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WO2015167990A1 - Injecteurs d'échantillons microfluidiques sans injection électrocinétique - Google Patents

Injecteurs d'échantillons microfluidiques sans injection électrocinétique Download PDF

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Publication number
WO2015167990A1
WO2015167990A1 PCT/US2015/027728 US2015027728W WO2015167990A1 WO 2015167990 A1 WO2015167990 A1 WO 2015167990A1 US 2015027728 W US2015027728 W US 2015027728W WO 2015167990 A1 WO2015167990 A1 WO 2015167990A1
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WIPO (PCT)
Prior art keywords
sample
channel
additional
separation
injection
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Application number
PCT/US2015/027728
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English (en)
Inventor
Keqi Tang
Ryan T. Kelly
Original Assignee
Battelle Memorial Institute
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Priority claimed from US14/264,385 external-priority patent/US20140291155A1/en
Application filed by Battelle Memorial Institute filed Critical Battelle Memorial Institute
Publication of WO2015167990A1 publication Critical patent/WO2015167990A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44743—Introducing samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F16—ENGINEERING ELEMENTS AND UNITS; GENERAL MEASURES FOR PRODUCING AND MAINTAINING EFFECTIVE FUNCTIONING OF MACHINES OR INSTALLATIONS; THERMAL INSULATION IN GENERAL
    • F16K—VALVES; TAPS; COCKS; ACTUATING-FLOATS; DEVICES FOR VENTING OR AERATING
    • F16K99/00—Subject matter not provided for in other groups of this subclass
    • F16K99/0001—Microvalves
    • F16K99/0034—Operating means specially adapted for microvalves
    • F16K99/0055—Operating means specially adapted for microvalves actuated by fluids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2205/00—General characteristics of the apparatus
    • A61M2205/02—General characteristics of the apparatus characterised by a particular materials
    • A61M2205/0244—Micromachined materials, e.g. made from silicon wafers, microelectromechanical systems [MEMS] or comprising nanotechnology
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M39/00—Tubes, tube connectors, tube couplings, valves, access sites or the like, specially adapted for medical use
    • A61M39/22—Valves or arrangement of valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F16—ENGINEERING ELEMENTS AND UNITS; GENERAL MEASURES FOR PRODUCING AND MAINTAINING EFFECTIVE FUNCTIONING OF MACHINES OR INSTALLATIONS; THERMAL INSULATION IN GENERAL
    • F16K—VALVES; TAPS; COCKS; ACTUATING-FLOATS; DEVICES FOR VENTING OR AERATING
    • F16K99/00—Subject matter not provided for in other groups of this subclass
    • F16K99/0001—Microvalves
    • F16K99/0003—Constructional types of microvalves; Details of the cutting-off member
    • F16K99/0015—Diaphragm or membrane valves
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44791—Microapparatus

Definitions

  • electrokinetic injection is used almost exclusively for microchip CE sample injection.
  • various electrodes in a first mode apply voltages in four intersecting channels to drive sample through the intersection to a waste reservoir.
  • the voltages induce injection of only a plug of sample occupying the small volume in the intersection towards a separation channel.
  • the voltage applied to the separation channel is different in the first mode, wherein sample is diverted to waste, compared to the second mode, wherein the plug of sample is injected into the separation channel. Except for the sample plug, the vast majority of sample is wasted.
  • electrokinetic injection can yield small sample plugs for improved separation efficiency and can minimize electrophoretic injection bias under certain conditions, it also has several significant limitations. For example, a considerable amount of time is required to achieve steady state in the first mode. Steady state is a necessary condition to avoid sample bias and/or injection bias caused by high mobility species arriving more quickly than low mobility species. During prolonged operation, the high mobility species can be depleted preferentially and prematurely from the sample supply. Sample utilization is extremely inefficient because the total volume required is very large compared to the actual injected plug volume, which is very small. Furthermore, the injection volume is fixed because it is determined by the geometry of the intersection. In order to change the injection volume, the geometry of the
  • intersection must typically be altered. Further still, the rate at which sequential injections can be analyzed, and the total number of sample plugs that can be injected into the separation channel, is limited by the steady-state flow requirements and by the changing voltages in the separation channel associated with electrokinetic injection.
  • the present invention is a microfluidic injector system based on a mechanical valve rather than electrokinetic injection.
  • the injector system comprises a port configured to interface with a discrete separation column connected to the injector system via the port.
  • the mechanical valve can be operated to provide rapid sequential sample injections and to eliminate the need for changing the electric field in an attached separation column to induce sample injection. Instead, sample injection is accomplished by pressure gradients and by opening the mechanical valve. A constant electric field can, therefore, be continuously applied along the separation channel. Varying the sample pressure and/or the duration of time that the valve remains in an open position can vary sample injection volume. Depending on the type and configuration of the separation column attached to the microfluidic injector, various kinds of separations and analytical techniques can be performed.
  • the terminal end of the separation column can also be configured to enable different analyses.
  • an electrospray ionization (ESI) emitter can be arranged at the end of the separation column.
  • mass spectrometry can be performed to analyze the sample after CE separation.
  • the introduced sample can be advantageous, though not required, for the introduced sample to displace fluid in the sample loading channel preferentially to the side of the sample channel opposite the separation column. This increases probability that the introduced sample does not disturb an ongoing separation in the separation column. If the introduced sample alternatively displaces fluid towards, or into, the separation column, it may degrade a separation already underway inside the separation column, as the pressure pulse from the injection may induce broadening of the separated peaks. This condition will likely automatically be met when the separation column contains packed particles, monolithic material, a cross-linked gel or a viscous polymer solution as these materials in the separation column increase its back pressure substantially.
  • the backpressure in the separation column is at least 10 times greater than the backpressure of the sample loading channel on the side opposite the separation column. This can be achieved by ensuring that the ratio of the length of the separation column to its cross-sectional area is at least 10 times greater than the ratio of the length of the sample loading channel on the side opposite the separation column to its cross-sectional area.
  • a microfluidic injector system comprises a flow layer having a first sample channel connected in a T-shaped arrangement to a loading channel at a first intersection, wherein the first sample channel has a source of pressure and is configured to maintain a sample pressure greater than that of the loading channel at the first intersection.
  • the loading channel has a terminus comprising a port configured to interface with a separation column.
  • the system further comprises a control layer comprising a valving channel, wherein the valving channel in the control layer crosses over the first sample channel in the flow layer at or near the first intersection.
  • the system also comprises a mechanical valve, not an electrokinetic-based injector, to control a sample injection from the first sample channel into the loading channel, the mechanical valve comprising a deformable membrane between the control layer and the flow layer and separating the valving channel and the first sample channel, wherein the membrane has a closed position to obstruct flow in the first sample channel, and an open position to permit flow in the first sample channel based on a first and second pressure, respectively, in the valving channel.
  • a mechanical valve not an electrokinetic-based injector
  • the system comprises one or more CE electrodes arranged along the loading channel and configured to continuously apply an electric field for CE separation.
  • Additional CE electrodes can be arranged along an attached separation column and can be configured to continuously apply an electric field for CE separation.
  • the continuous application of an electric field along the loading channel and/or separation column for CE separation is significant because any sample injection provided to the separation channel will be subject to a continuously applied CE separation field. There is no required change in voltage between an injection mode and a CE separation mode.
  • a sample channel is connected to the loading channel at an intersection and has a sample pressure that is greater than that which is present in the loading channel near the intersection.
  • the sample channel does not have electrodes that apply voltages for electrokinetic injection.
  • a sample injector in the sample channel, or at the intersection, comprises a mechanical valve to control sample injection from the sample channel to the loading channel. When the valve is opened for a short time, a small volume of sample solution is pushed into the separation channel under a low pressure. When the valve is closed, the sample solution is completely isolated from the run buffer in the loading channel such that there is no risk of sample leakage during the operation, and a discrete, well defined sample plug is injected with each valve opening event.
  • the system comprises a plurality of sample sources connected to the first sample channel via a manifold. Valves and/or fluid flow controllers can be utilized to select which sample source, or combination of sources, provides an injection through the sample channel.
  • the system comprises at least one additional sample channel connected in a T-shaped arrangement to the loading channel at the first intersection or at an additional intersection. The additional sample channel is configured to maintain a sample pressure greater than that of the loading channel at the first or additional intersection.
  • An additional valving channel for each additional sample channel can also exist. The additional valving channel in the control layer can cross over the additional sample channel in the flow layer at or near the first intersection or the additional intersection.
  • An additional mechanical valve not an electrokinetic -based injector, for each additional sample channel can control sample injection from the additional sample channel into the loading channel.
  • the additional mechanical valve comprises a deformable membrane between the control layer and the flow layer and separates the additional valving channel and the additional sample channel.
  • the additional membrane has a closed position to obstruct flow in the additional sample channel, and an open position to permit flow in the additional sample channel based on a first and second pressure, respectively, in the additional valving channel.
  • the first sample source can contain an analyte or an analyte precursor while the additional sample source can contain an analyte-derivatizing reactant or an analyte precursor.
  • the injection from the first and additional sample channels can mix and/or react in the loading channel. If the positions of the first and additional sample channels are offset, or they occur at differing positions of the loading channel, then the injections can be timed appropriately to facilitate the desired degree of mixing of the plurality of injections.
  • analyte-derivatizing reactant can include, but are not limited to radiolabels, fluorescent labels, labels to enhance electrospray ionization efficiency, and reactants that alter the charge state of the analyte.
  • a significant characteristic of the system is that the sample injection is independent of the separation technique. For example, during CE operation, a high voltage is applied only along the loading channel and/or separation column and no voltage switching is needed. The sample is directly provided into the loading channel and/or separation column for subsequent separation. There is no need to wait for production of a steady-state, stable sample plug as would be required in the traditional electrokinetic injection. Discrete sample plugs can be injected repeatedly over relatively long periods of time. The injection and separation frequency is only determined by the actuation of the mechanical valve. A valve having a high duty cycle makes it possible to perform continuous flow monitoring, high throughput analysis, and/or multiplexed separations. Systems having multiple sample channels with multiple sample sources and discrete valves can enable many combinations of sample injections and combinations.
  • Embodiments of the present invention can further comprise a plurality of discrete injections of samples from one or more sample channels to the loading channel in a rapid sequence.
  • the sequence can preferably be pseudo-random.
  • a detector at the end of a separation column attached to the loading channel through an interface can detect the discrete injections after CE-induced overlap, which comprises mixing of at least one component from at least one of the discrete injections to another discrete injection.
  • a processing device executes programming to deconvolute the CE-induced overlap in data collected by the detector so that a spectrum can be reconstructed.
  • Some embodiments can further comprise a plurality of separation columns as well as a manifold at or near the interface between the loading channel and the separation columns.
  • the manifold comprises a conduit that transitions from a single fluid flow line to multiple flow lines and distributes the flow of sample injections among the multiple flow lines.
  • Each flow line can proceed to a separation column.
  • an electrospray ionization (ESI) emitter is connected at the end of each separation column.
  • sample injections from the microfluidic injector systems have a volume less than 10 nL. In other embodiments, the volume is less than 1 nL. In still other embodiments, the volume is less than 200 pL.
  • the separation column can refer to a capillary or another tube. In one embodiment, the separation column can have an inner dimension that is as small as 10 micrometers and which may be as large as 5 millimeters. The separation column may be an open tube filled with an electrolyte solution for performing capillary zone electrophoresis separations.
  • the column may also be filled with a viscous polymer solution, a cross-linked gel or another medium for performing sieving-based separations (e.g., capillary gel electrophoresis).
  • the separation column may also be filled with chromatographic media in the form of particles or with a monolithic material having chemical properties such that, e.g., capillary electrochromatography can be performed using the column.
  • chromatographic media in the form of particles or with a monolithic material having chemical properties such that, e.g., capillary electrochromatography can be performed using the column.
  • Other suitable examples are encompassed by the present invention though they are not listed herein.
  • liquid chromatography (LC) separation is performed in conjunction with CE separation.
  • the system can further comprise a LC column connected to a sample channel and can provide LC separation prior to injection into the loading channel.
  • Embodiments of the present invention also include methods for analyzing a sample having a plurality of components using CE separation.
  • the method includes the steps of applying a sample pressure in the sample channel greater than the sample pressure in a separation channel.
  • the sample channel is connected to the separation channel at an intersection and lacks electrodes associated with electrokinetic -based injectors.
  • a continuous electric field for CE separation is applied along the separation channel.
  • Injection of the sample occurs by mechanically opening for a duration a mechanical valve, not an electrokinetic -based injector.
  • the mechanical valve is located in the sample channel or at the intersection. The electric field in the separation channel can then separate the components in the injection.
  • the method can further comprise repeating the mechanical opening in a rapid, pseudo-random sequence to provide a plurality of discrete injections of the sample from the sample channel to the separation channel.
  • CE-induced overlap can be the result of mixing at least one component from at least one of the discrete injections with another discrete injection.
  • the discrete injections are then detected at the end of the separation channel after CE-induced overlap.
  • the CE-induced overlap in data collected by the detector is deconvoluted so that a spectrum can be reconstructed.
  • the method can further include distributing one or more injections among a plurality of CE channels within the separation channel.
  • an electrospray can be generated at the end of each CE channel.
  • LC separations can be performed in conjunction with the CE separations. Accordingly, the method can further comprise separating the sample in a liquid chromatography column prior to providing an injection to the separation channel.
  • Figs. 1A and IB include diagrams of a microchip CE system A) having two layers according to one embodiment of the present invention and B) having three layers according to another embodiment.
  • Fig. 2 is a diagram depicting a microchip CE system using pressure injection and a pneumatic valve according to one embodiment of the present invention.
  • Fig. 3 is a sequence of micrographs depicting one cycle of sample injection.
  • Figs. 4A - 4D include graphs of fluorescence intensity as a function of time for four repeatable injection results at frequencies of 0.21 Hz, 0.43 Hz, 1.1 Hz, and 2.2 Hz, respectively.
  • Figs. 5A - 5D include graphs of peak width as a function of various operating parameters, including valve actuation time, sample injection pressure, valve control pressure, and valve actuation frequency, respectively.
  • Fig. 6 is a graph of fluorescence intensity as a function of time and shows a CE separation of four FITC-labeled amino acids using embodiments of the present invention.
  • Fig. 7 is a graph showing repeated CE separation of three FITC-labeled amino acids.
  • Figs. 8A - 8C include graphs showing portions of Fig. 7 in greater detail after varying numbers of runs.
  • Fig. 9 is a diagram depicting one embodiment in which the separation channel comprises a plurality of CE channels.
  • Figs. 10A - 10E are illustrations depicting various configurations and operational aspects of microfluidic sample injectors according to embodiments of the present invention.
  • Fig. 11 is a series of time-elapse images showing a pressure-driven injection sequence using one embodiment of a microfluidic sample injector described herein.
  • Fig. 12 is a graph showing the number of theoretical plates as a function of separation potential in CE separation following injection according to embodiments of the present invention.
  • Fig. 13 depicts a series of repeated separations of a three-peptide mixture containing kemptide, angiotensin II and leucine encephalin using embodiments of the present invention wherein the valve opening time is changed for each successive separation.
  • Figs. 14A - C present data depicting the tradeoff between separation efficiency and S/N as the injection volume is varied.
  • 14A three different pressures are applied at the sample loading channel to provide flow for electrospray ionization.
  • Fig. 15 is a graph depicting the peak area for three different peptides in a mixture over a range of injection times using embodiments of the present invention.
  • Figures 1-9 show a variety of embodiments and aspects of systems in which the separation column is integrated as a CE separation channel into a microchip that also contains the microfluidic injector.
  • Figures 10-15 show a variety of embodiments and aspects of systems in which the microfluidic injector is interfaced to a discrete separation column through a loading channel.
  • FIG 1A shows the outline of the microchip structure, which comprises two layers.
  • a flow layer (solid line) contains a CE separation channel 100 and a sample channel 103 with a T-shaped intersection 101.
  • the separation channel has electrodes for applying voltages 102 associated with CE, but the electrodes are not configured for electrokinetic injection.
  • the sample channel does not have electrodes, thereby precluding electrokinetic injection.
  • the control layer (dashed line) contains a microchannel 104 for valving.
  • An exemplary, but not limiting, width for the channels is 100 ⁇ .
  • Exemplary lengths for the separation and sample channels are 3 cm and 0.5 cm, respectively.
  • the channel 104 on the control layer crosses over the sample injection channel 103 and, therefore, the area at the T-shaped intersection 101 is 100 ⁇ x 100 ⁇ and is large enough for a mechanical injector comprising a pneumatic valve 105.
  • the pneumatic valve includes a thin elastomer membrane formed between the two layers. When a pressure is applied in the control channel, the membrane separating the two channels deforms into the flow channel to seal the sample channel (i.e., closed valve).
  • Integrated poly(dimethylsiloxane) (PDMS) microchips were fabricated using multilayer soft lithography techniques.
  • PDMS poly(dimethylsiloxane)
  • two kinds of silicon templates were produced using standard photolithographic patterning.
  • the silicon template for the control layer 107 was modified with hexamethyldisilazane (HMDS) using gas phase deposition method in order to assist in releasing the PDMS membrane from the patterned template.
  • HMDS hexamethyldisilazane
  • a 10: 1 weight ratio of PDMS base monomer to curing agent was then mixed, degassed under vacuum, poured onto the patterned wafer for the flow layer 106 to a thickness of 1-2 mm and spin-coated on the surface-modified wafer at 2000 rpm for 30 s to coat the control layer to a thickness of ⁇ 50 ⁇ , and cured in an oven at 75 °C for 2 h.
  • a small through-hole was created at the end of sample injection channel by punching the substrate with a manually sharpened syringe needle. Two holes were generated at the ends of separation channel for connections to buffer reservoirs.
  • the flow layer PDMS piece was then cleaned and treated with oxygen plasma for 30 s.
  • polyE-323 was used to coat the PDMS microchannel surfaces to provide anodic EOF in the same direction as electrophoresis for the negatively charged analytes.
  • PolyE-323 is a cationic polyamine, which can be absorbed on negatively charged surfaces through strong electrostatic interaction.
  • the polymer can support strong and stable anodic electroosmotic flow (EOF).
  • EEF electroosmotic flow
  • the PolyE-323 was synthesized by mixing 17.65 g l,2-bis(3-aminopropylamino)ethane with 20 g water and 9.3 g epichlorohydrine by strong stirring. Two days later, 100 g water was added and the reaction was allowed to continue for 1 week at room temperature. The polymer solution (5 mL) was then diluted in 25 mL of 0.2 M acetic acid, adjusting the pH to ⁇ 7. The diluted polymer solution was filtered using 0.2 ⁇ syringe filters and stored at 4 °C.
  • the diluted polyE-323 solution was pumped into the separation channel through the sample introduction hole for 10 min, and then the solution was left in the channel for 30 min. The channel was then flushed with 10 mM ammonium acetate (pH 7) for 10 min to remove excess polymer. Finally, the microchip was filled with a run buffer (10 mM carbonate buffer, pH 9.3) for CE separation.
  • FIG. 2 shows a diagram depicting a microchip CE system using pressure injection with the pneumatic valve described above.
  • the valve control sub-system comprises a valve manifold 202 and a controller box 201 controlling the valve manifold.
  • the valve manifold was connected to a second manifold 205 having manually controlled outputs.
  • the second manifold was connected with a regulated continuous N 2 gas pressure supply.
  • One output of the valve manifold was connected to the on-chip control line 104 with a tube to provide pneumatic operation and control of the valve.
  • the pneumatic valve 105 was operated and controlled automatically through a personal computer (PC) 203.
  • the valve actuation time and frequency were set in the software.
  • the valve control channel in the PDMS device can be filled with either water or air, but water was generally used to avoid introduction of bubbles into the flow channels.
  • the sample was contained in a sealed vial 204 with an air inlet to pressurize the sample and an outlet allowing sample to be transferred into the microchannel through a fused-silica capillary.
  • One end of the capillary was immersed into the liquid sample and the other end was inserted into a ⁇ 2 mm long section of Tygon tubing, which was then inserted into the through-hole of the sample injection channel 103 on the microchip. Before injection, any air bubbles trapped in the transfer line and microchannel were removed.
  • a high voltage (typically, 3 kV) was continuously applied along the separation channel 100 using a high- voltage power supply 207 via platinum electrodes 206 placed in the reservoirs.
  • the pneumatic valve was actuated to inject discrete sample plugs into the separation channel for subsequent CE separation.
  • LIF laser-induced fluorescence
  • Figure 3 is a sequence of micrographs depicting one cycle of sample injection controlled by the pneumatic valve embodiment described above.
  • the sample injection pressure was 1.5 psi and the valve actuation time and frequency were 33 ms and 2.2 Hz, respectively.
  • the valve control pressure was 30 psi.
  • the microchannel surface was dynamically coated with PolyE-323 and an electric field of 1000 V/cm was applied along the separation channel.
  • EEF electroosmotic flow
  • the sample injection can be operated at different frequencies in the present injection mode.
  • Figure 4 shows four repeatable injection results at frequencies of 0.21 Hz, 0.43 Hz, 1.1 Hz, and 2.2 Hz, respectively. The fluorescence intensity was recorded close to the T intersection.
  • the relative standard deviations (RSD) of the peak height and width shown in Figure 4 are listed in Table 1, which indicates good reproducibility has been achieved for repeated injections with RSD less than 3.6%.
  • the valve actuation time was the same (33 ms).
  • broader peaks were obtained as the valve control and sample injection pressures were set at 20 psi and 2 psi, respectively, and the injected sample volume was approximately 500 pL.
  • the valve control and sample injection pressures were 30 psi and 1 psi, respectively.
  • the valve control and sample injection pressures were set at 30 psi and 1.5 psi, respectively.
  • the pressure-injected sample volume can depend on several factors, including the mechanical injector open time, sample injection pressure, and the backpressure present in the separation channel.
  • the mechanical injector open time is determined by the actuation time, the valve control pressure, and the mechanical properties of the injector (e.g., the PDMS membrane in the pneumatic valve described previously).
  • Figure 5 depicts the relationships between those parameters and the peak width of injected fluorescein samples.
  • the peak width was recorded at approximately 400 ⁇ downstream of the intersection to represent the original injected sample plug size and minimize the influence of the plug migration in the channel and diffusion. All data shown were collected from the same pneumatic valve device.
  • Figure 5A shows that the peak width increases linearly with the valve actuation time, because the valve was opened for a longer period of time allowing more sample to enter into the separation channel.
  • the sample injection pressure was 1 psi and the valve control pressure was 30 psi.
  • the valve actuation frequency was 0.21 Hz.
  • Valve actuation times ranging from 33 ms to 167 ms were investigated.
  • the insets are the pictures of the injected fluorescein sample plugs corresponding to different valve actuation times. Based on the length of the sample plugs and the dimension of the channel, the injected sample volumes were estimated from less than 200 pL to approximately 1 nL.
  • the peak width increases with the sample injection pressure (Figure 5B).
  • the valve actuation time 33 ms
  • frequency 0.21 Hz
  • the valve control pressure 30 psi. Only low injection pressures (1-3 psi) were investigated.
  • the pulsed hydrodynamic flow induced in the separation channel and the longer injected sample plug would destroy the separation.
  • the higher injection pressure increased the back pressure present in the sample channel, increasing the resistance to closing the valve.
  • Figure 5C shows that the peak width linearly decreases with increase in the valve control pressure.
  • valve actuation time 33 ms
  • frequency (0.21 Hz)
  • sample injection pressure 1.5 psi
  • Valve control pressure was tested from 20 psi to 40 psi.
  • the response of the PDMS membrane to the pressure was slow because of its flexibility. Therefore, it took a longer time to close the valve. This behavior resulted in actual valve open times that were much longer. If the pressure was too low ( ⁇ 20 psi), sample leakage occurred or the injected sample plug was too long, because the valve was not completely closed after actuation.
  • the PDMS membrane When applying relatively higher control pressures, the PDMS membrane can stick on the channel surface and can take additional time to relax, and then open the valve. On the other hand, the PDMS membrane responds quickly to the pressure to close the valve. Thus, the actual time for the valve to reach the open state can still be shorter. If the pressure was too high, the PDMS membrane can stick to the channel surface too tightly and can take a very long time to open the valve or the valve may not even open during the valve actuation period. In some instances, pressures greater than 40 psi were too high.
  • Figure 5D shows that the injected sample plug size is independent of the valve actuation frequency.
  • the sample injection pressure wasl psi
  • the valve actuation time was 33 ms
  • the valve control pressure was 30 psi. Because both the sample and the run buffer were diluted aqueous solutions, the variance of the backpressure present in the separation channel was negligible.
  • the injected sample plug size varied little at different valve actuation frequencies.
  • Figure 6 shows a CE separation of four FITC-labeled amino acids using embodiments of the present invention.
  • the sample concentration of each amino acid was 250 nM.
  • the valve actuation time and frequency were set as 67 ms and -0.1 Hz.
  • the sample injection and valve control pressures were 3 psi and 40 psi, respectively.
  • the data acquisition system was activated to record the fluorescence intensity at the end of the separation channel.
  • the analyte migration distance was measured as approximately 2.4 cm. Only the first cycle of separation was shown in Figure 6.
  • Four major peaks were completely resolved in 15 s, and some minor peaks showed up due to the impurities present in the sample solution.
  • the theoretical plate numbers of each major peak are calculated and listed in Table 2.
  • the efficiency of the separation is higher than 9.2 x 10 plates for -2.4 cm long microchannel.
  • Figure 8A 7.4 x 10 3 1.0 x 10 4 1.3 x 10 4
  • Figure 7 shows a repeated CE separation of three FITC-labeled amino acids. Although only 100-second period of separation (10 runs) is shown, the data was recorded after running the microchip continuously for ⁇ 2 h, corresponding to more than 700 runs.
  • the average separation efficiency for each peak is listed in Table 2. Compared with the separation shown in Figure 6, the efficiencies obtained in Figure 7 decreased slightly due to some degradation of the PolyE-323 dynamic coating during the preceding hours of operation. However, the efficiency was higher than 2.9 x 10 5 plates/m. It should be pointed out that the peak positions at time scale were not the real peak migration times. The migration times of each peak can be estimated based on the intervals between injections and the first valve actuation time.
  • Figure 8 shows portions of Figure 7 in greater detail.
  • Figures 8A and 8B show the same separations performed earlier. Based on the recording time, if the separation shown in Figure 8A is defined as the first one, the separation in Figure 8B is approximately the 40 th run, and the separation in Figure 8C is about the 100 th run. It should be noted that all three separations are randomly selected to evaluate the pressure injection and microchip separation performance. All peak efficiencies are listed in Table 2. Apparently, the separation is reproducible although the separation becomes slightly worse with increase in the operation time, which is a normal phenomenon because of the dynamically coated surface.
  • SI, S2 and S3 represent the peak areas of 1 st , 2 nd and 3 rd peak, respectively, indicated in the Figures.
  • the fast, reproducible sample injections described above provide for multiplexed
  • multiplexed CE in which separations from multiple injections overlap in the separation channel and are subsequently analyzed in comparison and in combination.
  • the multiplexed CE separation can provide an increase in signal-to-noise ratio and a much higher duty cycle compared with discrete CE separations.
  • multiplexed CE is coupled with LC separations for two dimensions of orthogonal liquid phase separations, and for providing a much greater peak capacity without significant increase in the analysis time.
  • LC-CE has not been practical using electrokinetic injection, as the low sampling rate and the long delay time required between injections has led to almost all of the sample from the LC column being wasted.
  • the combination of the fast injections and the multiplexed CE can utilize at least 50% of the LC eluent for high- sensitivity, high-peak capacity analyses.
  • LC is coupled to fast, multiplexed microchip CE separations followed by high-performance electrospray ionization-mass spectrometry (ESI-MS).
  • ESI-MS electrospray ionization-mass spectrometry
  • a LC column is connected to the sample channel, which provides a plurality of sample injections to the separation channel through a sample injector.
  • the sample injector can include the pneumatic valve describe elsewhere herein or another mechanical valve providing fast, reproducible sample injections without the use of electrokinetic techniques.
  • the separation channel terminates in one or more ESI emitters directed at a mass spectrometer. This configuration can provide at least a 5-fold increase in proteome coverage for complex samples relative to a comparable LC-MS platform alone.
  • the pressure differential between the sample channel and the separation channel is such that upon briefly opening the mechanical valve, analyte is rapidly introduced for separation.
  • a potential is continuously applied in the separation channel enabling automatic separation of the sample injection.
  • the plurality of rapid injections occurs in a pseudorandom sequence resulting in multiple overlapping separations, which can be deconvoluted to reconstruct a spectrum.
  • Deconvolution can be achieved according to a modified Hadamard transform.
  • the achievable gain in signal-to-noise ratio relative to simple signal averaging can be proportional to the square root of the number of injections.
  • a preferred embodiment of the present invention includes a single pneumatic injector that spans multiple CE channels 901 within the separation channel 100.
  • the eluent from the LC column can be provided as a relatively large sample injection by the sample injector and can be distributed among a plurality of CE channels. In one instance, twenty or more CE channels can exist within the separation channel.
  • the injection volume can be increased in direct proportion with the number of CE channels, enabling high resolution separations while processing sample volumes compatible with typical nanoLC flow rates (e.g., 100-500 nL/min).
  • each CE channel will terminate with an electro spray 900 at its own ESI emitter.
  • the array of emitters can provide an order of magnitude sensitivity improvement relative to a single emitter analysis. The enhanced sensitivity, in
  • a discrete separation column is interfaced to the microfhiidic injector through a port configured for connecting the separation column to a loading channel.
  • a schematic illustration depicts one embodiment of a microfhiidic injector 1007 fabricated on a PDMS microchip
  • the PDMS microchip was created from three patterned templates: a control layer, a flow layer and a cover plate, using multilayer soft lithography similar to embodiments described elsewhere herein.
  • the control layer channel 1005 was 25 ⁇ tall and 100 ⁇ wide and was rectangular in cross-section.
  • the flow layer channels, which include the sample channel 1004 and the loading channel 1006, were -10 ⁇ tall, 100 ⁇ wide, and were rounded in cross-section to enable complete channel closure using the on-chip pneumatic valve.
  • the loading channel 1006 terminated at a port
  • Fig. 10A The embodiment in Fig. 10A is depicted with an optional ESI emitter 1003 at the terminal end of the separation column.
  • the port had a height of 160 ⁇ and a width of 310 ⁇ .
  • a cover plate contained a channel of rectangular cross-section that was 310 ⁇ wide channel and was 110 ⁇ thick.
  • FIG. 10B and IOC detailed illustrations depict the T-shaped intersection of the sample channel 1004 and the loading channel 1006 as well as the overlap of the valving channel 1005 and the sample channel.
  • the valving channel 1005 in the control layer crosses over the sample channel 1004 in the flow layer at or near the T-shaped intersection 1009.
  • a mechanical valve represented at location 1008, and shown in Fig. IOC comprises a deformable membrane between the control layer and the flow layer and separates the valving channel and the sample channel. Referring to Fig. IOC, the membrane has a closed position to obstruct flow in the sample channel, and an open position to permit flow in the sample channel based on a first and second pressure, respectively, in the valving channel.
  • an illustration depicts a configuration having two sample channels.
  • the first sample channel 1004 and valving channel 1005 intersect the loading channel 1006 at a different position than the second sample channel 1010 and valving channel 1011.
  • the two sample channels can intersect the loading channel at the same position along the length of the loading channel.
  • additional sample channels can intersect the loading channel at different positions.
  • the two sample channels interface the loading channel in a single plane.
  • sample channels can be fabricated in different planes. Developments in 3-D printing and other manufacturing technologies are well known for such fabrication capabilities.
  • a separation column 1002 interfaces with the loading channel at a port 1001.
  • CE electrodes and applied voltage 1012 are configured for separation along the continuous length of the loading channel and separation column.
  • FIG. 10E an illustration depicts a configuration having two sample channels, each having a plurality of sample sources.
  • a first sample channel 1004 is fed by four sample sources 1014 via a manifold 1015.
  • a second sample channel 1010 is fed by three sample sources 1013 via a manifold 1016.
  • Valves (not illustrated) and other fluid flow devices can be used to select and/or control the sample sources providing sample to a sample channel.
  • sample injection possibilities enabled by embodiments having multiple sample channels and/or multiple sample sources per sample channel enables a high degree of analytical flexibility.
  • on-column sample derivatization can enable or enhance chemical analyses.
  • one sample channel contains an analyte or a mixture of analytes (Sample Channel A).
  • sample Channel B contains the derivatization reagent or mixture of reagents.
  • Sample Channel A may contain derivatization reagent(s)
  • Sample Channel B may contain analyte(s). If the two sample channels are located in close proximity to one another, the valves separating the analyte(s) (Valve a) and derivatization reagent(s) (Valve b) may be opened simultaneously such that a mixture of analyte(s) and derivatization reagent(s) is formed in the sample loading channel.
  • the timing for opening Valves a and b may be staggered such that Valve a is opened first, sample from Sample Channel A moves to the proximity of Sample Channel B, at which time Valve b is opened to achieve mixing of A and B.
  • Multistep sample derivatization procedures may also be achieved beyond simply combining sample from Sample Channels A and B.
  • a and B may be located in close proximity to one another and opened simultaneously to initiate the first step of a derivatization reaction.
  • a third sample channel (C) and its corresponding valve (c) may be located downstream of A and B. The distance from Channels A and B to Channel C, and the velocity of the mixture of A and B as it travels to C will determine the amount of time that A and B have to react prior to the introduction of C by opening Valve c.
  • By adding additional sample channels D, E, etc., each with corresponding valves d, e, etc., on-column derivatization reactions having more individual mixing steps may be accomplished.
  • A contains analyte(s)
  • B contains an acid or a base that is used to alter the charge state of the analytes in A
  • C contains the derivatization reagent that only binds to A in the charge state achieved by prior combination with B.
  • sample derivatization procedures that may be employed to enable or enhance chemical analyses include, but are not limited to, (1) adding a radiolabel for detection of analytes based on the radioactive decay of the label; (2) adding a fluorescent molecule to an analyte that does not fluoresce natively for fluorescence detection; (3) attaching a hydrophobic moiety to an analyte that in its native state exhibits low ionization efficiency when detected using electrospray ionization mass spectrometry; (4) attaching a singly or multiply charged moiety to alter the charge state of the analyte. (4) may be used to impart charge to a mixture of natively uncharged analytes that cannot otherwise be separated by capillary electrophoresis.
  • sample derivatization procedures beyond the four examples provided here that can be used to enable or enhance chemical analyses.
  • PDMS for fabrication of the microchip was prepared by thoroughly mixing a silicone elastomer (Sylgard ® 184) base and curing agent at a 10: 1 ratio.
  • the PDMS was poured onto the control layer template and spin-coated at 2000 rpm for 30 s.
  • PDMS was also poured over the flow layer and cover plate templates to a thickness of 3-4 mm and degassed under vacuum. All substrates were cured at 70 °C for 2 h.
  • the flow layer substrate was then removed from its template and holes were formed using a 20 gauge catheter punch. Debris was removed from the substrate by applying compressed nitrogen followed by tape to both sides.
  • the surfaces of the flow layer and control layer substrates were activated in an oxygen plasma system at 50 W power and 200 mTorr pressure for 30 s. Following activation, the flow layer substrate was aligned at the sample
  • the irreversibly bonded assembly was placed in an oven at 70 °C for 1 h to improve bond strength and then the bonded flow and control substrates were cut and removed from the control layer template. It was necessary to remove the portion of the membrane that spanned the loading channel. This was accomplished by grasping the suspended membrane with a pair of fine-tipped tweezers and carefully pulling in such a way that the membrane tore along the loading channel walls. A hole was punched through both substrates as described above to provide access to the pneumatic valve, and the assembly was again cleaned using a combination of compressed nitrogen and tape.
  • the microchip injector was completed by aligning and bonding the flow and control layers to the cover plate as described elsewhere herein.
  • fused silica capillaries having an o.d. of 140 ⁇ and an i.d. of 30 ⁇ were passivated with HPC to suppress electroosmotic flow.
  • the coating was prepared by first flushing a -2.5 m long capillary with 1 mL of 1 M HCL solution by applying 15 psi N 2 back pressure to the solution reservoir followed by flushing the capillary with 200 ⁇ ⁇ 5% HPC in water using 20 psi N 2 back pressure. The capillary was then flushed with deionized water to remove excessive hydroxypropyl cellulose.
  • the treated capillary was subsequently cut into 10 equal lengths and ⁇ 3 cm at the end of each length was chemically etched in 49% HF to render it porous for electrical contact as described previously.
  • the etching of all 10 capillaries took place in a single batch using an approach adapted from previous work.
  • the distal end (inlet) of the capillary was inserted into a ⁇ 5 cm length of 360 ⁇ m-o.d., 150 ⁇ m-i.d. capillary and sealed in place with epoxy.
  • This same end was cut a few mm from the inlet using a dicing saw (SYJ-400, MTI Corp., Richmond, CA) to provide a clean interface at the loading channel-capillary interface.
  • An assembly consisting of a PEEK tee and an inserted metal tube (.04" i.d., 1/16" o.d) 27 was then slid onto the capillary from the inlet end to form an optional, sheathless ESI interface.
  • the emitter end of the etched capillary protruded 1-2 mm from the metal tube.
  • the capillary inlet end was then inserted under a microscope into the 3-mm-long port that to connect the capillary to the loading channel on the microchip and a small amount of PDMS was applied at the loading channel- capillary interface.
  • the PDMS was cured by placing the assembly in an oven at 110 °C for 20 min.
  • High voltage for CE operation was applied to a platinum wire inserted into the buffer solution using a Glassman High Voltage power supply (High Bridge, N. J.)- A transfer fused silica capillary (360 ⁇ o.d., 50 ⁇ i.d.) with one end inserted into the buffer solution and the other end press-fitted into the microfluidic chip using a short length of Tygon tubing as a sheath was used to provide the CE run buffer to the device.
  • the N 2 back pressure controlling the flow through the CE capillary referred to as the eluting pressure, was regulated using a second digital pressure controller.
  • a second voltage of ⁇ 2 kV was applied to the metal tube at the capillary outlet through a Bertan power supply (Hauppauge, N. Y.) to provide for stable electrospray operation.
  • Mass spectra were acquired in full scan mode covering an m/z range from 300 to 1000 at an acquisition rate of 2 Hz.
  • Leucine encephalin, kemptide, angiotensin II, methanol, acetic acid, hydrofluoric acid, ammonium acetate and hydroxypropyl cellulose (HPC, average MW -100,000) were utilized for demonstrating the injection, CE separation, and mass spectrometry analysis.
  • Peptide stock solutions were prepared individually in water at a concentration of 1 mg/mL. A 10 ⁇ mixture of the three peptides was then prepared by dilution from the stock solutions into the run buffer.
  • the run buffer was 9: 1 20 mM aqueous ammonium acetate: methanol, adjusted to pH 4 with acetic acid. Colored dye was used for visualizing pressure-driven injection and transfer of the sample plug to the capillary.
  • a pressure-driven injection sequence is shown in Fig. 11 using an aqueous dye in place of the sample and in the absence of an electric field.
  • the eluting and sample pressures were 2.0 and 2.5 psi, respectively, and the valve opening time was 65 ms.
  • the interfaced capillary had a 30 ⁇ i.d. and was 75 mm long.
  • To estimate the volume of this and other sample plugs it was necessary to know the cross-sectional area of the rounded microchannel. This was determined by filling the separation channel in both the microchip and capillary with perfluorodecalin, an immiscible oil, injecting colored dye from the sample channel, and comparing the length of the plug in the microchannel and inside the capillary of known diameter.
  • the microchannel had a cross-sectional area of 450 ⁇ , equivalent to a 24 ⁇ m-diameter capillary.
  • the injection volume shown in Figure 11 was approximately 400 pL, a plug size typical of microchip electrophoresis, and the volume could easily be tuned larger or smaller by adjusting the valve opening time and the sample injection pressure.
  • the back pressure was such that when an injection took place, the sample traveled mostly upstream, thereby minimizing any pressure shock that could affect an ongoing separation.
  • the flow rate was found to range from -20 to 100 nL/min. These flow rates are in the nanoflow regime, enabling high ionization efficiency for improved MS detection, when utilized.
  • Figure 13 shows a series of repeated separations of a three-peptide mixture containing kemptide, angiotensin II and leucine encephalin.
  • the valve was opened every 1.25 min to inject the sample, and the valve opening time was varied from 0.1 s to 3 s throughout the series. The trend of increasing peak intensity with injection time is clear.
  • FIG 14A The tradeoff between separation efficiency and S/N as the injection volume is varied is shown in Figure 14.
  • Figure 14A the number of theoretical plates as a function of injection time is shown for three different elution pressures. Values were calculated for the leucine encephalin peak. Higher flow rates resulted in reduced plate counts due to increased Taylor dispersion, and in each case, smaller injection plugs produced narrower detected peaks and thus greater plate counts.
  • the modest separation efficiency achieved here is due to the pressure-assisted mode of operation, as even at the lowest pressure used (1 psi providing a flow rate of -20 nL/min), Taylor dispersion degraded separation performance.
  • Figure 14B shows the S/N for kemptide for three replicate measurements using 2 psi for both the sample injection and the elution pressure. Noise was calculated from the data points in the range of 0.4 to 0.2 minutes before the apex of each peak.
  • Figure 14C shows two overlaid kemptide peaks, one acquired from a 0.3 s injection and the other from a 3 s injection. The peak resulting from a 0.3 s injection is clearly narrower than that from the 3 s injection, but the S/N is also substantially reduced as reflected in the baseline adjacent to the peak. This demonstrates the ease with which separation performance can be assessed and optimized over a range of conditions using this approach.
  • sample injection is pressure based, and expected to avoid quantitative biases inherent in electrokinetic injection strategies of the prior art.
  • the elution and sample injection pressure was 2 psi for all data points.
  • the peak area for each analyte increases linearly with injection time and as such, the proportionality between peptides is maintained, even across this range of injection times spanning more than a factor of 20.

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Abstract

L'injection d'échantillons microfluidique, qui est basée sur une soupape mécanique plutôt que sur une injection électrocinétique dans un canal de séparation intégré ou une colonne de séparation distincte, peut permettre d'obtenir de meilleures injections d'échantillons, aboutir à des capacités accrues, et éviter d'avoir à changer le champ électrique dans le canal de séparation pour provoquer l'injection d'échantillons. Une interface permettant l'utilisation d'une colonne de séparation distincte permet d'employer très facilement cet injecteur microfluidique avec des techniques d'analyse existantes. Plusieurs canaux d'échantillons et/ou sources d'échantillons peuvent être utilisés avec cet injecteur d'échantillons microfluidique.
PCT/US2015/027728 2014-04-29 2015-04-27 Injecteurs d'échantillons microfluidiques sans injection électrocinétique WO2015167990A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0620432A1 (fr) * 1993-04-15 1994-10-19 Ciba-Geigy Ag Méthode pour contrôler l'introduction d'un échantillon en séparation par micro colonnes et appareil d'échantillonage
WO2001035088A1 (fr) * 1999-11-05 2001-05-17 New Mexico State University Technology Transfer Corporation Dispositifs microfluidiques avec detection electrochimique a film epais
WO2001094635A2 (fr) * 2000-06-05 2001-12-13 California Institute Of Technology Dispositifs et procedes microfluidiques a flux actif integre
WO2012050642A2 (fr) * 2010-09-23 2012-04-19 Battelle Memorial Institute Électrophorèse capillaire sur puce sans injection électrocinétique

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0620432A1 (fr) * 1993-04-15 1994-10-19 Ciba-Geigy Ag Méthode pour contrôler l'introduction d'un échantillon en séparation par micro colonnes et appareil d'échantillonage
WO2001035088A1 (fr) * 1999-11-05 2001-05-17 New Mexico State University Technology Transfer Corporation Dispositifs microfluidiques avec detection electrochimique a film epais
WO2001094635A2 (fr) * 2000-06-05 2001-12-13 California Institute Of Technology Dispositifs et procedes microfluidiques a flux actif integre
WO2012050642A2 (fr) * 2010-09-23 2012-04-19 Battelle Memorial Institute Électrophorèse capillaire sur puce sans injection électrocinétique

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