allow trace components stay in solution; Another kind is trace components is separated and allowed key component stay in solution.In order to improve separation, concentration effect, conventionally apply macking technique.Another object of sample pretreatment is trace components to be changed into be most appropriate to the last form of measuring.Conventional separation, enrichment method have volatilization, precipitation and co-precipitation, electrolysis, liquid-liquid extraction, ion-exchange, chromatogram, extraction-chromatography, electrophoresis etc.In separation, enrichment process, to be given one's full attention to for the loss of pollution and trace components.
The method of measuring trace components, depends on sensitivity, accuracy, precision and the selectivity of analyzed object and assay method and rationality economically.The trace analysis methods such as the instrumental analysis from classical colourimetry and spectrophotometric method to various modern ages, more conventional method has: 1. optical means.Comprise spectrophotometric method, atomic emission spectrometry, atomic absorption spectrophotometry, atomic fluorescence spectrometry, molecular fluorescence and phosphorimetry, chemoluminescence method, Laser Enhance Ionization Spectrometry etc.2. electrochemical method.Comprise polarogarphy, coulometry, potential method and chronoptentiometry etc.3. x-ray method.Comprise electron microprobe method, x ray fluorescence spectrometry etc.4. radiochemical method.Comprise activation analysis, isotope dilution method, radioactive label analytic approach etc.5. mass spectroscopy.Comprise secondary ion mass spectrum analysis, spark source solid mass spectrum.6. chromatography.Comprise vapor-phase chromatography, liquid phase chromatography, the chromatography of ions etc.
Blood Biochemical mark conventional sense method has radioimmunoassay method RIAs, high pressure liquid chromatographic analysis method, electrochemical methods, protein analyzer, various diagnostic kit and immunity-chromatography test strip etc. clinically at present, these method ubiquity radioactive contamination, need large-scale instrument, length consuming time, the shortcoming such as can not quantitatively detect, application clinically has certain limitation.
Summary of the invention
The object of the invention is for overcoming above-mentioned the deficiencies in the prior art, provide a kind of operation terse, the operational phase is consuming time few, can quantitatively detect the detection method of measuring for trace target substance that detection efficiency is high.
For achieving the above object, the present invention adopts following technical proposals:
A detection method of measuring for trace target substance, the sampling and analyzing device that the pick-up unit that uses comprises sample adding device and sample that sample adding device obtains is detected, described detection method comprises:
1) get a sample adding device, and be fixed for catching the biochip of marker substances to be detected in detection zone;
2) close application of sample raceway groove and assist gallery, pump into damping fluid (as PBS damping fluid) to detecting raceway groove, at least fill the detection raceway groove between application of sample raceway groove and assist gallery;
3) close detection raceway groove, pump into and contain sample to be tested by application of sample raceway groove, assist gallery earial drainage, sample to be tested is at least filled the detection raceway groove between application of sample raceway groove and assist gallery, sample to be tested in this section forms detection segment, and wherein sample to be tested is the sample that contains described band detection marker substances;
4) close application of sample raceway groove and assist gallery, pump into damping fluid to detecting raceway groove, detection segment is pushed to detection zone, and covers detection zone;
5) the first association reaction, the time that the sample to be tested in detection segment and biochip reaction first are given;
6) after the first association reaction, use damping fluid that described detection segment is released to detection zone, then close detection raceway groove;
7) utilize step 3) and 4) identical mode, use semiochemicals liquid to substitute sample to be tested and produce signal segment, cover detection zone;
8) the second association reaction, makes material that semiochemicals and detection zone produce through association reaction for the first time carry out association reaction for the second time within second given time, produces sample;
9) close application of sample raceway groove and assist gallery, utilize damping fluid detection zone is completed after association reaction for the second time to remaining material liq to release and detect raceway groove by detecting raceway groove;
10) utilize acquisition and analysis device to carry out collection analysis to the sample of detection zone.
Described detection zone is modified to the nanometer thin rete of coupling organism-absorbing chip.
Avidin is further modified on described nanometer thin rete surface, with biotinylated probe molecule coupling, and increase binding site by stem grafting method, formation storage unit.
Described semiochemicals is signal antibody or signal aptamer, and both are all marked with probe molecule, and probe molecule is enzyme molecule or fluorescence molecule.
Described sample adding device comprises:
Detect raceway groove, one section of raceway groove in its downstream is configured to detection zone, and at least leaves detection window at detection zone place;
Application of sample raceway groove, be connected in detect raceway groove Upstream section and form application of sample bypass;
Assist gallery, is connected on the detection raceway groove between application of sample raceway groove tie point and detection zone, is configured for the bypass of application of sample earial drainage; And the detection channel length between application of sample raceway groove and assist gallery is greater than the length of detection zone;
Signal piping unit, is coupled to detection raceway groove, and is provided with operation valve;
Application of sample pipe-line cell group, is coupled to application of sample raceway groove and assist gallery, and is provided with operation valve group;
Testing pump, by the upstream extremity of described signal piping unit and corresponding operation valve access detection raceway groove, detects auxiliary liquid with pumping;
Sample-adding pump group, by described application of sample pipe-line cell group and corresponding operation valve group access application of sample raceway groove, with corresponding pumping sample adding liquid group; And
Control module, connects testing pump, sample-adding pump and operation valve and operation valve group, with the sequence of movement of logic control testing pump, sample-adding pump group and the operation valve that matches, operation valve group.
The invention has the beneficial effects as follows, according to the present invention, by means of simply constructed raceway groove, then coordinate the analysis of sample, the Whole Equipment compactness that uses, the accurate and whole operation of application of sample amount is relatively terse, and what institute's elapsed time was more was embodied on the reaction time, operational phase holding time is few, and the detection efficiency of entirety is higher.Such as using sample adding device continuous sample-adding 10 times, the single sampling time was less than for 2 seconds, measured application of sample amount volume each time, and the coefficient of variation is less than 1%.
Brief description of the drawings
Fig. 1 a is an embodiment raceway groove structural texture schematic diagram, and is expressed as the front view of application of sample;
Fig. 1 b is along a-a ' raceway groove filling damping fluid view;
Fig. 1 c is for to pass into sample to be tested view along b-c-d-b ' raceway groove;
Fig. 1 d, for to pass into damping fluid view along a-a ' raceway groove, pushes over cd section sample to be tested at the position that covers detection zone e;
Fig. 2 a is the structural representation of sandwich structure in an embodiment;
Fig. 2 b is the structural representation of sandwich structure in another embodiment;
Fig. 3 is that a kind of nano-probe storage unit forms flow process;
Fig. 4 is the usual sandwich structure that carries out as an example of luminescence probe example, modifies high loose nanometer thin rete and modify the comparison that further uses the responsive effects of three kinds of situations of nano-probe storage unit on the basis of high loose nanometer thin rete at detection zone e at detection zone e;
Fig. 5 is the sample of different target material concentrations detects the trace target substance of realizing quantitative detection design sketch by embodiment of the present invention method;
Fig. 6 is three kinds of biochip test result comparison diagrams in embodiment;
Fig. 7 is the raceway groove schematic diagram of structure of parallel detection in an embodiment;
Fig. 8 is raceway groove schematic diagram of structure in another embodiment;
Fig. 9 is raceway groove schematic diagram of structure in another embodiment;
Figure 10 is a kind of raceway groove structural texture schematic diagram;
Figure 11 is another kind of raceway groove structural texture schematic diagram;
Figure 12 is optical detecting module structural representation;
Figure 13 is the principle of work schematic diagram that light path is covered plate;
In figure: 1. detect raceway groove, 2. application of sample raceway groove, 3. detection zone, 4. substrate, 5. assist gallery, 6. seizure antibody, 7. target substance, 8. signal antibody, 9. probe molecule, 10. catch aptamer, 11. signal aptamers, 12. upper cover plates, 13. first type raceway grooves, 14. Second-Type raceway grooves, 15. cover plate, 16. lens, 17. 2 to spectroscope, 18. pin holes, 19. first optical filters, 20. photoelectric commutators, 21. data processing modules, 22. displays, 23. light sources, 24. second optical filters, 25. ball screw assembly,s, 26. effective fluorescence, 27. exciting lights, 28. invalid fluorescents, 29. biochips.
Embodiment
Below in conjunction with drawings and Examples, the present invention is further described.
In order to make object, technical scheme and the advantage of embodiments of the invention clearer, below in conjunction with embodiment and accompanying drawing, the technical matters to many levels of the present invention and technological means used are elaborated.At this, illustrative examples of the present invention and explanation be only for explaining the present invention, but not as to concrete restriction of the present invention.
In such a concept, as detect raceway groove 1, it is a fluid passage in essence, and not for detection or raceway groove specifically limit.As the fluid passage of runoff, can distinguish positions different on fluid circulating direction with upstream, midstream and downstream, those skilled in the art should be expressly understood this.
Be to be understood that, if the core word of detection window is " window ", but be not expressed as a window of offering, can be used in detection and be expressed as, be one with " can " term that is limited, as using telescope to look far into the distance in fixed position, it also meets the condition that can look far into the distance around, establishes separately window but be not illustrated in position of telescope.
Should be appreciated that herein, as application of sample pipe-line cell, be not a pipeline, and should be understood to application of sample pipe system.
A pick-up unit of measuring for trace target substance 7, is configured to 29, one sample adding devices of a biochip and a detection module for Detection of Weak Signals.
About biochip 29, claim again DNA chip or genetic chip, they are crystallizations that DNA hybridization probe technology combines with semi-conductor industry technology.After this technology means a large amount of probe molecules 9 is fixed on holder be with fluorescently-labeled DNA sample molecule to hybridize, by detecting the hybridization signal intensity of each probe molecule 9 and then obtaining quantity and the sequence information of sample molecule.In this article, substrate 4 and raceway groove add that biomolecule is configured to a biochip 29.
Biochip technology originates from making nucleic acid molecular hybridization.So-called biochip refers generally to high density and is fixed on the microarray hybridization cake core (micro-arrays) of the biological information molecule (as genetic fragment, DNA fragmentation or polypeptide, protein, glycan molecule, tissue etc.) on mutual supporting dielectric, in array, the sequence of each molecule and position are known, and are pre-set sequence dot matrix.Micro-fluidic chip (microfluidic chips) and liquid phase biochip are than the biochip new technology developing after micro-array chip, and biochip technology is the substance of Systems biotechnology.
Say more intuitively, biochip 29 is put biological sample exactly on the materials such as glass sheet, silicon chip or a nylon membrane, then collects signal by a kind of instrument, uses Computer Analysis data result.
Although people may be easy to a biochip and electronic chip connects, and in fact, both truly have a common ground the most basic: the data message in microsize with magnanimity.But they are diverse two kinds of things, on electronic chip, Boulez is semi-conductor electricity subelement one by one, and on biochip Boulez be bioprobe molecule 9 one by one.
Therefore, in the structure being made as Figure of description 1a, detection zone 3, the region that namely in figure, e represents, for fixed trapped material molecule, for catching the biomolecule of application of sample liquid.
For the measurement of trace target substance 7, in such a sample adding device, its basic configuration should comprise:
Detecting raceway groove 1, in the structure at figure as shown in 1a, is a straight raceway groove, and two ends are identified by a-a ', and a end is fluid upstream end, and the other end is fluid outflow end.One section of raceway groove in its downstream is configured to detection zone 3, and at least leaves detection window at 3 places, detection zone.
Ying Zhi, in the time detecting raceway groove 1 bending or while there is bending structure, little on overall fluid pumping impact, the detection raceway groove 1 being not specifically limited on this basis can be understood as to meet arbitrarily and moves towards.
From flow resistance angle, preferably adopt straight channel structure, flow resistance is little, for identical pump, nominally can have larger lift.
Detecting raceway groove 1 can get on to understand it from another one concept, predetermined substance can be transported to detection zone by the use that detects raceway groove.
So specific material is just carried by application of sample raceway groove 2, and it is connected in the Upstream section of detection raceway groove 1 and forms application of sample bypass, and the various fluids of application of sample are all carried by application of sample raceway groove 2.
Be to be understood that, because said various Fluid Volumes are very little, so relevant raceway groove cross section is also not too large, while contact with liquid according to solid because of not infiltrating and infiltration phenomenon that surface tension produces, thereby can ensure application of sample liquid in cross section smaller passage carried reliably by entirety.
Ying Zhi, does not infiltrate when showing as solid and contacting with liquid, and surface of contact is tending towards dwindling, liquid can not be attached to the phenomenon on solid.As mercury can not be attached on glassly, mercury to be poured in glass container, the angle of mercury and glass wall contact position is greater than 90 °, shows that surface of contact is tending towards dwindling, and mercury does not infiltrate glass.Water can not be attached on paraffin, illustrates that water does not infiltrate paraffin.
Concept on the other side is to infiltrate, as is placed on a water on clean glass plate, can be attached to and on glass plate, form thin layer.A clean glass sheet is entered in water and taken out, and glass surface can be stained with one deck water.This phenomenon is called infiltration.Concerning glass, water is to infiltrate liquid.
So associated as capillarity, infiltrate liquid and in kapillary, rise in (as water-glass), liquid level becomes concave moon surface type, and liquid is attached to wall; Do not infiltrate liquid and in kapillary, decline in (as mercury-glass), liquid level becomes gibbous moon face type, and liquid is non-cohesive at wall, and surface tension makes its protrusion.
According to above-mentioned principle, can be to one section of liquid being carried out to overall conveyance in the smaller raceway groove in cross section.
For sample adding device, in order to ensure normally carrying out of application of sample, further configure assist gallery 5, as shown in Figure 1a, assist gallery 5 and actual another fluid passage of formation of application of sample raceway groove 2, linked by the cd section sense channel shown in Fig. 1, forms b-c-d-b ' passage.Need to assist like this ditch to 5 to be configured to be connected on the detection raceway groove 1 between application of sample raceway groove 2 tie points and detection zone 3, be configured for the bypass of application of sample earial drainage; And detection raceway groove 1 length between application of sample raceway groove 2 and assist gallery 5 is greater than the length of detection zone 3.
The requirement of length is inevitable diffusion phenomena between different liquid levels, may cause cross section fuzzy, and the while is about the amount of pumping liquid, control that can not be desirable.In order to carry out given reaction in detection zone 3, need the liquid segment of application of sample can cover detection zone 3 completely, go forward side by side and may eliminate the impact of control accuracy and different liquids diffusion.
In order to obtain liquid cross-sectional relatively clearly, in the structure shown in Fig. 1 a, application of sample raceway groove 2 is all vertical with detection raceway groove 1 with assist gallery 5.
Arranging according to the simple above-mentioned raceway groove of introducing, is to meet sending into of liquid so as to the channel system of setting up, further need to be to its pipe arrangement.Due to as shown in Figure 1a, 4 interfaces, and control mode are altogether also uncomplicated, needn't as " pipe arrangement databook ", carry out complicated PIPING DESIGN, and only need to also distinguish and fluidly carry out pipe arrangement according to needed control mode.
Pipe arrangement is divided into two large parts, is respectively for detection of detection raceway groove Unit 1 of raceway groove pipe arrangement with for the application of sample pipe-line cell group of application of sample raceway groove 2 and assist gallery 5 pipe arrangements.Then configure relevant operation valve, for detection of the break-make control of raceway groove 1, application of sample raceway groove 2 and assist gallery 5, wherein two raceway grooves below, because unitary construction is a fluid passage, should carry out synchro control.
In following content visible it, a end that detects raceway groove 1 is mainly used in pumping into a kind of liquid, application of sample raceway groove 2 need to add two kinds of liquid.For this reason, the b end of application of sample raceway groove 2 can configure solenoid directional control valve, as three-position three-way valve, and the intervention for two kinds of liquid in the different periods, and close down control.Also can utilize a threeway to connect two pipelines, on each pipeline, configure an operation valve.
Simple with regard to pipe arrangement, generally also comprise the configuration of valve, therefore, for simple control, those skilled in the art can configure accordingly, and because the pipeline configuration that the application addresses is relatively simple, its piping method does not repeat them here.
And then relate to the configuration of pump about described sample adding device, and be also configured to testing pump and sample-adding pump group according to above-mentioned pipe arrangement, wherein the latter is preferably made into multiple according to the kind of liquid.
Testing pump detects the upstream extremity of raceway groove 1 by described signal piping unit and corresponding operation valve access, detect auxiliary liquid with pumping.
Sample-adding pump group is by described application of sample pipe-line cell group and corresponding operation valve group access application of sample raceway groove 2, with corresponding pumping sample adding liquid group.
And then in order to control better, configuration control module, connects testing pump, sample-adding pump and operation valve and operation valve group, with the sequence of movement of logic control testing pump, sample-adding pump group and the operation valve that matches, operation valve group.
Known according to aforesaid content, steering logic is relatively simple, and those skilled in the art can realize relevant control algolithm without paying creative work.
About raceway groove, from structure, herein, more excellent selection similarly is more open channel, its foundation structure can be with reference to open channel structure, and the runoff xsect that shows as detection raceway groove 1, application of sample raceway groove 2 and assist gallery 5 is identical, and is plane towards the face of checkout equipment, be to be understood that accordingly, the plane is here mainly effectively carrying out of being conducive to detect, and the identical structure of xsect is conducive to form stable liquid section.
In certain embodiments, possible piecewise configures different structures, as part raceway groove is configured to pipe, and is configured to open channel structure in liquid section tectonic province and the liquid section district that flows through.
Embody to some extent about the content more than content of liquid section, and have concrete application in content below, do not repeat them here.
In as Figure 10 and structure as shown in figure 11, detect raceway groove 1, application of sample raceway groove 2 and assist gallery 5 correspondences and comprise the channel groove that is formed on substrate 4 upper surfaces and the upper cover plate 12 that is covered in corresponding raceway groove upper surface, wherein the runoff xsect of channel groove is the square of semicircle or lower surface drift angle arc, wherein semicircle raceway groove groove is expressed as the first type channel groove 13, and another kind of channel groove is expressed as Second-Type channel groove 14.
Channel groove is molded over the upper surface of transparent substrate 4, controls if adopted manually, can check flowing of fluid by the different colours of liquid, can facilitate manually and control.
About upper cover plate 12, due to needed substrate 4 upper surface area little, upper cover plate 12 can be one-piece construction, covers the upper surface of whole substrate 4.The upper cover plate 12 that also can mate according to channel groove tendency arranges.
About the specification of channel groove, limit according to its width of rebate, the width of rebate of described channel groove is 100 μ m～1000 μ m.Concrete specification should adapt with the sensitivity of detecting instrument, and detecting instrument sensitivity is enough high, and micro-channel width can reduce.Concrete specification also has relation with promotion liquid by the pump of micro-raceway groove in addition, and micro-channel width size is less, and resistance is larger, and the expulsive force of the pump needing is also just larger.Therefore, the specification of channel groove should consider the driving force of pump and the sensitivity of detecting instrument, and the width of rebate of channel groove should not be less than 100 μ m.
Because raceway groove is unsuitable excessive, the amount that comes from various liquid is also little again, and too large cross section also can affect the quality that liquid section obtains simultaneously, and for this reason, the width of rebate of channel groove should not be greater than 1000 μ m.
In actual application, we can face such problem, sometimes need multiple blood to analyze, or the multiple samples to same blood sample are analyzed, in order further to improve detection efficiency, one detects raceway groove 1, an application of sample raceway groove 2 and an assist gallery 5 forms an application of sample unit, and the detection raceway groove of multiple application of samples unit is connected in series or and connects detection when can carrying out accordingly as multiple blood sample.
As shown in Figure 7, there are multiple detection zone e in the longer detection raceway groove that aa ' indicates, and then can configure multiple application of sample raceway grooves and assist gallery, like this, as a total detection raceway groove can once cover three detection zones, can save on the whole the consumption of sample, and reduce the effect consumption to auxiliary liquid, reduce follow-up processing load.On the other hand, can also reduce the pipeline quantity of pipe arrangement, simplify overall structure.
Structure is as shown in Figure 7 a kind of version of serial, and structure is as shown in FIG. 8 and 9 parallel version, wherein the structure shown in Fig. 8 is a kind of simple overlaying structure, is that one-piece construction is reduced to some extent in the direction perpendicular to a-a ', has reduced the application of base material.
Fig. 9 has expressed another kind of parallel organization, and all detection raceway grooves that connect are centered by inlet and be common junction circular array, have public node, can reduce pipeline and connect quantity.And if adopted same pump, the problem of different processes in the parallel pipeline that the difference of intermediate duct length causes could be reduced.
About the problem that is related to of detection raceway groove 1 length between application of sample raceway groove 2 and assist gallery 5 and detection zone 3 length, in aforesaid content, have related, find through long-term experiment, when detection zone 3 length be detection raceway groove 1 length between application of sample raceway groove 2 and assist gallery 5 be not less than 1.5 times time, substantially there will not be that the fuzzy and control accuracy of liquid surface produces can not effectively produce interface clearly liquid section just cover the problem of detection zone completely, to ensure that target substance to be measured is trapped in detection zone 3 completely, guarantees to detect quality.Nature, this needs the control accuracy of pump valve to coordinate with it, but pump valve type selecting does not belong to the content that this case need to be considered, and under the pump valve configuration condition of degree of precision, above-mentioned multiple proportions is suitable for.
But detection zone 3 is unsuitable long, so can produce excessive structure and the loss of reagent, for this reason, getting detection zone 3 length is the upper limit for detecting 3 times of raceway groove 1 length.
Sample adding device is main improvement, as for pick-up unit, is containing under the condition of sample adding device, configures relevant checkout equipment, and checkout equipment can adopt the checkout equipment of the known final thing in matching detection district.
Mated, one detects raceway groove 1, an application of sample raceway groove 2 and an assist gallery 5 forms an application of sample unit, the detection raceway groove serial connection of multiple application of samples unit or and connect;
Correspondingly, described acquisition and analysis device is furnished with and drives it to move in turn the driving mechanism of each 3 detection positions, detection zone.
In content as previously shown, the area of substrate is very little, correspondingly, distance between each detection zone also can be very little, and driving mechanism must have enough driving precision so, preferably, described driving mechanism is ball-screw screw mechanism, has reached needed control.
In addition reference, if array chip micro-raceway groove location when the fluoroscopic examination is a key factor that affects testing result, adopt the secondary key feature as location of precision lead screw, precise ball screw pair 25 can reach in the displacement of 0.5 meter and only have the positioning error of several microns, can meet the accurate positioning requirements of multiple micro-raceway grooves detection zone on biochip.
As shown in figure 12, in certain embodiments, a kind of acquisition and analysis device comprises:
Monochromatic source, according to given light path outgoing, the light source that light source 23, the second optical filters 24 as shown in Figure 12 form, wherein light source 23 can directly adopt laser diode, also can adopt other laser pump (ing) to produce laser, and monochromaticity is relatively good.Also can produce based on described the second optical filter 24 laser of single wavelength.
And then configuration shaping mirror group, be configured in the light path of monochromatic source, be given light beam by beam shaping; After shaping mirror group is packed, be commonly called beam shaping mirror or beam shaping, mainly for generation of suitable light beam, the especially good light beam of linearity, can ensure the controllability of light path can obtain again suitable light intensity.
Further, plate 15 is covered in configuration, has via hole, for described given light beam by being irradiated to selected detection zone 3, and passing through for detection of district's 3 folded light beams; In structure as shown in figure 13, make detection zone to be measured 3 be exposed to exciting light 27 times by covering plate 15, and other and its parallel detection district is covered and can not be excited, even or excited on a small quantity its fluorescence to pass, thereby the problem of fluorescence phase mutual interference while effectively solving the parallel detection of many raceway grooves.The reflected light in the namely target detection district 3 between clearly can taking a fancy in figure can outgoing from cover the wish perforate of plate 15, form effective fluorescence 26, and the reflected light of other two detection zones is invalid fluorescence 28, can not be caught by corresponding optical device.
And then configuration pre-service optical element, is configured in folded light beam light path, for the pre-service of folded light beam, pre-service is limited to follow-up optical converter 20, is mated both collaborative type selectings.
Mated, a photoelectric commutator 20, receives through pretreated described folded light beam, converts light signal to electric signal.
Data processing module 21, input connects described photoelectric commutator 20, and the electric signal obtaining is calculated according to predetermined algorithm; And
Display 22, connects described data processing module 21, for output display result of calculation.
So, according to said structure, first light source sends exciting light, exciting light is reflected to dichronic mirror by two after mating plate purifying after filtration, arrive micro-raceway groove detection zone e through lens focus, excite the fluorescent molecular probe on signal antibody 8 or the signal aptamer 11 of staying detection zone e, make it send fluorescence signal, this fluorescence signal sees through two to dichronic mirror after light path is covered plate, lens, after pin hole and optical filter purifying, changed into electric signal by electrooptical device, pass through again data processing, will be presented on liquid crystal display with target amount of substance to be measured.
About two to spectroscope 17, concrete configuration is that folded light beam is vertical with the light path of monochromatic source outgoing, and then described acquisition and analysis device also comprises that the light path junction that is configured in folded light beam and monochromatic source outgoing is furnished with two to spectroscope 17, to the plain coil reflection of monochromatic source outgoing, and to folded light beam total transmissivity;
Correspondingly, two to spectroscope 17 between described pre-service optical element and described shaping mirror group.
Also is furnished with a punctured element at pre-service optical element and two between spectroscope 17, pin hole 18 as shown in figure 12, for seeing through the light beam to uniform section, and pre-service optical element is the first optical filter 19, gathers the light beam of required wave band to obtain matching optics converter 20.
And the detection method on basis, we are defined as a biochip substrate 4 and the raceway groove being molded in substrate 4, and like this, the each end of substrate 4 respectively coupling is provided with the inlet and the waste liquid outlet that communicate corresponding to each raceway groove.As shown in Figure 1a.By controlling micro-raceway groove liquid flow, can on biochip, realize the accurate application of sample of liquid.
Micro-raceway groove is provided with detection zone e, in the e of detection zone, be fixed with the seizure antibody 6 of target material to be measured or catch aptamer 10, can form in detection zone the sandwich structure to target material to be measured according to application of sample step, utilize probe molecule 9 and nanometer sensitization to convert trace target substance signal to optics or electrical signal in detection zone.
Specifically comprise the following steps:
1) get a sample adding device, and be fixed for catching the biochip of marker substances to be detected in detection zone 3.
2) and then close application of sample raceway groove 2 and assist gallery 5, pump into damping fluid to detecting raceway groove 1, at least fill the detection raceway groove 1 between application of sample raceway groove 2 and assist gallery 5; As shown in Figure 1, generally whether there is damping fluid to flow out the filling state that judges damping fluid from liquid outlet by observing, this situation need to be filled it up with whole detection raceway groove, certainly, can be by flow control, carry out the capacity matching of flow and raceway groove and control, under such controlled condition, relevant liquid needn't be full of whole raceway groove.
3) close and detect raceway groove 1, pump into and contain sample to be tested by application of sample raceway groove 2, assist gallery 5 earial drainages, sample to be tested is at least filled the detection raceway groove 1 between application of sample raceway groove 2 and assist gallery 5, sample to be tested in this section forms detection segment, and wherein sample to be tested is the sample that contains described band detection marker substances; As shown in Fig. 1 c, the damping fluid of cd section is released by sample to be tested, and sample to be tested is trapped in bcdb ' pipeline, and wherein cd section row becomes needed liquid section.
Should be appreciated that being described in herein only explanation about the keying of raceway groove closes, and opens and can clearly know those raceway grooves by context.
4) and then, as shown in Figure 1, close application of sample raceway groove 2 and assist gallery 5, to detect raceway groove 1 pump into damping fluid, detection segment is pushed to detection zone 3, and covers detection zone 3; In figure, different profile lines has represented different liquid, has clearly performance in figure.
5) the first association reaction, the time that the sample to be tested in detection segment and biochip reaction first are given, about the reaction time between relevant liquid, by those skilled in the art is known, to this, repeats no more.
6) after the first association reaction, use damping fluid that described detection segment is released to detection zone, then close detection raceway groove.
7) utilize step 3 and 4 identical modes, use semiochemicals liquid to substitute sample to be tested and produce signal segment, cover detection zone 3; Because the acquisition pattern of signal segment liquid is consistent with the acquisition pattern of detection segment liquid, again repeat no more.
8) the second association reaction, make material that semiochemicals and detection zone produce through association reaction for the first time carry out association reaction for the second time within second given time, produce sample, form a kind of sandwich structure, as the sandwich structure of " fixing seizure antibody 6 or seizure aptamer 10-testing protein-signal antibody 8 or signal aptamer 11 ".
9) close application of sample raceway groove 2 and assist gallery 5, utilize damping fluid detection zone is completed after association reaction for the second time to remaining material liq to release and detect raceway groove 1 by detecting raceway groove 1.
10) utilize acquisition and analysis device to carry out collection analysis to the sample of detection zone.
Micro-raceway groove detection zone e on described biochip, is fixed with the seizure antibody 6 of target substance 7 to be measured or catches aptamer 10.In the time having target substance 7 to be measured in sample, be fixed on the seizure antibody 6 of detection zone e or catch aptamer 10 and will catch target substance 7, adding meeting after signal antibody 8 or signal aptamer 11 form the sandwich structure to target substance 7 in detection zone.As shown in Figure 2 a and 2 b.
Those skilled in the art can be familiar with sandwich structure by the relevant document of reference antibody prize law, owing to not relating to the improvement of this type of scheme herein, does not repeat them here.
Described signal antibody 8 or signal aptamer 11 refer to be marked with probe molecule 9 can target substance 7 specific bindings to be measured antibody or aptamer, described probe molecule 9 can be enzyme molecule, fluorescence molecule or other probe molecule 9.
In certain embodiments, we are referred to as nanometer sensitization concept, mainly comprise following two parts content:
(1) at detection zone e decorated nanometer thin layer, utilize nano-material surface effect to realize at detection zone e and fix more antibody 6 or the seizure aptamer 10 of catching, improve target material capture ability to be measured in sample, thus the sensitivity of increase biochip.
The common concept of nano thin-film is nanometer film, membrana granulosa and dense film.Membrana granulosa is that nano particle sticks together, and there is the film in very tiny gap centre.Dense film refers to rete densification but crystallite dimension is nano level film.
The development of nanofiltration (NF) film and application are compared with reverse osmosis membrane approximately 20 years evenings.20 century 70 JECadotte research NS-300 films, are the beginning of research NF film.
Nanometer film can be held back the molecule that relative molecular weight is 300～100000 (separated material particle diameter is equivalent to 0.3～100 nanometer), can be used for this case.
" nanometer storage unit " can be expressed as " multiple probe molecules 9 are modified at the cell cube forming on nano particle ".Wherein the quantity of probe molecule 9 is relevant with the surface area of the nano particle functional group surperficial with it.In general, the surface area of nano particle is larger, more in the functional group of its finishing, and to be fixed on the quantity of nanoparticle surface more for probe molecule 9 so.But nano particle is unsuitable excessive, the cell cube that excessive nano particle can make the adhesion between signal antibody 8 or signal aptamer 11 and target substance 7 be not enough to signal antibody 8 or signal aptamer 11 to form is stayed detection zone, makes on the contrary detection signal reduce.
(2) design nanometer storage unit increases to the probe molecule 9 of mark on signal antibody 8 or signal aptamer 11.First at nanoparticle surface modified Avidin, and with biotinylated probe molecule 9 couplings, design different stem grafting methods and increase binding site, make probe molecule 9 in nanoparticle surface enrichment, each nano particle forms and comprises dozens of or the storage unit of multiprobe molecule 9 (as shown in Figure 3) more.
Nano-probe molecule 9 storage units are modified on the antibody or aptamer with target material specific binding to be measured, than common single or a small amount of probe molecule 9 is modified on antibody or aptamer, under the same conditions, the former can significantly increase the fixed amount of probe molecule 9 on sensor, sensor output signal is amplified, thereby obviously improve transducer sensitivity.
Fig. 4 is the responsive effect comparison (only taking luminescence probe as example) of three kinds of situations: in Fig. 4, (a) is common sandwich structure; (b) be to modify high loose nanometer thin rete at detection zone e, utilize nanometer layer specific surface effect can fix more seizure antibody 6 or catch aptamer 10 at detection zone e, increase the sample material capture probability that hits, improve detection sensitivity; (c) be to modify on the basis of high loose nanometer thin rete at detection zone e, adopted the nano-probe storage unit shown in Fig. 3, increased signal intensity, improve detection sensitivity.
Fig. 5 is the testing result that the sample of different target material concentrations obtains by above-mentioned detection method, and detected object is that brain is received peptide.It is an important indicator of reflection heart failure degree that brain is received peptide BNP (Brain natriuretic peptide), and BNP content in blood only has nanograms/milliliter magnitude, detects as example taking trace BNP in sample, and label probe is luminescence probe.
As can be seen from Figure 5,, according to said method, can obtain higher accuracy of detection.
In another embodiment, get 3 biochips, wherein No. 1 chip, at fixing 10, No. 2 chips of aptamer and No. 3 chips fixing aptamer 10 that catches again after detection zone e modifies high loose nanometer thin rete that catches of detection zone e, carries out application of sample according to the above-mentioned sample loading mode that adds to biochip.
The first, 3 biochip passes into damping fluid at its a-a ' passage respectively;
The second, 3 biochip passes at its b-b ' passage the serum sample that contains BNP to be measured respectively;
The 3rd, 3 biochips pass into damping fluid at its a-a ' passage respectively, and serum sample is pushed to detection zone, with the fixing seizure aptamer 10 in detection zone, specific binding occur under certain condition;
The 4th, after specific binding has reacted, 3 biochips continue to pass into damping fluid at its a-a ' passage respectively, and serum sample is released to detection zone;
The 5th, what the b-b ' passage of No. 1 chip and No. 2 chips passed into that the b-b ' passage of common 11, No. 3 chips of signal aptamer passes into is the signal aptamer 11 of decorated nanometer probe storage unit;
The 6th, 3 biochips pass into damping fluid at its a-a ' passage respectively, and signal aptamer 11 is pushed to detection zone, and signal aptamer 11, with the BNP that is trapped in detection zone, specific binding occurs under certain condition;
The 7th, 3 biochips pass into luminescence reagent at its a-a ' passage respectively, on optical detector, biochip test district are carried out to luminous detection.
The testing result of 3 biochips as shown in Figure 6, is 1. No. 1 chip; 2. be No. 2 chips; 3. be the testing result of No. 3 chips.
The sample of different target material concentrations as shown in Figure 5 detects the quantitative detection design sketch of the trace target substance 7 of realizing by embodiment of the present invention method, as can be seen from the results, at biochip test district decorated nanometer thin layer fixing antibody 6 or the seizure aptamer 10 of catching again, then use the signal of the biochip of the signal aptamer 11 of decorated nanometer probe storage unit obviously to be amplified, thereby realize the quantitative measurment to trace target substance 7.
By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendments that creative work can make or distortion still in protection scope of the present invention.
Claims (5)
1. a detection method of measuring for blood sample trace target substance, is characterized in that, the sampling and analyzing device that the pick-up unit that uses comprises sample adding device and sample that sample adding device obtains is detected, and described detection method comprises:
1) get a sample adding device, and be fixed for catching the biochip of marker substances to be detected in detection zone;
2) close application of sample raceway groove and assist gallery, pump into damping fluid to detecting raceway groove, at least fill the detection raceway groove between application of sample raceway groove and assist gallery;
3) close detection raceway groove, pump into and contain sample to be tested by application of sample raceway groove, assist gallery earial drainage, sample to be tested is at least filled the detection raceway groove between application of sample raceway groove and assist gallery, sample to be tested in this section forms detection segment, and wherein sample to be tested is the sample that contains described marker substances to be detected;
4) close application of sample raceway groove and assist gallery, pump into damping fluid to detecting raceway groove, detection segment is pushed to detection zone, and covers detection zone;
5) the first association reaction, the time that the sample to be tested in detection segment and biochip reaction first are given;
6) after the first association reaction, use damping fluid that described detection segment is released to detection zone, then close detection raceway groove;
7) utilize step 3) and 4) identical mode, use semiochemicals liquid to substitute sample to be tested and produce signal segment, cover detection zone;
8) the second association reaction, makes material that semiochemicals and detection zone produce through association reaction for the first time carry out association reaction for the second time within second given time, produces sample;
9) close application of sample raceway groove and assist gallery, utilize damping fluid detection zone is completed after association reaction for the second time to remaining material liq to release and detect raceway groove by detecting raceway groove;
10) utilize acquisition and analysis device to carry out collection analysis to the sample of detection zone.
2. detection method as claimed in claim 1, is characterized in that, described detection zone is modified to the nanometer thin rete of coupling organism-absorbing chip.
3. detection method as claimed in claim 2, it is characterized in that, at nanoparticle surface modified Avidin, and with biotinylated probe molecule coupling, design different stem grafting methods and increase binding site, make probe molecule in nanoparticle surface enrichment, each nano particle forms the storage unit that comprises dozens of or more nanometer probe molecules; Nano-probe molecule storage unit is modified on the antibody or aptamer with target material specific binding to be measured.
4. the detection method as described in as arbitrary in claims 1 to 3, is characterized in that, described semiochemicals is signal antibody or signal aptamer, and both are all marked with probe molecule, and probe molecule is enzyme molecule or fluorescence molecule.
5. detection method as claimed in claim 1, is characterized in that, described sample adding device comprises:
Detect raceway groove, one section of raceway groove in its downstream is configured to detection zone, and at least leaves detection window at detection zone place;
Application of sample raceway groove, be connected in detect raceway groove Upstream section and form application of sample bypass;
Assist gallery, is connected on the detection raceway groove between application of sample raceway groove tie point and detection zone, is configured for the bypass of application of sample earial drainage; And the detection channel length between application of sample raceway groove and assist gallery is greater than the length of detection zone;
Signal piping unit, is coupled to detection raceway groove, and is provided with operation valve;
Application of sample pipe-line cell group, is coupled to application of sample raceway groove and assist gallery, and is provided with operation valve group;
Testing pump, by the upstream extremity of described signal piping unit and corresponding operation valve access detection raceway groove, detects auxiliary liquid with pumping;
Sample-adding pump group, by described application of sample pipe-line cell group and corresponding operation valve group access application of sample raceway groove, with corresponding pumping sample adding liquid group; And
Control module, connects testing pump, sample-adding pump and operation valve and operation valve group, with the sequence of movement of logic control testing pump, sample-adding pump group and the operation valve that matches, operation valve group.
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| CN109939751B (en) * | 2017-12-21 | 2023-06-27 | 东莞东阳光科研发有限公司 | Microfluidic chip, detection device and detection method for whole blood detection |
| CN111013677B (en) * | 2019-12-23 | 2022-07-08 | 东莞市东阳光诊断产品有限公司 | Microfluidic chip, detection device and detection method |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004187678A (en) * | 2002-11-29 | 2004-07-08 | Kawamura Inst Of Chem Res | Method for analyzing polynucleotide |
| CN1712540A (en) * | 2004-06-15 | 2005-12-28 | 中国科学院大连化学物理研究所 | Detecting miniflow controlled chip from single-cell wilting DNA fragmentation |
| CN101034064A (en) * | 2006-03-06 | 2007-09-12 | 中国科学院理化技术研究所 | Microfluidic chip and application thereof |
| JP2007240413A (en) * | 2006-03-10 | 2007-09-20 | Konica Minolta Medical & Graphic Inc | Micro fluid chip |
| CN101046448A (en) * | 2007-05-08 | 2007-10-03 | 湖南大学 | Process of detecting gene mutation in cell with one-dimensional microflow controlled biochip |
| CN101109759A (en) * | 2006-07-17 | 2008-01-23 | 财团法人工业技术研究院 | Fluidic device and control method thereof |
| WO2009066236A1 (en) * | 2007-11-22 | 2009-05-28 | Koninklijke Philips Electronics N.V. | Methods and systems for coupling a bio-cartridge with an optical reader |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7670556B2 (en) * | 2001-07-10 | 2010-03-02 | Wisconsin Alumni Research Foundation | Surface plasmon resonance imaging of micro-arrays |
-
2013
- 2013-10-24 CN CN201310507020.3A patent/CN103529195B/en not_active Expired - Fee Related
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004187678A (en) * | 2002-11-29 | 2004-07-08 | Kawamura Inst Of Chem Res | Method for analyzing polynucleotide |
| CN1712540A (en) * | 2004-06-15 | 2005-12-28 | 中国科学院大连化学物理研究所 | Detecting miniflow controlled chip from single-cell wilting DNA fragmentation |
| CN101034064A (en) * | 2006-03-06 | 2007-09-12 | 中国科学院理化技术研究所 | Microfluidic chip and application thereof |
| JP2007240413A (en) * | 2006-03-10 | 2007-09-20 | Konica Minolta Medical & Graphic Inc | Micro fluid chip |
| CN101109759A (en) * | 2006-07-17 | 2008-01-23 | 财团法人工业技术研究院 | Fluidic device and control method thereof |
| CN101046448A (en) * | 2007-05-08 | 2007-10-03 | 湖南大学 | Process of detecting gene mutation in cell with one-dimensional microflow controlled biochip |
| WO2009066236A1 (en) * | 2007-11-22 | 2009-05-28 | Koninklijke Philips Electronics N.V. | Methods and systems for coupling a bio-cartridge with an optical reader |
Non-Patent Citations (2)
| Title |
|---|
| 刘剑等.基于酶催化发光的便携式痕量cTnI分析仪研究.《仪器仪表学报》.2010,第33卷(第1期),201-206. |
| 基于酶催化发光的便携式痕量cTnI分析仪研究;刘剑等;《仪器仪表学报》;20100131;第33卷(第1期);201-206 * |
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