CN100458434C - Analyte injection system - Google Patents

Abstract

This invention provides methods and systems for injection of analytes into a separation channel for resolution and detection. Samples can be preconditioned and concentrated by isotachophoresis (ITP) before the injection is triggered by a detected voltage event. Separation of analytes from other sample constituents can be enhanced using skewing channel ITP.

Description

Analyte injection system

The cross reference of related application

The right of priority of the U.S. Provisional Application that the application requires to submit on September 5th, 2006 U.S. Provisional Application is submitted to number on November 7th, 60/500,387 and 2003 number 60/518,169.

Background of invention

Invention field

The present invention relates to analytical electrophoresis system and method field.The present invention can comprise high resolving power and highly sensitive isotachophoresis (ITP) and Capillary Electrophoresis (CE) mensuration.

Association area is described

Generally speaking, electrophoresis is charged molecule moving in electric field.Analytical approach based on electrophoresis has extensive use, especially in albumen and nucleic acid analysis.The sample that contains charged analyte molecule interested can be placed selective dielectric, as size exclusion media, Ion Exchange Medium or have in the medium of pH gradient, they can be because of differential migration and other sample molecule high-resolution in medium.Can detect the molecule of separation identifies with quantitative.

Kapillary and microfluidic scale electrophoretic be separated in need in small samples or the high-throughout analysis especially commonly used.For example, can make plastics or the glass baseplate chip that contains micron order application of sample passage, split tunnel and sense channel.Can sample be transferred to from microwell plate in the application of sample passage by the sample collection tube that robot handles.The bootable all sample compositions of electromotive force move by the selective dielectric in the split tunnel, and sequence detection is eluted to all compositions the sense channel from split tunnel.The micron order size of this pilot system can provide the express-analysis of adopting micron order or nanometric sample volume.Yet for complex sample or dilute sample, its resolution or sensitivity may be not enough.

Improving the resolution of Capillary Electrophoresis (CE) method and a kind of method of sensitivity is with isotachophoresis (ITP) predecomposition and pre-concentration sample before CE separates.In ITP, with sample be added to electrophoretic mobility greater than the leading electrolyte (LE) of sample and electrophoretic mobility less than in the passage between the tailing electrolyte (TE) of sample.Under electric field influence, analytes of interest analytes can be passed through sample bolus (bolus) migration, accumulates on the interface of LE and/or TE solution.By this way, analytes of interest analytes and some other sample composition can be separated, and be concentrated into the higher detection level.Therefore, but concentrating sample and desalination so that improved injection material to be provided, are used for further carrying out with high resolving power the capillary electrophoresis separation of high-sensitivity detection.For example, " improving the detection sensitivity of microfluid system " (TandemIsotachophoresis-Zone Electrophoresis via Base-Mediated Destacking forIncreased Detection Sensitivity in Microfluidic Systems) at Vreeland etc. by the depalletizing of the alkali mediation isotachophoresis-zone electrophoresis of connecting, Anal.Chem. in (2003) ASAP article, further decompose and detect the sample that concentrates through ITP with capillary zone electrophoresis (CZE).In the Vreeland article, sample is subjected in electrophoretic mobility carry out ITP between the TE of pH control of Tris damping fluid and the LE.When passing through the ITP concentrating analysis, the hydrolytic action at the cathode terminal place of split tunnel forms hydroxyl ion (OH).This hydroxyl ion by split tunnel migration finally can in and the Tris damping fluid eliminated mobility difference between LE and the TE solution.Be transformed into the CZE separating medium among the Tris and with the ITP separating medium.Then, because the analyte that the sample active volume reduces and the ITP test procedure causes concentrates, can come separate analytes than higher sensitivity and the resolution of same sample standard C ZE method.The Vreeland method is subject to the ITP based on pH of compatibility sample, may be because neutralization procedure and very consuming time and because buffer preparation or the aborning variation of hydroxidion and inconsequent.

In ITP and another program that CE combines, electric field is switched to before the capillary electrophoresis separation that split tunnel carries out analyte, analytes of interest analytes is with the migration of ITP pattern, reaches point of interface with the CE split tunnel up to them.For example, " in microfluidic device by isotachophoresis pre-concentration sample " (SamplePre-concentration by Isotachophoresis in Microfluidic Devices) at Wainright etc., in J.Chromat.A979 (2002) the 69-80 pages or leaves, pre-concentration sample in the ITP passage is up to the point of interface of their arrival with the CE passage.Accept the photomultiplier (PMT) of light by the copolymerization focus objective lens that focuses on this point of interface and monitor this point of interface with microscope.

Can pass through, for example fluorescence or light absorption detect the analyte that enters point of interface, and the manual switchover electric field injects the CE passage with analyte.Yet problem is that manual switchover may inconsequent, some analytes with PMT may detect less than, micron-sized PMT detects and may bother and costliness.

As mentioned above, need to improve sensitivity, consistance and the resolution of kapillary and micron order electrophoresis method.The system that need between electrophoretic, as one man switch automatically.Can will be appreciated that these and other feature provided by the invention by reading following content.

Brief summary of the invention

The invention provides, for example, based on trigger voltage analyte being linked up as one man is injected into the system and method for separating medium.Can in its passage, pass through isotachophoresis (ITP) and pile up pre-service and concentrating analysis, when detecting voltage in this passage, the analyte of piling up be put on separation channel segments then.

The inventive method can provide the analysis result repeatably of the height with high sensitivity, speed and resolution.This method can comprise, for example, in piling up channel section, pile up one or more analytes and carry out analyte injection, detect the electromotive force in this passage, with when detecting selected voltage, the analyte of piling up is applied in the separation channel segments by apply the electric field differential pressure along separation channel segments.This passage can be for example to have the application of sample of formation channel section, pile up channel section and/or the intersection of separation channel segments or the micron order passage of shared pathway section.

Stacked analyte can occur in piles up in the channel section, analytes of interest analytes can be clipped between the selected damping fluid in this section, makes analyte be gathered into concentrated band during ITP.The typical analyte that injects for example comprises: protein, nucleic acid, carbohydrates, glycoprotein, ion etc.Pile up channel section and can contain different tailing electrolyte of mobility and/or leading electrolyte.For example, the mobility of leading electrolyte can be faster than tailing electrolyte or analytes of interest analytes under electric field influence.In many embodiments, the pH of tailing electrolyte and leading electrolyte, viscosity, conductivity, size exclusion, ionic strength, ion composition, temperature and/or can influence other parameter of electrolytical relative migration can be different.Can adjust the mobility of tailing electrolyte, make it, so that be accumulated on the hangover interface at ITP period analysis thing less than analyte.Randomly, can adjust the mobility of leading electrolyte, make it greater than one or more analytes, so that in that they are accumulated in advance on the interface between the ITP separation period.By straitly adjusting the mobility of hangover and leading electrolyte, analyte can be concentrated in advance and between the tailing electrolyte, and the sample composition of loseing interest in is moved to other district of piling up channel section and is with.Promptly, can adjust the mobility of tailing electrolyte, make it, maybe can adjust the mobility of leading electrolyte greater than one or more sample compositions of loseing interest in, they make it less than one or more sample compositions of loseing interest in, so that can not accumulate between the electrolyte with analytes of interest analytes.

When the passage of analyte injection method comprises accumulation separately and separation channel segments, can be by electric field be switched to separation channel segments and switches to separation channel segments from piling up passage from the accumulation channel section, for example, when the analyte of piling up enters the point of interface of accumulation and separation channel segments.For example, electric field is put on separation channel segments can comprise lacking substantial current flows and pile up from separation channel segments that electric current is arranged in the channel section, switching to has electric current in the separation channel segments and piles up failure of current in the channel section.Can flow in this channel section to prevent electric current by applying floating voltage, or only by the high resistance electric current that (for example, not allowing to export any obvious electric current from this channel section) cuts off this channel section is provided in this channel section.Randomly, can switch by applying the pressure reduction of striding separation channel segments.

The distinguishable analyte of separation channel segments in the injecting method and other analyte or sample composition.This resolution characteristic can be identified or the quantitative measurement analytes of interest analytes.Separation channel segments can have selective conditions or separating medium, with the migration of impact analysis thing and sample composition.For example, split tunnel can contain medium, hydrophobic medium of pH gradient, big or small selective dielectric, Ion Exchange Medium, raising viscosity etc.

Can detect the analyte of being differentiated in the separation channel segments, with evaluation and/or quantitative.Can make detecting device concentrate in the monitoring separation channel segments analyte or when its check and analysis thing during wash-out from separation channel segments.Can be by the correlation parameter of monitoring analysis thing, for example conductivity, fluorescence, absorbance, refractive index wait the check and analysis thing.

For example, available various technology with the sample solution application of sample in the passage of these methods, so that enough sensitivity and speed to be provided.For example, when the application of sample passage can not hold the enough sample analyte of carrying out required detection, but continuous several times application of sample and accumulation are merged repeatedly deposit, the analyte that provides small size concentration to improve then.Can pile up two or more analyte sample by the following method, for example: first sample is added in the application of sample passage; Stride sample and apply electric field, thereby pile up this sample; Second sample is added in the application of sample passage; Pile up sample and second sample and apply electric field piling up second sample with striding, and make these two to pile up samples and between hangover and leading electrolyte, flock together.Before application of sample second sample, allow first sample flow of piling up to help this multiple accumulation technology to remove too much electrolyte and to remove empty sample solution to the application of sample passage.The another kind of method of concentrating analysis sample can be, for example, analyte sample is added in the application of sample passage, and the xsect of this application of sample passage is greater than the xsect of piling up channel section, so that the analyte of bulk sample needn't be moved at a distance on the interface of hangover or leading electrolyte and accumulated.

Mobility can be added between sample and/or the stacked analyte in the spacer electrolyte between tailing electrolyte and the leading electrolyte, sample separation is become two or more analytes of interest analytes.In one embodiment, pile up to be included in and add mobility between two or more analyte sample segments greater than tailing electrolyte but less than one or more spacer electrolyte of leading electrolyte.In another embodiment, one or more in two or more analyte sample segments are previous analyte sample of piling up, and insert this spacer electrolyte during repeatedly piling up the application of sample step.Can adjust the mobility of this spacer electrolyte, be located between two or more analyte mobilities, with separate analytes in ITP.Can wait the adjusting of carrying out this spacer electrolyte by selecting suitable electrolyte pH, spacer electrolyte composition, spacer electrolyte viscosity, spacer electrolyte conductivity.

In some injecting methods, can prepare electrolyte cleverly, carry out ITP with analyte and separate injection.For example, if by experiment or calculate to determine the pK of certain analyte, can adjust in advance and the pH value of tailing electrolyte, make it comprise pK, reduce so that clamp-on the electrically charged minimizing of analyte institute and the movability of leading electrolyte, and/or electrically charged the increasing with movability of analyte of clamp-oning tailing electrolyte improved.Before the analyte of injection of stacked, carry out this adjustment and can improve selectivity and the concentrating capacity of ITP.

Can go in the separation channel segments by detecting the analyte injection that selected voltage causes accumulation.The voltage of all places in this passage can be monitored, the voltage on the preferred opportunity of injecting can be determined accurately to show.For example, detect voltage and can comprise that monitoring keeps the necessary no-load voltage of separation channel segments zero current (or electric current of other definition) condition.Being used to trigger the exemplary voltages of separating beginning can comprise, for example voltage peak, voltage groove, predefined voltage, relative voltage or voltage changing rate (for example zero slope that arrives in the voltage graph top view).The stacked analyte that is transformed into to the separation channel segments injection from ITP can be the electric field differential pressure that applies automatically along this channel section when detecting this voltage.

The system that is used for injection of analytes of the present invention is the analyte of injection of stacked automatically, so that the sensitive analysis of making peace that links up reliably to be provided.Analyte injection system can comprise, for example: be deposited in analyte in the passage, electrically contact and the voltage-level detector of communicating by letter with controller with passage, so that controller can start electric current in the channel separation section when this voltage-level detector detects selected voltage, or produce pressure reduction along this channel section.General this passage is the micron order passage that has the application of sample channel section, piles up channel section and separation channel segments.

Usually, according to isotachophoresis method accumulation channel section in this system is set with tailing electrolyte (TE) and/or leading electrolyte (LE).Each electrolyte can have different adjustable mobilities.For example, each electrolyte can have different pH values, viscosity, conductivity, size exclusion cutoff value, ionic strength, ionic composition or temperature.The analyte that is deposited in this passage can comprise molecule, as protein, nucleic acid, carbohydrates, glycoprotein, derived molecules, ion etc.Customizable electrolyte repels other sample composition with the selectivity stack analytes of interest.For example, can prepare tailing electrolyte and make its mobility,, become to separate TE and lose interest in so that analyte is accumulated in the TE leading edge less than the mobility of analytes of interest analytes with greater than the mobility of the sample composition of loseing interest in.Can prepare LE, make its mobility greater than the mobility of analytes of interest analytes with less than the mobility of the sample composition of loseing interest in, so that analyte is accumulated on the LE interface, and the composition of loseing interest in is moved to the leading edge away from the LE interface.

The separation channel segments of this system can contain the conditioned disjunction selective dielectric of the separating piled analyte in piling up post of energy and the composition of loseing interest in.For example, this separating column can comprise the pH gradient, the medium of big or small selective dielectric, Ion Exchange Medium, hydrophobic medium, raising viscosity etc.

Controller can be accepted the output of voltage-level detector, to start injection when detecting selected voltage.This controller can be, for example logical device or Systems Operator.In some embodiments, injection can be to switch to from accumulation passage ITP current field condition stacked analyte is inserted the required driving force of separation channel segments.For example, when detecting voltage, injection can be from the ITP current switching to during piling up channel section, having eliminated electric current substantially, and in separation channel segments, start electric field or voltage.

Each channel section of this system can comprise and pile up the application of sample channel section that the channel section fluid contacts.Can adopt various application of sample schemes to satisfy the needs of concrete analysis.In one embodiment, the xsect of application of sample channel section can be greater than the xsect of piling up channel section, so that can accumulate the analyte sample of larger volume in piling up channel section in the short period, promptly average analysis thing molecule is shorter than the migration distance in the long application of sample channel section of equal volume by the migration distance in the big xsect application of sample channel section.At application of sample on the other hand, in a plurality of accumulation schemes, can before adding second sample, the first stacked analyte sample be entrained back into the application of sample channel section, to improve analyte concentration and assay sensitivity.Can pass through, for example, stride the accumulation channel section pressure reduction is provided, realize this " towing back to " so that the first accumulation sample refluxes to the application of sample channel section.Can be from, the hole on the micro-fluid chip for example, or by fluid handling system, as filling up the application of sample channel section from the sample of microarray by collection tube (suction pipe) acceptance.

Spacer electrolyte can be used for this system, for example, and to improve the resolution between two or more analytes of interest analytes.For example, can will contain between the sample section of analyte in the spacer electrolyte introducing accumulation channel section of mobility between two or more analyte mobilities.The slow analyte of mobility ratio spacer electrolyte can be separated after spacer electrolyte, and mobility faster analyte can separate in spacer electrolyte the place ahead.In another embodiment, analyte sample can be mixed with spacer electrolyte, for example, separate in the analyte district band that separates under the influence of or steady-state condition instantaneous at ITP.

System of the present invention can have the voltage-level detector of communicating by letter with controller, reacts with the voltage in the sense channel with to voltage.Voltage-level detector can detect voltage and reference voltage such as the ground voltage between the contact point of any position in voltage between two or more electrical pickofves of striding all sections of passage or the passage.In some embodiments of this system, voltage-level detector can be monitored the voltage in the separation channel segments in banking process.Can monitor separation channel segments between accumulational stage and the point of interface of piling up channel section or along any locational voltage of separation channel segments, for example, when not having substantial current flows in the separation channel segments, as when by the no-load voltage regulator when separation channel segments applies no-load voltage, do not have electric current this moment from the output of an end of this channel section, or this moment this channel section gauge tap in the closed position.

When detecting selected voltage, controller can switch to clastotype with this system from accumulation mode automatically, and the analyte injection of piling up is gone in the separation channel segments.This voltage can be that for example, voltage peak, selection voltage, voltage groove, relative voltage, voltage change speed etc.Automatic switchover can be that for example, electric current flows, strides the relative voltage change of channel section or the pressure reduction that applies along this channel section in this channel section, and induces the analyte of accumulation to move along separation channel segments.

The analyte detection of available this system detects the analyte that separates in the separation channel segments, to identify and/or quantitative analytes of interest analytes.Analyte detection can be set, with the analyte of monitoring in the separation channel segments, or from the analyte of separation channel segments wash-out.Analyte detection can comprise photofluorometer, spectrophotometer, refractometer, diagometer etc.

System of the present invention is fit to microfluidic applications very much.Can be with for example, the application of sample channel section, pile up channel section, separation channel segments, sensing chamber etc. and be combined in the micro-fluid chip.The micron order size of microfluidic device is compatible with many systems of the present invention.Microfluid system known in the art can be provided for implementing voltage, pressure, liquid handling, communication and the detecting device etc. of system of the present invention.

Brief Description Of Drawings

Figure 1A-1C is the isotachophoresis system schematic.

Fig. 2 A and 2B are the synoptic diagram that instantaneous ITP concentrates on the electrolyte interface of going ahead of the rest.

Fig. 3 A-3C is the steady state (SS) ITP synoptic diagram arranged side by side that instantaneous ITP separates analytes of interest analytes and analyte.

Fig. 4 A-4C is the synoptic diagram of selective removal sample composition during ITP.

Fig. 5 A-5C is the synoptic diagram of exemplary sample solution application of sample technology.

Fig. 6 A-6E is a sequential schematic of describing repeatedly the accumulation technology of loads of sample analytes.

Fig. 7 A-7C shows the synoptic diagram that utilizes xsect to improve the sample solution volume greater than the application of sample channel section of piling up the channel section xsect.

Fig. 8 A-8D is the synoptic diagram that detects voltage on the channel section contact point piling up.

Fig. 9 A-9D is the synoptic diagram that the caused analyte band of ramp way tilts of flowing through.

Figure 10 A-10E is that sample composition tilts and is scattered among the ramp way ITP and analyte of interest band is kept the synoptic diagram of gathering.

Figure 11 A-11C is the synoptic diagram that puts on the stacked analyte of separation channel segments.

Figure 12 A and 12B have the micro-fluid chip synoptic diagram that sample solution is offered the collection tube of application of sample channel section.

Figure 13 A and 13B are the analyte injection system synoptic diagram, wherein pile up channel section and separation channel segments and share total passage.

Figure 14 A-14C is the synoptic diagram that has added the analyte injection system of spirality and snakelike ramp way.

Figure 15 A-15C is the synoptic diagram that makes the ramp way of outside displacement and the increase of medial movement distance proportion by turning.

Figure 16 A-16C is the ramp way synoptic diagram, on one side its inclination is the bigger translational surface distance of ratio another side that provides by this passage.

Detailed Description Of The Invention

Unless this paper or the following description book remainder have special definition, all technology used herein and scientific terminology have the common connotation of understanding of one skilled in the art of the present invention.

Before describing the present invention in detail, should understand concrete method or the system of the invention is not restricted to, they can be different certainly.Also should understand term used herein and only be used to describe embodiment, not be intended to restriction.

Used term in this instructions and the claims, singulative " ", " a kind of " and " this " comprise plural connotation, unless this content spells out other connotation.Therefore, for example, mention the combination that " a kind of composition " can comprise two or more compositions; Mention " all analytes " and can comprise a kind of analyte etc.

Can adopt and similar, modified or suitable many methods and material described herein though implement the present invention, and need not to carry out additional experiments, described herein is preferable material and method.In instructions of the present invention and claims, use following term according to following listed definition.

Term used herein " accumulation " refers to that one or more analytes are accumulated on the electrolyte interface different with mobility in electric field, for example, and during isotachophoresis.The accumulation of analyte also for example can occur in, and the composition that is present under instantaneous (dynamically) or the steady-state condition in the test specimen is modified other the band place, district that is produced because of migration.

In " selectivity isotachophoresis ", analytes of interest analytes that can certain mobility is known is by providing leading electrolyte and/or tailing electrolyte and other sample composition of mobility between this analyte and sample composition mobility, and the sample composition of for example loseing interest in is separated.

Term used herein " analyte " refers to by the detected sample composition of analyte detection." analytes of interest analytes " used herein refers to and need detect and/or quantitative analyte in certain test.

Term used herein " passage " refers in the inventive method and system to flow and/or the pipeline of liquid hold-up.Passage for example can be: pipe, post, kapillary, microfluidic channel etc.Passage can for example comprise various channel section in the different piece of this passage, the shared portion of this passage and/or the part that intersects with other section of this passage.Channel section is the funtion part of passage normally, for example application of sample channel section, accumulation channel section and separation channel segments.

" ramp way " can be the channel section that causes that the sample composition that flows in the passage tilts among the present invention.For example, the inner surface configuration of ramp way can make during through this ramp way band or peak that inclination trend with respect to this channel axis takes place in sample flow.

Term used herein " mobility " refers to charged molecule, the migration rate under the passage electric field influence as analyte in the solution or electrolyte.

Term used herein " no-load voltage " refers to that the required electric current that prevents substantially flows through this section or set up the voltage of required steady current in this section in the channel section.

Term used herein " micron order " refers to the size of about 1000 μ m-0.1 mu m ranges.

The present invention relates to analyte injection is gone into the method and system of split tunnel.The higher analyte concentration of the accumulation of analyte sample in can less volume injected provides the electrophoretic separation with improved assay sensitivity and resolution.

In many cases, can by before injecting in ramp way stacked analyte improve sensitivity and separating effect.By detecting the consistance that can improve result between the test run opportunity that voltage triggers automatic injection.

The inventive method and system can high-caliber sensitivity be used to separate with resolution, evaluation and/or quantitative measurement analyte.Analyte of the present invention can be, for example, and charged molecule such as protein, nucleic acid, carbohydrates, glycoprotein, ion, derived molecules etc.

Analyte injection method

That the inventive method can be is sensitive, repeatably, high-resolution test provides the analyte injection of accumulation to go into the accurate opportunity of split tunnel.The inventive method generally includes, for example, sample is added to the application of sample channel section, in piling up channel section, carry out isotachophoresis (ITP) then, detection shows that the analyte sample of accumulation has been positioned at the voltage on the injection position, apply the electric field differential pressure the stacked analyte sample is put on separation channel segments, the analytes of interest analytes of separating with detection.ITP can comprise that analyte moves by ramp way.Can estimate detection signal, to determine existing or measuring of analyte.

Stack analytes of interest

Available isotachophoresis (ITP) is stacked into analytes of interest analytes in the volume less than the original analysis matter sample.For example, sample bolus can be added in the passage between two kinds of different Laemmli buffer system Laemmlis, apply electric current, produce the stable transition state of solute district band, to reduce mobility.In steady state (SS), these district's bands can take identical concentration to move along passage with the speed identical with leading electrolyte.Perhaps, sample bolus can be added near the electrolyte,, for example, need not to reach the homeostasis between the ITP electrolyte dynamically being deposited in the injection interface under (instantaneous) condition.

Can, for example, pile up in the passage of micro-fluid chip, application of sample is between the passage area of tailing electrolyte and leading electrolyte in the chip.Shown in Figure 1A, can analyte sample 10 be added to application of sample channel section 11 by the pressure reduction between vacuum hole 12 and the sample well 13.Pile up channel section 14 when applying electric field when striding, (for example, electric charge/mass ratio height the analyte 16 of) leading electrolyte 15, medium mobility and the tailing electrolyte 17 of low mobility carry electric current, shown in Figure 1B by high mobility.Carry out with ITP, can set up a kind of steady state (SS), this moment, the volume concentration that is reduced to charged analyte 16 of analyte 16 equated with the concentration of leading electrolyte 15.In this steady state (SS), stacked analyte solution is along piling up channel section 14 migrations, and is identical with the speed of going ahead of the rest with tailing electrolyte, shown in Fig. 1 C, and the electrolyte of per unit volume and the electric current that charged analyte is carried same amount in piling up channel section.During ITP, some factors, as analyte and electrolytical electric density and different instantaneous migration rates, trend accumulates in analyte and electrolyte in each district's band.Accumulation channel section of the present invention can be a virtually any size, comprises being of a size of for example about 1000 μ m-0.1 μ m of wide or dark scope, or about 100 μ m-1 μ m, or the micron order passage of about 10 μ m.

Also can realize piling up in instantaneous state.Shown in Fig. 2 A, the analyte molecule 20 of initial dilution and dispersion for example can be accumulated on the leading electrolyte interface 21, shown in Fig. 2 B.Analyte this dense long-pending on the interface can occur in the analyte of setting up the steady state (SS) homogeneous and electrolyte carrier dense long-pending before.Randomly, for example, can in ITP, begin to apply during the electric field, on tailing electrolyte interface 22, accumulate certain analyte with instantaneous state.In other embodiment or instantaneous ITP, can other district's band beyond the ITP electrolyte interface in dense long-pending analyte.

Multiple analytes of interest analytes can steady state (SS) or the instantaneous state accumulation, for example, is accumulated on one or both electrolyte interfaces.Shown in Fig. 3 A-3C, the sample solution 30 that contains first kind of analytes of interest analytes 31 and second kind of analytes of interest analytes 32 can be added between tailing electrolyte solution 33 and the leading electrolyte solution 34.When the mobility of first kind of analyte was lower than second kind of analyte but is higher than tailing electrolyte, first kind of analyte can be accumulated on the tailing electrolyte interface in the presence of electric field.Simultaneously, in instantaneous state, shown in Fig. 3 B, second kind of analyte of a little higher than first kind of analyte of mobility can be accumulated on the interface of leading electrolyte along mobility faster at the other end of sample bolus.This situation can offer an opportunity respectively sequentially or abreast first kind and second kind of analyte be put on one or more separation channel segments, as skilled in the art to understand.In case in ITP, set up steady state (SS), shown in Fig. 3 C, charged first kind and second kind of analyte can be overstock in narrow adjacent ribbons, for example, be used from separation channel segments with one and separate.

In the methods of the invention, the mobility of scalable tailing electrolyte and leading electrolyte is carried out the selectivity pre-concentration to analytes of interest analytes, simultaneously this analyte and the sample composition of loseing interest in is separated.Shown in Fig. 4 A, the sample solution 40 of the sample composition 43 of loseing interest in that contains the lose interest in sample composition 42 and the high mobility of analytes of interest analytes 41, low mobility can be added between tailing electrolyte 44 and the leading electrolyte 45.When this passage was applied electric field, the sample composition 42 of loseing interest in of low mobility can drop on after the tailing electrolyte, and the sample composition 43 of loseing interest in of high mobility can run before leading electrolyte, shown in Fig. 4 B.Carry out continuously ITP when the steady state (SS) also can, for example, this analyte and the sample composition of loseing interest in are separated, shown in Fig. 4 C.From analytes of interest analytes, remove the sample composition of loseing interest in improved injection material can be provided, be used for separating in separation channel segments.After the sample composition of loseing interest in removal with the ITP pretreatment sample, analysis puts on the analytes of interest analytes of separation channel segments, can have, the background noise of Jiang Diing for example, owing to reducing, volume injected has high-resolution, because preferable baseline and less overlap peak and can be quantitative more accurately etc.

Can customize tailing electrolyte and leading electrolyte according to means known in the art, keep and stack analytes of interest, remove the sample composition of loseing interest in simultaneously by adjusting electrolyte rate high special ground.In an embodiment of this method, select electrolytical pH, make its pK that comprises analytes of interest analytes, so that the lose interest in sample composition of pK outside this scope is removed in ITP.For example, can be rule of thumb or determine the pK of analytes of interest analytes according to the known molecular structure of analyte.In other embodiments, analytes of interest analytes can closely be squeezed in selected known mobility and is lower than and be higher than between the hangover and leading electrolyte composition of analyte.Can adopt many ions and buffering agent in the electrolyte, to comprise analyte, for example chloride, TAPS, MOPS and HEPES.Randomly, can be by the viscosity of adjusting sample solution, tailing electrolyte solution and/or leading electrolyte solution or the mobility that the size exclusion feature is regulated electrolyte and/or analyte.The another kind of the mobility of mobility, analyte solution and/or the electrolyte solution of adjustment ITP solution is selected, by adjust concentration, ionic strength or the conductivity of these solution during instantaneous ITP migration.In other is selected, can select the temperature of solution to adjust the mobility of analyte, electrolyte or ITP solution.

Various sample solutions add quadrat method can make the analysis of the inventive method benefit.Pile up passage and can utilize single sample solution application of sample, sample solution application of sample and utilize spacer electrolyte application of sample between the sample solution application of sample repeatedly, such as detailed in the following.

Can single application of sample sample be added to the sample pipetting volume channel section according to technology known in the art, shown in Fig. 5 A-5C.Sample solution 50 can be put on application of sample channel section 51, for example utilize electroosmotic flow (EOF) differential pressure that sample solution is flowed through from sample well 52 and flow through waste fluid channel 53 crossings after the application of sample channel section is come out, again along the shunting of application of sample channel section, shown in Fig. 5 A.Perhaps, under the effect of the pressure reduction between sample well 52 and the waste liquid hole 54, sample solution 50 can be added to bifurcation and enter application of sample channel section 51, shown in Fig. 5 B.In Fig. 5 A and 5B, the voltage that must adjust currentless other hole guarantees zero current.Add in the sample loading mode at another, the relative vacuum on the waste liquid hole 54 can be dragged sample solution 50, tailing electrolyte and leading electrolyte become " pinching " stream, shown in Fig. 5 C, is used for accurately and working sample volume as one man.

The sample solution that available multiple accumulation technology adds additional quantity carries out ITP.First kind of sample can be added in the application of sample channel section 60, as shown in Figure 6A.Can stride accumulation channel section 61 and apply electric field, with stacked analyte sample 62, shown in Fig. 6 B.The analyte sample 62 of piling up can be back to the application of sample channel section, with the second application of sample sample solution 63 be added to first stacked analyte near, shown in Fig. 6 C.Can stride the accumulation channel section and apply electric field to pile up second kind of analyte sample 64 for the second time, shown in Fig. 6 D.When begin between accumulational stage the second time, can have basically the separating area belt of forming by the hangover damping fluid 65, but it can be separated in electric field and drops on stacked analyte tailing electrolyte afterwards for the second time.Finally, can make under electric field influence for the first time and the analyte of piling up for the second time mixing, form multilayer deposit 66, its amount doubles the analyte of piling up for the first time, shown in Fig. 6 E.The amount that increase again that number wheel deposit is towed back to, application of sample and accumulation can further increase repeatedly analyte in the deposit.Randomly, the sample solution of large volume can be added to xsect greater than in the application of sample channel section of piling up the channel section xsect.Shown in Fig. 7 A, for example, can utilize the pressure reduction of striding sample well 72 and waste liquid hole 73 that sample solution 70 is added in the application of sample channel section 71 of big xsect.Under electric field influence, analyte sample 74 can densely be amassed near accumulation channel section inlet, shown in Fig. 7 B.Have the application of sample channel section of bigger xsect can be in the short period concentrating analysis, this is that the axial distance 75 that analyte moves reduces because the application of sample channel section that xsect is less with volume is similar is compared.Tailing electrolyte 76 randomly can be brought into and analyte sample 74 position adjacent that concentrate, to carry out follow-up ITP, shown in Fig. 7 C by providing pressure reduction to wash the application of sample channel section with tailing electrolyte.

The inventive method can be by obtaining advantage between the analyte sample segments that spacer electrolyte is placed ITP.The mobility of spacer electrolyte can be between tailing electrolyte and leading electrolyte.The mobility of spacer electrolyte can be between two or more analytes of interest analytes.Spacer electrolyte can improve the resolution between the multiple analytes of interest analytes.In one embodiment, spacer electrolyte can be present in the sample solution of adding, so that the band of the spacer region between the analyte to be provided when applying electric field.In another embodiment, can repeatedly add spacer electrolyte between the accumulation circulation.For example, can be as mentioned above, but utilize the spacer electrolyte that exists in the initial accumulation of leaving over, utilize the spacer electrolyte that exists in one or more application of sample sample solution sections, or, repeatedly pile up by between the circulation of application of sample sample solution section, adding spacer electrolyte.Can adjust spacer electrolyte as mentioned above,, regulate the migration interval between the analytes of interest analytes to adjust the mobility of hangover and leading electrolyte.

Detect voltage

Detection can provide with solution, analyte and/or the electrolyte voltage that migration is relevant in piling up channel section, for example, is applied to the corresponding to enabling signal of separation channel segments with the analyte that will pile up.During ITP, stride the voltage of piling up channel section or measurable voltage can change in time on any point along piling up on the channel section.Operate running next time from an ITP, have the corresponding to voltage that detects between each running, its effect can be to be used for linking up as one man triggering injection and the time stamp that is transformed into different separation schemes from ITP.

In the exemplary embodiment that detects voltage, piling up in the channel section mobile at tailing electrolyte, analyte and leading electrolyte during the ITP.Tailing electrolyte is to the impedance ratio leading electrolyte height of electric current.For example, with the voltage of voltmeter monitoring along accumulation channel section halfway point, as shown in Figure 8, can be with this voltage that detects of ITP.Put on the sample solution 80 of piling up column inlet (flow) with application of sample at the beginning, leading electrolyte 81 has filled up the accumulation channel section, is being about half of ITP voltage of electric field along detected voltage on the contact point 82 of this channel section halfway.Along with analyte and tailing electrolyte 83 move from piling up channel section, the entrance side resistance of piling up channel section increases, and causes detectable voltage rising in the voltmeter contact, shown in Fig. 8 B.When stacked analyte arrived at the voltmeter contact point, along with detecting voltage (rising), the resistance difference of contact point both sides reached maximal value, shown in Fig. 8 C.At last, pile up the channel section end along with analyte is approaching, this section has been full of tailing electrolyte basically, makes the resistance of contact point both sides equate that detected voltage is returned to half that is about the ITP voltage of electric field, shown in Fig. 8 D.In this example, the detection of voltage can comprise starting potential value, voltage begin to raise, voltage raises or reduce rate of change (slope), maximum voltage (voltage peak), observe slope to be zero, turn back to starting potential at the maximum voltage place, the relative voltage in any predetermined voltage, this channel section between each position etc.For example, one or more voltmeters observations of difference link up consistent but different change in voltage figure on the channel section along piling up can to utilize contact.Can select these consistent detected voltages that link up to trigger the conversion of electric current differential pressure in each channel section, and the analyte of piling up is put on separation channel segments.

If split tunnel be not entire circuit a part (for example, " dead end " of Lian Jieing) with not having if or no-load voltage put on separation channel segments, with pile up the separation channel segments that channel section electrically contacts will be without any substantive electric current.In detecting the preferred structure of voltage, the voltmeter contact point can and be piled up on certain point between the channel section in separation channel segments, or is positioned at along on any position of separation channel segments.In a preferred implementation, can detect voltage by monitoring separation channel segments no-load voltage.

Improve the separating effect of ramp way

Can be during and/or improve separating of analytes of interest analytes and other sample composition by stacked analyte afterwards by the ramp way section.For example, in the isotachophoresis method, disperse and analytes of interest analytes when continuing to be assembled, can improve the sensitivity of test by electrolyte when the sample composition of loseing interest in becomes through number wheel (electrophoresis).

When passage during from the straight line path bifurcated, variable must the dispersion of analyte band of in the analytic system passage, flowing.Shown in Fig. 9 A-9D, shorter than analyte in the turning flows outside in analyte 90 displacements of 91 flows inside of turning.Band becomes the band that tilts and disperse along the length of this passage closely at the beginning, shown in Fig. 9 C.The axial diffusion of inclined strip can be diluted this band, and stops band to be reset, shown in Fig. 9 D.Tilt and diffusion after, the band peak signal that detecting device detected that focuses on this band among Fig. 9 A than focus among Fig. 9 D this band that detecting device detected was stronger narrower.In many stratographic analyses, because the peak broadening that is produced and shortening, it may be debatable that this band disperses.Yet, but the inclination that the present invention's coupling ITP technology have a mind to strengthen improves separating effect by the stack analytes of interest sample composition that disperses simultaneously to lose interest in.

For example, in one embodiment, what usable sensitivity improved is separated analytes of interest analytes in a small amount and the relatively large sample composition of loseing interest in improved quantivative approach.In not having the ITP system of ramp way, for example shown in Figure 10 A synoptic diagram, the analytes of interest analytes 100 of piling up can be moved between hangover of for example selecting and leading electrolyte in a small amount, and relatively large mobility is similar to the sample composition 101 of loseing interest in of tailing electrolyte, moves near the tailing electrolyte leading edge.The detecting device 102 that focuses on this passage can not resolved analysis thing and sample composition peak, as shown in detector output signal table 103.Can improve analyte sensitivity and quantitation capabilities by for example one of a plurality of ramp way sections being introduced the accumulation passage.The inclination that can become of analyte 100 that in piling up passage, moves and sample composition 101 (Figure 10 B), and be scattered in (Figure 10 C) in the ramp way 104.Withdraw from ramp way after a period of time, in advance and the accumulation force of tailing electrolyte the analyte peak in this passage is assembled and rearrangement, and inclination is kept at the sample composition peak that does not have a guiding, and the disperse that becomes.The detecting device that focuses on this passage can weakening and existence and amount that low invasive comes the check and analysis thing by the sample composition background.

Can assemble by the accumulation of selecting hangover and/or leading electrolyte to strengthen analyte, the mobility difference that improves simultaneously between electrolyte and the sample composition improves the benefit that ITP separates in the ramp way.In selectivity ITP, select in advance and the mobility of tailing electrolyte makes it near the known mobility of certain analyte and/or improve electrolyte and one or more mobility difference between the sample composition of loseing interest in.For example, in these cases, in the mobility of analytes of interest analytes when loseing interest in sample composition, can select the mobility of tailing electrolyte, make it than (uninterested) sample composition, more approach analyte, so that this analyte is subjected to the tight guiding sample composition of loseing interest in simultaneously and lags behind and experience and tilt and dispersion.If the mobility of analytes of interest analytes is lower than the sample composition of loseing interest in, can select the mobility of leading electrolyte in a similar manner, it is between this analyte mobility and this sample composition mobility, to improve the ITP separating effect of ramp way.In a preferred embodiment, select certain electrolytical mobility, make between its mobility that is in analytes of interest analytes and one or more sample compositions of loseing interest in, but more approach the mobility of this analyte.In another embodiment, can select in advance and tailing electrolyte, make its known mobility near analytes of interest analytes.Near very fast and slow sample composition is moved to this analyte and/or during ITP, prove effective when instantaneous accumulation, special benefit can be provided.

The validity of ramp way ITP can great changes will take place because of various factors, these factors for example, the flowing velocity and the viscosity of the shape of the radius of turn that relates to, the internal diameter of this passage, conduit wall, the xsect of ramp way, solution.For example, as with described in the ramp way ITP system of lower part, can utilize short radius of turn, on equidirectional repeated turns, can improve difference between the length surface of relative conduit wall and improve the degree of tilt of passage perpendicular to the channel shape of the wideer channel cross-section of turning axis.For example, can be by calculating and/or test the condition that is suitable for concrete grammar or system that produces.

In order to consider how diffusion influences the caused tilt quantity of turning, can consider the advection-diffusion equation (also referring to " analytical chemistry " (Analytical Chemistry), the 73rd volume, the 6th phase, 1350-1360, March 15 calendar year 2001) of two-dimensions, non-dimensionization:

Wherein, L is the length of turning channel, and w is the inner width of turning channel, Pe ' _wIt is the Peclet number that disperses; U ', c ', t ', x ' and y ' are respectively standardized speed, concentration, time, axial passage size and interconnection size.After measured, Pe ' in the present invention _w, L and these three parameters of w are to the inclination of analyte under the influence of ramp way and disperse particular importance.

Peclet number (Pe) is no dimension factor, represents the advection (or propulsion) of certain analyte and the ratio of diffusion.If Pe is big, the inclination peak by first ramp way can enough keep stable tilted shape for a long time, and allows rightabout second turning that it is turned to.If Pe is little, the inclination peak in the ramp way can be transformed into the peak that disperse is widened with the inclination peak by this width of channel diffusion in the short period.In the methods of the invention, the sample composition of loseing interest in is tilted and from analytes of interest analytes, spread, for example, when the Peclet number that condition provided that exists in the ramp way greater than the ratio of ramp way length and ramp way inner width (when being Pe＞L/w).When Peclet number that these conditions provided is higher about 0.01 times, 0.1 times, 1 times, 10 times, 100 times or more for a long time than the ratio of ramp way length and ramp way width, can in ramp way medium dip, diffusion and dispersion, obtain remarkable benefit at the sample of loseing interest in.

The condition that influences Peclet number can be for example, to influence the advection of molecule in this passage and/or the condition of diffusion, as is known to the person skilled in the art.For example, the speed that the existence of solution viscosity, gel, temperature, molecular conecentration, molecule move along this passage, this channel diameter etc. can influence Pe.The condition of adjusting control advection and diffusion can provide and can cause sample composition during by ramp way section of the present invention and/or be dispersed to the Peclet number of desired level afterwards.

The analyte of piling up is put on split tunnel

Can pass through, the electric field differential pressure that for example applies the analyte of striding separation channel segments and accumulation is gone into the analyte injection that ITP piled up in the separation channel segments.Electric field and/or voltage can make the analyte migration or flow in the separation channel segments.As mentioned above, can trigger and apply electric field or voltage by detecting voltage so that to be provided the injection opportunity of consistance and functional selection thing.The electric field differential pressure that puts on separation channel segments can carry out simultaneously with the electric current elimination of piling up in the channel section.Can determine the opportunity between voltage and the injection, so that the concrete shape of passage, point of interface and solution segments is consistent.Can play a key effect this opportunity in the resolution of determining the peak and signal intensity, transfers the time that the instantaneous isotachophoresis in back continues because it can influence.

The condition that separation channel segments can provide electrophoretic separation of analytes and/or separate by selective dielectric.In a preferred embodiment, separation channel segments has micron order size (for example, the degree of depth or width range are about 1000 μ m-0.1 μ m, or are about 1000 μ m-1 μ m), for example, can separate the small size analyte sample fast.Separation channel segments can contain the separating medium that can work to the separation of analyte, for example medium of pH gradient media, big or small selective dielectric, Ion Exchange Medium, raising viscosity, hydrophobic medium etc.

Separation channel segments (and piling up channel section) can contain the medium such as the gel that improve viscosity, to reduce electroosmotic flow (EOF) in the clastotype that does not need EOF.Separation channel segments can be independent of other channel section, or can with other channel section, for example application of sample channel section and pile up an all part or the parts of channel section sharing channel.In preferred implementation, separation channel segments is independently, but crossing along contacting with liquid on some points of piling up channel segment length.

In exemplary embodiment, when detecting peak voltage on the point of interface of piling up channel section and separation channel segments, the stacked analyte that derives from the ITP separation is injected in the separation channel segments.For example, that no-load voltage in the separation channel segments 110 reaches is maximum (speed that changes with voltage or the slope vanishing of voltage graph), because be clipped in the stacked analyte 111 between tailing electrolyte 112 and the leading electrolyte 113, migration is by separation channel segments and the voltmeter contact point of piling up the channel section intersection, shown in Figure 11 A in ITP.The voltage maximum can trigger the elimination of piling up ITP electric field in the channel section and apply the electrophoresis electric field and induce analyte 111 migrations (applying) of accumulation to enter in the separation channel segments in separation channel segments, shown in Figure 11 B.The analyte migration can make the common migration of analytes of interest analytes 114 and this analyte be separated (resolution) by the sample composition 115 of loseing interest in of piling up channel section during ITP by the selective dielectric of separation channel segments, shown in Figure 11 C.In some embodiments, during ITP, for example can make the multiple analytes of interest analytes that is deposited in the separation channel segments together or presses close to mutually disconnected from each other by capillary zone electrophoresis.

Those skilled in the art will know that and determine the injection another program on opportunity.This scheme can be according to calculating or model, or can determine by rule of thumb.For example, time delays can be building up to and trigger in the reaction, this reaction is based on the geometric configuration of channel volume, passage, voltmeter contact position, the selection of voltage, the analyte position relevant with the solution properties that influences voltage etc.In one embodiment, analyte is deposited in tailing electrolyte near interface among the instantaneous ITP (also not reaching steady state (SS)), and remaining sample solution group has high resistance, the suitable triggered time can be certain time behind the voltage peak, makes the analyte of accumulation that the extra transit time arrival and the point of interface of separation channel segments be arranged.

Can apply electric field (promptly not needing manual switchover) automatically along separation channel segments.Can pass through, for example electronic installation and algorithm known in the art are realized this electric field that applies automatically.For example, voltmeter can be set, make and when the voltage of contact point reaches the setting level, skip switch.In a preferred embodiment, can be to logical unit, for example, integrated circuit or computing machine are programmed, to start the switch of gearing according to the parameter of setting (voltage of setting for example, takes place).

The check and analysis thing

The analyte that can detect in the separation channel segments and/or separate with the inventive method behind the separation channel segments sequentially eluting.Can fix suitable detecting device, for example, the analyte of monitoring in the sense channel, the analyte in all channel section of sequential scanning, or the continuous shooting of whole passage is provided.

Suitable detecting device is usually determined by the type of analyte to be detected.Usually can pass through, for example the specific absorbing light wavelength of spectrophotometric method monitoring detects albumen and nucleic acid.Can detect many ion-type analytes of interest analytes by the change of monitoring electrical conductivity of solution.Many analytes have fluorescence or available fluorescent marker comes mark, detect with photofluorometer.Can detect many analytes, especially carbohydrates in the solution with refractometer.

In an exemplary embodiment, the light source that photomultiplier (PMT) monitoring that can utilize micro objective to focus on this passage sees through separation channel segments detects.How to it will be understood by those skilled in the art that by adding suitable excitation source, the filter light of laser or bulb for example, this arrangement is set to fluorescence detector.Randomly, object lens can be contained on the X-Y scanning machine any position on the monitoring micro-fluid chip.By this arrangement, can scan the analyte in the separation channel segments length along the pH gradient separations.In another embodiment, can stride the split tunnel outlet and settle the diagometer sensor, to monitor them when wash-out comes out from channel section in charged analyte.

Detecting device can link to each other with data memory device and/or logical unit, with the operation of record experiment.Can will get self-detector, supply with the chart draught machine, be retained on the paper with trace with the analyte separated graphics as the simulation output of PMT and diagometer.Analog to digital converter can pass to detection signal logical unit providing of data storage, separated graphics and/or test evaluation are provided.By making comparisons with the respective standard curve of regretional analysis, digital logic arrangement can go far towards the quantitative of analyte.

Analyte injection system

Moving electrical analyte injection system as herein described can provide sensitive detection of analytes, has the high resolving power of highly consistent mode.Can be according to the detection of voltage in all passages determined accurate opportunity, selectivity is piled up in the analyte injection (applying) piled up in the channel section in separation channel segments.Can improve this accuracy by automatic injection subsystems is provided.

System of the present invention generally includes, stacked analyte in passage, communicate by letter with controller and with the contacted voltage-level detector in the one or more positions of passage, when voltage-level detector detects selected voltage, in passage, set up the electric current differential pressure and this electric current differential pressure be transferred to controller.This passage can comprise crossing accumulation channel section and separation channel segments, forms continuous passage or sharing of common channel part.Put on the separated analyte of separation channel segments and can use the detecting device that links to each other with logical unit to detect, to measure existing or the amount of evaluation analysis thing of specific analyte.

Passage

Passage of the present invention can be for example, to comprise the application of sample section, pile up the single multifunctional channel of section, separate sections and/or detector segments.Randomly, this passage can comprise with application of sample channel section, accumulation channel section and the separation channel segments separately of liquid contact on the interface point.In a preferred embodiment, shown in Figure 11 synoptic diagram, the application of sample channel section is the extension of piling up channel section, and separation channel segments contacts with liquid phase with the point of interface of accumulation channel section by analyte injection.All passages of this system can be any passages known in the art, for example, and pipe, post, kapillary, microfluidic channel etc.In a preferred embodiment, for example, this passage is the micron order passage on the micro-fluid chip.

Can be embedded on the substrate surface by the passage with microfluidic device such as model injection, photoetching, etching, laser ablation.These passages can have the micron order size, and for example, dark or wide scope is about 1000 μ m-0.1 μ m, or are about 100 μ m-1 μ m.For example, can liquid be flowed in passage by electroosmotic flow, capillarity (surface tension), pressure reduction, gravity etc.Passage can be in (for example) solution hole and/or with the point of interface of other passage or chamber on stop.Can have and electrically contact on each end of passage, to provide electric field and/or electric current to come separate analytes or to induce EOF.Detecting device can link to each other with channel function, with monitoring parameters of interest, for example voltage, conductivity, resistance, electric capacity, electric current, refractive index, light absorption, fluorescence, pressure, flow velocity etc.Micro-fluid chip can have function information communication connection and be connected with purposes to support the use of instrument, as electric power connection, vacuum source, pneumatic supply, hydraulic power source, analog-and digital-communication line, optical fiber etc.

Passage can comprise that the application of sample channel section is to introduce this passage with the sample solution of one or more volumes.Can mode understood by one of ordinary skill in the art settle this application of sample passage, for example the syringe ring comprises the collection tube 120 that leads to micro-fluid chip, as shown in figure 12, and/or the irrigation channel section, shown in the synoptic diagram of Fig. 5 A-5C.The xsect of application of sample channel section can be greater than the xsect of piling up channel section, as shown in Figure 7, and so that dense fast the amassing of the analyte in the bulk sample solution piled up near the channel section inlet.

The native system passage can comprise spawn, and it can influence the migration and the flow performance of passage valuably.Gel can be incorporated in the passage,, provide better electrophoretic characteristic for separation simultaneously to reduce the bad electroosmotic flow of solution.Gel can come impact analysis thing and/or electrolytical relative mobility by the more macromolecular swimming of slowing down.The gel instrument that can be used as helps to regulate analyte and the electrolytical migration area of ITP band in the accumulation channel section.For example, analytes of interest analytes is usually greater than ITP electrolyte commonly used.Pile up channel section by gel is placed, can make express-analysis thing (big but specific charge height) slack-off, leading electrolyte small molecule salt or damping fluid after, move.Randomly, gel can make analyte slack-off, and its migration is only fast slightly than tailing electrolyte.Can change gel type, gel-type vehicle concentration and gel-type vehicle crosslinking degree by (for example) and regulate the impedance of gel big molecular migration.In accumulation or separation channel segments, gel can improve the concentration and/or the resolution of analyte.In piling up channel section or separation channel segments, can there be one or more different gels.

The function of piling up channel section is, for example, by ITP selectivity stack analytes of interest, is injected into separation channel segments, further separates and detects.That piles up channel section respectively can have electrical pickoff on the end, is suitable for the electric field that analyte is piled up to apply.Pile up channel section can with for example, the contact of the pneumatic or hydraulic branch pipe liquid phase of external drive, thus can implement the driven stream of the technology of repeatedly piling up described in above-mentioned " stack analytes of interest " chapters and sections, load or tow back to as electrolyte.Pile up channel section and for example can contain, be applicable to the electrolyte of isotachophoresis (ITP), as tailing electrolyte, spacer electrolyte and/or leading electrolyte, described in above-mentioned method chapters and sections.Pile up channel section and can have tailing electrolyte hole 18, as shown in Figure 1 and leading electrolyte hole 19, so that electrolyte is introduced in the channel section.

Separation channel segments can be accepted from piling up the stacked analyte of channel section injection, by various isolation technics, for example increase ITP round, ion-exchange, size exclusion, hydrophobic effect, reversed phase chromatography, isoelectric focusing, capillary zone electrophoresis etc. and further separate.Separation channel segments can comprise and be used for applying electrically contacting of electric field and/or driving outside connection of voltage source of liquid flow along channel section.Separation channel segments can be (for example) with pile up channel section that channel section intersects, with the channel section of piling up the total passage of channel section and/or with the channel section of piling up channel section sharing channel part on function.In exemplary embodiment, separation channel segments intersects on some points on the edge accumulation channel segment length with the accumulation channel section, as shown in figure 11.In this embodiment, stacked analyte interested is injected into separation channel segments after, the sample composition of loseing interest in can be retained in separately the accumulation channel section part.In other embodiments, for example, accumulation and separation channel segments can functionally reside in the total passage, and do not have the point of interface of insertion.As shown in FIG. 13A, accumulation can continue up to detecting voltage in channel section.In case detect this voltage, can change the condition in the passage, to carry out the transition to clastotype.This transition can comprise that (for example) applies pressure reduction on passage two ends 130, flows in the size exclusion resin 131, shown in Figure 13 B to cause analyte.To before big molecule, pass through detecting device 132 by wash-out than micromolecule.Other example that carries out the transition to clastotype can comprise the change of the change of (for example) direction of current flow, liquid flow direction, dissociating buffer is injected into the change of passage, voltage of electric field etc.

Ramp way ITP system

Isotachophoresis of the present invention system can be included in piles up among the passage and/or ramp way section before, to improve separating of analytes of interest analytes and the sample composition of loseing interest in.Can disperse this sample composition, assemble analytes of interest analytes by in ramp way, piling up simultaneously.For example, can turn to the bigger ramp way of wide-angle, zig zag, cross-sectional width by accumulation, have different length opposed surface the ramp way shape and/or have and make Peclet number improve separating effect greater than the ramp way system of the condition of the ratio of ramp way length and ramp way width.

Inclination in the raising ramp way section and a kind of method of dispersion are that big corner is provided in passage.In two dimensional surface, can utilize (for example) continuous spiral turns or conversion snake turn to accumulate corner, shown in Figure 14 A-14C.The advantage of spiral turns is can accumulate corner by the big measuring angle on the direction, accumulation inclination concomitantly.The shortcoming of spirality ramp way may be intrinsic the expanding to continuously in the lower curvature range of validity of radius of turn.The difficulty of spirality ramp way structure also may be the path problem that is connected in the inner passage end.Providing in spirality ramp way structure and enter a kind of method that can reach channel end can be the helical duct side by side with the center that can pass in and out, as shown in Figure 14B.Perhaps, can third dimension mode, for example, provide the path that enters the spirality channel end by the back passage in suction pipe or another plane, shown in Figure 14 A.Another restriction to spirality channel length is to optimize the required Peclet number that tilts to increase with spirality channel length.Snakelike ramp way shown in Figure 14 C can easily lead to channel end, but the turning of giving can be eliminated the inclination that previous turning causes, especially under the situation that the Peclet number between turning is big or the time is short.Randomly, can adopt the three-dimensional tilt passage, as spiral and curling.

Owing to tilt and be dispersed in that have can be more obvious with respect in the passage of the zig zag of passage internal diameter by the ramp way section.For example, for the high ramp way of passage internal diameter and turning width ratio, can increase inclination.In one embodiment, when passage along the xsect of radius of turn (inner width of ramp way) greater than perpendicular to the xsect of radius of turn (degree of depth of ramp way) time, increased the inclination of ramp way section with turning.

The shape of ramp way section can influence the inclination and the dispersion of migration analyte.For example, increase along the displacement in the outside of turning and along the channel surface profile of the ratio between the displacement of turning medial and can increase inclination.Can increase inclination with respect to the channel interior width of channel depth by increasing turning point.As shown in figure 15, have the surperficial turning of bolbus outside turning by flowing through and to make analyte 150 high inclination.Can increase the inclination in the ramp way, wherein in the surperficial displacement on the ramp way first side 151 greater than the surperficial displacement on ramp way second side 152, even on whole ramp way, do not have bending, as shown in figure 16.For example, can be from opposite surfaces displacement difference scope more than 500%, to 100%, to 50%, to 10% or tangible inclination is provided more for a short time.

In selectivity stack analytes of interest in advance and between the tailing electrolyte is an importance of ramp way ITP of the present invention system.Can be during tilting and/or analytes of interest analytes is met again continuously combine between this two electrolyte afterwards, the sample composition of loseing interest in the simultaneously dispersion that becomes.Can understand the mobility of analytes of interest analytes by calculating or empirical data.Can select mobility at analytes of interest analytes and invasive lose interest in hangover and/or leading electrolyte between the sample composition.Be to improve the gathering of analyte and the dispersion of sample composition, can select mobility for for this sample composition more near the electrolyte of analytes of interest analytes.

The ITP channel section that tilts can be attached in the system and method for the above-mentioned analyte of injection.After disperseing other sample composition by ramp way ITP, can higher degree analytes of interest analytes be injected into split tunnel.In case detect voltage, but with regard to the start injection analyte.

Voltage-level detector

Voltage-level detector in the system of the present invention can be contacted with passage, give the voltage of controller with detected transmission.The type and the complicacy of voltage-level detector can be depending on the configuration of (for example) channel hardware and the type of voltage to be detected.

The scope of voltage-level detector can comprise that for example simply because of the relay switch of shunt tripping, with the simulation galvanometer, the device of chart recorder is equipped with in simulation, has the voltmeter of numeral output, estimates by logical unit.Voltmeter can detect between two position electrode usually, for example, and the voltage potential in the contact position in the passage and ground or the passage between two diverse locations.Can change with the position of the contacted voltage electrode of passage and to pile up detected voltage graph during the running.Yet, usually can according to the contact of voltmeter on each channel position (for example, the voltmeter contact not necessarily pile up and separation channel segments between point of interface on) and link up and as one man and clearly trigger injection and determine the good voltage of determining.

In one embodiment, the voltmeter contact position can be positioned at the passage two ends.Because the tailing electrolyte that resistance is high has relatively replaced leading electrolyte in passage, keeping the selected electric current required voltage that flows through this passage may increase.In this case, triggering the voltage of injecting can be the voltage that (for example) set.

In another embodiment, the voltmeter contact point can be positioned on any point last and the crossing separation channel segments with piling up channel section of ground (or other voltage reference).If do not allow electric current to flow through separation channel segments when not being complete circuit a part of (when for example no-load voltage is kept zero current in separation channel segments or separation channel segments), the accumulation channel section voltage on the point of interface will be reflected in any position in the separation channel segments.Along with the TE/LE interface by point of interface, detected voltage can be elevated to peak value and reduces then in separation channel segments, its mode is similar to the voltage graph of Fig. 8, as understood by one of ordinary skill in the art.

No current with pile up the separation channel segments that channel section contact in during monitoring voltage, lack electric current can by the adjusting of (for example) no-load voltage or circuit separate cause.The no-load voltage regulating device can be an electronic equipment known in the art, and it detects electric current flowing in channel section, and this channel section is applied voltage, with any voltage potential of striding this channel section that neutralizes, thereby prevents that electric current from flowing.The no-load voltage regulator can randomly be set,, in this channel section, provide the steady current of selection to regulate the pressure reduction of this channel section.Prevent that the another kind of method that electric current flows from being to guarantee that this channel section is not the part of complete circuit in channel section.For example, electric switch can be present in an end of this channel section, with selectively opened or close any interlock circuit.

Voltmeter can be connected with controller, analyte be applied (injection) in separation channel segments to start.Starting injection can be manually or automatic.For example, voltmeter can be Systems Operator (controller) visual voltage readings is provided, when making it in a single day observe voltage as the voltage selected or voltage peak, and just manually ALT-CH alternate channel electric field or liquid flow.In another embodiment, in case controller is the digital logic device that automatically analyte of piling up is applied to separation channel segments when being electrically connected and being set to detect selected voltage with voltmeter.

Analyte detection

Suitable analyte detection can be joined in the system of the present invention with the check and analysis thing.The type and the configuration of detecting device can be depending on the type and/or the channels designs of (for example) analyte to be detected.Analyte detection and logical unit can be linked to each other to come storage of analyte detection profiles and evaluation analysis result.

The scope that is used for the analyte that detects at native system can be very wide, and many is charged molecules or modified and have the molecule of electric charge.For example, analytes of interest analytes can be protein, nucleic acid, carbohydrates, glycoprotein, ion etc.Though available another kind of mechanism produces accumulation as size exclusion, in many systems of the present invention, drives accumulation by the migration of charged analyte in electric field.It will be understood by those skilled in the art that uncharged analytes of interest analytes can accept electric charge, by suitably adjusting pH or the analyte with charged chemical group of deriving carries out electrophoresis and piles up.

Analyte detection in the native system can be any suitable detecting device known in the art.For example, detecting device can be photofluorometer, spectrophotometer, refractometer, diagometer etc.The analyte that unavailable available detecting device detects usually serviceable indicia molecule is derived, thereby gives its detectability.Can settle detecting device or focus on the monitoring channel section, comprise the analyte in point of interface and/or the separation channel segments.Detecting device can be when analyte flows out separation channel segments, for example, and monitoring analysis thing in the chamber sense channel.

But analyte detection monitoring channel position, the sequential scanning passage length, or the consecutive image of the analyte of separation is provided.In one embodiment, the spectrophotometric detector that leaves standstill can be the photomultiplier that focuses on concrete channel position or point of interface.In another embodiment, analyte detection can be the photofluorometer that focuses on the microchannel, by the analyte that separates in the passage that is placed in the Laser Scanning Confocal Microscope object lens sequential scanning microfluidic device on the X-Y conveyor.In another embodiment, analyte detection can be electric coupling device (CCD) array that the separating for several times image can be provided in a plurality of separation chambers at once.

Analyte detection can be linked to each other with logical unit and store and the evaluation analysis result.The logical unit of this system can comprise, for example chart recorder, transistor, circuit board, integrated circuit, central processing unit, computer monitor, computer system, computer network etc.Computer system can comprise for example having the data set of the software systems of entering and the digital computer hardware of instruction group.This computing machine can link to each other with detecting device, with the existing of evaluation analysis thing, identity, quantity and/or position.This computing machine can be, for example PC (Intel x86 or Pentium chip-with DOS , OS2

, WINDOWS

Operating system is compatible), MACINTOSHO

, Power PC or SUN

Workstation (compatible) or other commercially available computing machine known to the skilled with LINUX or UNIX operating system.The software of explaining sensor signal or monitoring detection signal can obtain from the market, or technician's available standards programming language such as Visualbasic, Fortran, language such as Basic, Java easily make up.Computer logic system can (for example) be accepted Systems Operator's input, designated samples sign and starting is analyzed, the order robot system with sample transfer in system's application of sample channel section, the monitoring of controlling liquid disposal system, control detection device, accept detector signal, preparation standard sample result regression curve, determine the amount of analyte and/or store analysis result.

Should be understood that the just explanation of purpose of embodiment described herein and embodiment, various modifications or change that those skilled in the art make according to these contents should be included in the application's the spirit and scope and in the scope of claims.

Though for the purpose of illustrating and understand has described foregoing invention in detail, it will be understood by those skilled in the art that by reading content disclosed herein, the change of various forms and details made, and do not deviate from true scope of the present invention.For example, can variously be used in combination above-mentioned many technology and device.

With all that quoted among the application deliver thing, patent, patented claim and/or other document in fit into and be used for all purposes as a reference, its degree is included in and is used for all purposes as a reference as will respectively delivering thing, patent, patented claim and/or other document individually.

Claims (60)

1. one kind puts on the method for channel section with the analyte of piling up, and described method comprises:

In piling up channel section, pile up one or more analytes and carry out the analyte injection accumulation;

Detect the voltage potential in this accumulation channel section; With,

When detecting selected voltage, by apply the electric field differential pressure along separation channel segments;

The analyte of piling up is applied in this separation channel segments.

2. the method for claim 1 is characterized in that, piles up to comprise instantaneous accumulation or stable state accumulation.

3. the method for claim 1 is characterized in that, described passage comprises the micron order passage.

4. the method for claim 1 is characterized in that, described accumulation channel section contains tailing electrolyte or leading electrolyte.

5. the method for claim 1 is characterized in that, described accumulation channel section contains tailing electrolyte and leading electrolyte, and these electrolyte have different mobilities.

6. method as claimed in claim 5 is characterized in that, following one or more attributes of described tailing electrolyte and leading electrolyte are different: pH, viscosity, conductivity, size exclusion, ionic strength, ion are formed or temperature.

7. method as claimed in claim 5 also comprises: adjust the mobility of described tailing electrolyte, make it less than one or more analytes, or adjust the mobility of described leading electrolyte, make it greater than one or more analytes.

8. method as claimed in claim 5 also comprises: adjust the mobility of tailing electrolyte, make it greater than one or more sample compositions of loseing interest in, or adjust the mobility of leading electrolyte, make it less than one or more sample compositions of loseing interest in.

9. the method for claim 1 is characterized in that, described passage is included in the described accumulation channel section and/or the ramp way section before described accumulation channel section.

10. method as claimed in claim 9 is characterized in that, described ramp way section contains: serpentine curve, spiral, curl, angle or spiral.

11. method as claimed in claim 9 is characterized in that, it is the condition more than 0.1 times of ramp way length and ramp way width ratio that described ramp way section has the Peclet number of dispersion.

12. method as claimed in claim 9 is characterized in that, the ramp way inner width that described passage comprises is greater than the degree of depth of ramp way.

13. method as claimed in claim 9 is characterized in that, the translational surface distance on the ramp way first side that comprises in the described ramp way section is greater than the translational surface distance on this ramp way second side.

14. method as claimed in claim 9 is characterized in that, described accumulation comprises the selectivity isotachophoresis.

15. the method for claim 1, it is characterized in that the described electric field that applies comprises and lacks substantive electric current and pile up that current conversion is arranged in the channel section is electric current to be arranged in the separation channel segments and pile up in the channel section to lack substantive electric current from separation channel segments.

16. method as claimed in claim 15 is characterized in that, describedly lacks substantive electric current and comprises no-load voltage or lack complete circuit.

17. the method for claim 1 is characterized in that, described separation channel segments comprise following one or more: pH gradient, big or small selective dielectric, Ion Exchange Medium, hydrophobic medium or improve the medium of viscosity.

18. the method for claim 1 also comprises the analyte that detects in the separation channel segments or detects the analyte that goes out from the separation channel segments wash-out.

19. method as claimed in claim 18 is characterized in that, described check and analysis thing comprises monitoring: conductivity, fluorescence, light absorption or refractive index.

20. the method for claim 1 is characterized in that, described accumulation is included in piles up two or more analyte sample continuously in the passage.

21. method as claimed in claim 20 is characterized in that, two or more samples of described accumulation comprise:

First kind of sample is added in the application of sample channel section;

Stride sample and apply electric field, thus the stacked analyte sample;

First kind of analyte sample of piling up is back to described application of sample channel section;

Second kind of sample is added in the described application of sample channel section; With,

Analyte sample and second kind of sample of striding accumulation apply electric field.

22. the method for claim 1 is characterized in that, described accumulation is included in xsect greater than adding analyte sample in the application of sample channel section of piling up the channel section xsect.

23. the method for claim 1 is characterized in that, described accumulation comprises one or more spacer electrolyte is added between two or more analyte sample that the mobility of this spacer electrolyte is greater than tailing electrolyte but less than leading electrolyte.

24. method as claimed in claim 23 is characterized in that, one or more in described two or more analyte sample are the analyte sample of piling up.

25. method as claimed in claim 23 also comprises the mobility of spacer electrolyte is adjusted between the mobility of two or more analytes.

26. method as claimed in claim 23 also comprises by one or more that select following parameter and adjusts the mobility of spacer electrolyte: the conductivity of the pH of spacer electrolyte, the viscosity of spacer electrolyte or spacer electrolyte.

27. the method for claim 1 also comprises the pK that determines analyte.

28. method as claimed in claim 27 also comprises the pH that adjusts tailing electrolyte or leading electrolyte, makes it be higher or lower than definite pK.

29. the method for claim 1 is characterized in that, described detection comprises the monitoring no-load voltage.

30. the method for claim 1 is characterized in that, described voltage comprises: voltage peak, voltage groove, predefined voltage, relative voltage or voltage changing rate.

31. the method for claim 1 is characterized in that, describedly applies the electric field differential pressure along channel section and carries out automatically when detecting voltage.

32. the method for claim 1 is characterized in that, described analyte comprises one or more of following material: protein, nucleic acid, carbohydrates, glycoprotein, the molecule of deriving or ion.

33. one kind puts on the system of channel section with stacked analyte, described system comprises:

Comprise the passage of piling up channel section and separation channel segments;

Be deposited in the analyte in the described accumulation channel section; With,

The voltage-level detector that electrically contacts and communicate by letter with described accumulation channel section with controller;

Wherein, when voltage-level detector detected selected voltage, controller caused that electric current flows or causes pressure reduction along this separation channel segments in separation channel segments.

34. system as claimed in claim 33 is characterized in that, described analyte comprises one or more of following material: protein, nucleic acid, carbohydrates, glycoprotein, the molecule of deriving or ion.

35. system as claimed in claim 33 is characterized in that, described passage comprises the micron order passage.

36. system as claimed in claim 33 is characterized in that, described accumulation channel section contains tailing electrolyte and leading electrolyte, and these electrolyte have different mobilities.

37. system as claimed in claim 36 is characterized in that, following one or more attributes of described tailing electrolyte and leading electrolyte are different: pH, viscosity, conductivity, size exclusion, ionic strength, ion are formed or temperature.

38. system as claimed in claim 36 is characterized in that, the mobility of described tailing electrolyte is less than the mobility of analytes of interest analytes or greater than the mobility of the sample composition of loseing interest in.

39. system as claimed in claim 36 is characterized in that, the mobility of described leading electrolyte is greater than the mobility of analytes of interest analytes or less than the mobility of the sample composition of loseing interest in.

40. system as claimed in claim 33 is characterized in that, described separation channel segments comprise following one or more: pH gradient, big or small selective dielectric, Ion Exchange Medium, hydrophobic medium or improve the medium of viscosity.

41. system as claimed in claim 33 is characterized in that, described controller comprises logical device or Systems Operator.

42. system as claimed in claim 33 also is included in when detecting voltage, the basic electric current of piling up in the channel section of eliminating.

43. system as claimed in claim 33 is characterized in that, described passage also comprises and piles up the application of sample channel section that channel section liquid contacts.

44. system as claimed in claim 43 is characterized in that, the xsect of described application of sample channel section is greater than the xsect of piling up channel section.

45. system as claimed in claim 43 also comprises and strides the pressure reduction of piling up channel section, can allow the sample flow of piling up to the application of sample channel section by it.

46. system as claimed in claim 43 also comprises the controller pipeline, analyte sample can flow into the application of sample channel section by it.

47. system as claimed in claim 33 also comprises and piles up the spacer electrolyte between two or more analyte sample segments in the channel section.

48. system as claimed in claim 47 is characterized in that, between the mobility of two or more analytes of the mobility of described spacer electrolyte in sample segments.

49. system as claimed in claim 33 also comprises no-load voltage regulator or the switch that electrically contacts with this passage.

50. system as claimed in claim 33 is characterized in that, described voltage comprises following one or more: the voltage peak, select voltage, voltage groove, relative voltage or voltage changing rate.

51. system as claimed in claim 33 is characterized in that, when detecting voltage, allows electric current flow in this channel section automatically or applies pressure reduction along this channel section automatically.

52. system as claimed in claim 33 also comprises analyte detection, be used for monitoring in the separation channel segments analyte or from separation channel segments the analyte of wash-out.

53. system as claimed in claim 52 is characterized in that, described analyte detection comprises: photofluorometer, spectrophotometer, refractometer or diagometer.

54. system as claimed in claim 33 also comprises micro-fluid chip.

55. system as claimed in claim 33 is characterized in that, described passage is included in the described accumulation channel section and/or the ramp way section before described accumulation channel section.

56. system as claimed in claim 55 is characterized in that, described ramp way section comprises: serpentine curve, spiral, angle or spiral.

57. system as claimed in claim 55 comprises that also disperseing Peclet number is the ramp way section condition more than 0.1 times of ramp way length and ramp way width ratio.

58. system as claimed in claim 55 is characterized in that, the ramp way inner width of described ramp way section is greater than the degree of depth of this ramp way.

59. system as claimed in claim 55 is characterized in that, the translational surface distance on the ramp way first side that comprises in the described ramp way section is greater than the translational surface distance on this ramp way second side.

60. system as claimed in claim 55 is characterized in that, described accumulation comprises the selectivity isotachophoresis.

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