WO2006078968A2 - Systemes bioanalytiques microfluidiques planaires integres - Google Patents

Systemes bioanalytiques microfluidiques planaires integres Download PDF

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WO2006078968A2
WO2006078968A2 PCT/US2006/002157 US2006002157W WO2006078968A2 WO 2006078968 A2 WO2006078968 A2 WO 2006078968A2 US 2006002157 W US2006002157 W US 2006002157W WO 2006078968 A2 WO2006078968 A2 WO 2006078968A2
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channel
microfluidic
sacrificial material
channels
hollow
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PCT/US2006/002157
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WO2006078968A3 (fr
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Aaron R Hawkins
Bridget Peeni
John Barber
Adam T. Woolley
Holger Schmidt
Milton L. Lee
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Brigham Young University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • 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
    • B01L3/502707—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 the manufacture of the container or its components
    • 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
    • B01L3/502715—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 interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81C—PROCESSES OR APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OR TREATMENT OF MICROSTRUCTURAL DEVICES OR SYSTEMS
    • B81C1/00—Manufacture or treatment of devices or systems in or on a substrate
    • B81C1/00015—Manufacture or treatment of devices or systems in or on a substrate for manufacturing microsystems
    • B81C1/00222—Integrating an electronic processing unit with a micromechanical structure
    • B81C1/00246—Monolithic integration, i.e. micromechanical structure and electronic processing unit are integrated on the same substrate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/12—Specific details about manufacturing devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • 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
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0418—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic electro-osmotic flow [EOF]
    • 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
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0421—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic electrophoretic flow
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81B—MICROSTRUCTURAL DEVICES OR SYSTEMS, e.g. MICROMECHANICAL DEVICES
    • B81B2201/00—Specific applications of microelectromechanical systems
    • B81B2201/05—Microfluidics
    • B81B2201/058—Microfluidics not provided for in B81B2201/051 - B81B2201/054
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81C—PROCESSES OR APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OR TREATMENT OF MICROSTRUCTURAL DEVICES OR SYSTEMS
    • B81C2203/00—Forming microstructural systems
    • B81C2203/07—Integrating an electronic processing unit with a micromechanical structure
    • B81C2203/0707—Monolithic integration, i.e. the electronic processing unit is formed on or in the same substrate as the micromechanical structure
    • B81C2203/0735—Post-CMOS, i.e. forming the micromechanical structure after the CMOS circuit

Definitions

  • This invention relates generally to microfabrication processes for microfluidics . More specifically, the invention is a system and method of microfabrication that use planar, thin- film microfabrication techniques from which microfluidic and microelectronic components are combined on a substrate to perform bioanalytical microfluidic operations .
  • a maj or interest in bioanalytical chemistry - is the separation and identification of proteins .
  • the distribution of proteins in biological materials is sensitive to cellular conditions , and consists of proteins having abundances that are dependent on age , disease state (s) , and environmental conditions (e . g . , nutrients , medicines , temperature, stress , etc . ) .
  • Marker proteins whose expressions change during the progression of a disease, have been associated with, certain human ailments such as cancer, Alzheimer' s disease, schizophrenia, and Parkinson' s disease , to name a few. Measurements of such target proteins are becoming increasingly important in clinical assays for human disorders and disease .
  • biphasic column containing reversed-phase packing followed by a strong cation exchanger .
  • a 3 -phase column having an additional segment packed with reversed phase particles , enabled sample desalting on column.
  • the 3-phase LC system provided a greater number of protein identifications than the "biphasic" column in analyzing a protein mixture from bovine brain .
  • micellar electrokinetic capillary chromatography separation provided the first dimension
  • capillary zone electrophoresis (CZE) was the second separation dimension.
  • Analyses of serum albumin, ovalbumin and hemoglobin tryptic digests were performed. While only 50-80 baseline-resolved fragments were observed in these separations , a maximum peak capacity of -4 , 000 peptide fragments was proj ected for this approach, based on the widths of the peaks in each of the separation dimensions .
  • the analysis time for this approach was very fast (10-15 min) , illustrating one of the benefits of miniaturized separation methods .
  • polymeric substrates While typically providing simplified and low-cost device fabrication, polymeric substrates are disadvantageous in terms of compatibility with conventional Si processing methods that may require elevated temperatures , for example in thin-film deposition, which would hamper the direct integration of planar polymeric devices with some electronics , detection instrumentation, etc . Moreover, polymeric materials tend to be more compatible with stacked, rather than planar thin- film designs . To date , little success has been seen in fabricating functional microfluidic systems on silicon substrates .
  • Valves and micropumps that are driven by the actuation of flexible elastomeric membranes in controlled sequences have also been demonstrated.
  • the need for external pressure and vacuum sources to actuate the membranes makes the entire system difficult to miniaturize .
  • the use of PDMS membranes in direct contact with pumped fluids is problematic for LC, because PDMS acts as a hydrophobic stationary phase in chromatography.
  • the present invention is a system and method for performing rapid, automated and high peak capacity separations of complex protein mixtures through the combination of fluidic and electrical elements on an integrated circuit, utilizing planar thin-film micromachining for both fluidic and electrical components .
  • Figure 1 is a block diagram showing the embodiment of design principles for silicon electronic circuits and microfluidic circuits .
  • Figures 2A, 2B, 2C, 2D are fabrication steps used to create microfluidic channels based on removal of a sacrificial core .
  • Figures 3A, 3B, 3C are SEM images of hollow waveguides formed by the removal of sacrificial (a) aluminum, (b) SU-8 , and (c) reflowed photoresist .
  • Figure 4 is a cross-sectional SEM of a hollow microchannel structure consisting of a single layer of silicon dioxide surrounding a hollow core, fabricated using aluminum as the sacrificial material .
  • Figure 5A is a graph showing the length of aluminum etched versus etch time for microchannels fabricated using aluminum as the sacrificial material . The temperatures of the etchant and the width of the structure are indicated on the graph .
  • Figure 5B is a graph showing the percentage of hollow microchannel intact after the etching process as a function of channel width for four different overcoating silicon dioxide thicknesses .
  • Figure 6A shows top view optical micrograph of two crossing fluid channels built using sacrificial core etching
  • 6B is a close-up SEM image of the crossing point for the channels
  • Figure 7A shows SEM view of the intersecting channels that form the key element of an electrophoresis separation system .
  • Figure 7B is an SEM view of the cross section of • a channel .
  • Figure 8 is a graph showing electrophoretic separation of an amino acid mixture made using a hollow channel T structure on a quartz substrate .
  • Figure 9 is a photograph of four PMMA reservoirs attached to a thin film microfluidic system on a substrate .
  • Figure 10 is an illustration of an on-chip electroosmotic pumping device using closed channels formed from sacrificial etching .
  • Figure HA is a top view optical micrograph of the critical part of an electroosmotic pumping system built using sacrificial core etching .
  • Figure HB is an SEM cross-section picture of the channels .
  • Figure 12 is a graph of liquid linear velocity and flow rate as a function of applied voltage in an electroosmotic pump .
  • Figure 13A is an SEM image of an ARROW waveguide with a 3.5 x 10 ⁇ m hollow core and polarization of incident laser indicated .
  • Figure 13B is an optical mode profile measured using a CCD camera .
  • the inset shows a false color representation of the light intensity of the mode .
  • Figure 13C is a comparison between experiment (symbols) and theory (lines) of transverse and lateral mode cross sections .
  • Figure 14A is a block diagram of a fluorescence setup .
  • Figure 14B is a graph of a fluorescence spectrum.
  • Inset Fluorescence emitted at end facet of ARROW waveguide .
  • Figure 15 is a graph showing fluorescence power vs . dye concentration.
  • Figure 16A is an illustration indicating a hollow ARROW waveguide being intersected by a solid- core waveguide .
  • Figure 16B is an SEM image of fabricated waveguide intersections .
  • Figure 17 is a schematic of a proposed complex microfluidic system for protein analysis . This system is designed to fit on a 3 cm x 3 cm chip .
  • Figure 18 is an alternative schematic to Figure 17 for a proposed complex microfluidic system for protein analysis . This system is designed to fit on a 3 cm x 3 cm chip .
  • Figure 19 is a schematic diagram of a device layout for testing the integration of sample pretreatment/concentration/desorption with CGE in a planar format .
  • Figure 20 is a schematic diagram of a device layout for testing integrated capillary IEF with CGE in a planar format .
  • Figure 21 is a zoom view of the interface between the IEF channel and CGE channels .
  • Figure 22 is a schematic diagram of a device layout for coupling CGE of proteins with enzymatic digestion and peptide CZE .
  • Figure 23 is a schematic diagram of a microfabricated system that integrates planar optical detection based on liquid waveguides .
  • Figure 24 is a schematic diagram of a microfabricated system to demonstrate the integration of all components developed for the analysis of proteins .
  • the present invention is a system and method for integrating microfluidics directly onto electronic systems , thereby making it possible that logic and electric power elements can be built into a microfluidic analysis system .
  • Figure 1 illustrates the fundamental design concepts of the present invention .
  • Figure 1 is provided as an illustration of a cross section of a substrate showing how microfluidics are combined on the same substrate with microelectronics using thin-film microfabrication techniques .
  • electronic circuitry 12 is made in standard silicon foundries , leaving a flat surface of silica covering active components 14 and routing layers 16.
  • active microfluidic circuitry 18 in the form of microfluidic devices (preferably using silica-based materials) 20 and silica-based fluidic routing layers 22 are made .
  • metal interconnects 24 relay information and power signals where needed.
  • CAD programs also include automatic wire routing routines , design rules for placement and size requirements , and performance models that provide very accurate predictions of how a circuit will work before it is ever made in silicon.
  • CAD tools allow teams of designers to work on the same large circuit divided into functional groups .
  • Successful design using CAD tools relies on very robust fabrication processes and devices . A transistor must look and act the same anywhere on a chip .
  • the design of complex microfluidic circuitry can follow much of the same approaches used to do CAD layout in microelectronics .
  • many of the existing CAD programs can be adapted to allow for user-specified devices and design rules .
  • the first step in building a microfluidics toolbox is identifying robust designs for active microfluidic components including fluid pumps , separation devices , and detectors .
  • the second step is identifying design rules for spacing and widths of fluid channels for routing and connecting, including branching elements .
  • active devices and connections have the same performance no matter where they are placed in an on-chip network .
  • Another method of detection that fits in with the planar design philosophy of the present invention is the direct electrical measurement of biological species in a microfluidic channel . This can be done by attaching electrodes across a channel and monitoring the impedance of a fluid as it flows by the electrodes . Biological material passing through the detection window will be indicated by a change in impedance . These measurements can be very sensitive and are aided by the small geometry of microfluidic channels .
  • the first step of the present invention is to select a suitable substrate material .
  • a silicon substrate will be used in the following example .
  • any suitable substrate material known to those skilled in the art of integrated circuit fabrication can also be used in the present invention.
  • these substrate materials include semiconductors , insulators , polymers , ceramics , metals and glasses .
  • the next step is to create hollow tubes by surrounding a sacrificial core with silicon dioxide or silicon nitride .
  • the sacrificial core is then removed with etching .
  • This fabrication process of the present invention is depicted in Figures 2A, 2B, 2C and 2D, and relies on two well-known microelectronics-based processes .
  • the first process is the chemical removal of a material applied to a substrate
  • the second is chemical vapor deposition (CVD) of silicon-based thin films .
  • a substrate 30 is coated with silicon dioxide and/or nitride layers 32 using plasma enhanced chemical vapor deposition (PECVD) . This process takes place at approximately 250 0 C .
  • PECVD plasma enhanced chemical vapor deposition
  • silicon, quartz , and glass substrates have been used. Tolerance to the temperatures used in the vapor deposition process limits the materials that can be used for the substrate .
  • PECVD compatible solid films such as amorphous silicon can also be used.
  • evaporated dielectric thin films like alumina and silicon monoxide can be used in other embodiments .
  • a thin layer of sacrificial material 34 is then deposited and defined into a thin line using photolithography and etching techniques .
  • sacrificial materials may be used, including photosensitive polymers and metals . What is important is that the sacrificial material be capable of being removed through some process , such as acid etching, without damaging the underlying substrate or other layers of materials on the substrate , if any.
  • An overcoat layer of PECVD oxide 36 or nitride is then grown which covers the sacrificial material 34. The conformal nature of this process is important to ensure that the sacrificial material 34 is completely enclosed .
  • the final step of the process is to expose the sacrificial material 34 to an etch from either end of the channel 38. Upon completion of the acid etch, the result is a hollow tube 40 with walls composed of either silicon dioxide or silicon nitride .
  • the process above describes the creation of a single, straight, hollow tube .
  • one of the important advantages of the present invention is the ability to create tubes that can bend, tubes that can cross over other tubes without being in communication, tubes that can intersect and j oin with other tubes , and the creation of multiple parallel tubes , to name just a few.
  • the present invention is able to create complex interactions and intersections of tubes for the flow of fluids .
  • sacrificial materials have been investigated in the context of the fabrication process described above . These sacrificial materials include aluminum, SU8 (a photosensitive epoxy) , and photoresist . Aluminum is most quickly removed using a nitric and hydrochloric acid etching solution while SU8 and photoresist are removed using a sulfuric acid and hydrogen peroxide solution.
  • the different sacrificial materials result in different shaped hollow core cross sections as illustrated in figures 3A, 3B, 3C, thus providing the advantage of additional flexibility when designing microfluidic devices .
  • the important factors in choosing the sacrificial materials are the shape of the resulting channel , and the ease with which the sacrificial material can be removed .
  • the hollow channels 40 , 42 , 44 shown in figures 3A, 3B and 3C depict openings that are very small , down to approximately 3 ⁇ m across . These diameters are more than an order of magnitude smaller than typical on-chip fluid channels produced by other prior art methods .
  • the process can also be used to produce a much wider channel 46 as shown in Figure 4.
  • This image shows a cross section of a hollow structure 50 ⁇ m wide with walls only 1 ⁇ m thick .
  • the hollow microchannel structure 46 consists of a single layer of silicon dioxide 48 surrounding the hollow core , fabricated using aluminum as the sacrificial material .
  • the silicon dioxide layer is 1.0 ⁇ m. thick and the hollow core is 3.0 ⁇ m thick .
  • the channels In order for structures made using the new planar, thin-film technology of the present invention to be useful for fluid and light guiding, the channels must have smooth inner walls , be of reasonable length, and mechanically strong .
  • the first criterion is important to prevent optical scatter or interruptions in fluid flow and is met by the conformal CVD coating as evident in SEM micrographs of the channel structures . Experiments to determine ultimate channel length and strength were conducted and were compared to physical models .
  • Etch times were investigated for aluminum sacrificial cores 700 nm thick patterned on silicon. Core width varied between 10 and 300 ⁇ m. A single layer of silicon dioxide 3.0 ⁇ m thick was deposited over the aluminum, the silicon substrate was cleaved, and then the samples were placed in an aqua regia (3 : 1 mixture of hydrochloric and nitric acid) solution. Samples were periodically removed from the acid solution, and the amount of aluminum that was etched was measured using an optical microscope .
  • Figure 5A shows a graph of the total length of aluminum etched versus time in the etchant for tubes that are 10 and 100 ⁇ m wide and at solution temperatures of 55 0 C and 70 0 C . Because the structures were cleaved twice , aluminum was removed from both ends of the structure simultaneously during etching . The numbers in the graph represent the total length of aluminum etched from both sides . The etch length follows the equation:
  • a nearly 5 mm structure can be etched in 24 hours , which is a manageable fabrication time for most devices .
  • Total etch times can be reduced by raising the temperature of the etch solution or increasing the nitric acid concentration (increasing the constant c o ) .
  • a 2 : 1 hydrochloric to nitric acid mix at 85°C can clear a 10 mm channel in less than 24 hours .
  • Another aspect of the present invention with regards to the creation of integrated microfluidic devices is the ability to generate networks of fluid channels that can route liquids over the surface of a chip much like dense electrical signals are routed in integrated circuits . This requires the creation of cross-over elements and T-branches just like in macro-plumbing.
  • Figure 6A is a microscope picture of a hollow channel 50 passing over the top of another hollow channel 52 , which was made by completing the process for creating a single hollow tube , and then repeating the process for a tube laid out perpendicular to the first one .
  • the sacrificial materials for both cores were removed simultaneously .
  • fluid was placed in both channels 50 , 52 and electrophoretic flow was initiated . There was no detectable leakage or cross-talk between the channels .
  • Figure 6B is a close-up of the crossover point where the channels 50 , 52 cross .
  • FIG. 7A shows a top view electron micrograph of these channel structures 60.
  • the core used in this case was a combination of aluminum and photoresist .
  • This hybrid core structure takes advantage of the fast etch rate achievable when removing aluminum and the smooth, half dome geometry possible when using photoresist and reflowing it at a high temperature .
  • a cross-section of these channels 60 is shown in figure 7B .
  • the intersecting hollow channels shown in Figure 7A were used as the critical element of an electrophoresis separation device . These structures were fabricated on a quartz substrate with a separation channel extending away from the intersection region. Three amino acids , arginine , phenylalanine , and glycine , were labeled by reacting fluorescein 5-isothiocyanate (FITC) with their respective amine groups . After labeling, the amino acids were diluted to 500 nM in 100 mM carbonate buffer, pH 9.2. Pipetting 10 ⁇ L of the buffer solution into the reservoirs caused the channels to be filled by capillary action.
  • FITC fluorescein 5-isothiocyanate
  • reservoirs 1 , 2 and 3 were filled with the buffer solution and reservoir 4 was filled with 10 ⁇ L of the prepared sample .
  • reservoirs 1 , 3 and 4 were electrically grounded, and -600 V was applied to reservoir 2.
  • the loaded sample was separated by grounding reservoir 3 , applying -600 V to reservoirs 2 and 4 , and applying -750 V to reservoir 1.
  • Confocally filtered laser-induced fluorescence detection was accomplished using an Ar ion laser for excitation and a photomultiplier tube detector . Fluorescence was probed approximately 0.65 cm away from the junction region shown in Figure 7A.
  • the separation was completed in under 30 s ( Figure 8 ) . Interfacing microfluidic devices to external fluid sources and reservoirs is another important consideration for an integrated system because every analytical system must interact with the macro world. A number of schemes have already been investigated that would provide large fluid reservoirs on chip and be compatible with hollow core devices .
  • a maj or aspect of microfluidics is the manipulation of fluid flows in small on-chip channels .
  • One of the most attractive ways of doing this is by using electrical forces (electroosmotic flow) .
  • An electroosmotic pumping device can be built by directing the fluid flow generated from a large number of small diameter channels from one reservoir into another . The design of such a pump is illustrated in Figure 10. Voltages 80 , 82 are applied at both ends of the device , and the generated electric field produces fluid flow 90 in the small diameter channels 84. Pressure in Reservoir 2 86 then pushes fluid into the larger diameter channel 88 to the right .
  • An electroosmotic pump with enclosed channels is implemented as follows .
  • a sacrificial core was applied and then photodefined into the pump geometry.
  • Conformal PECVD oxide was grown over the core, and then the sacrificial layers were removed.
  • Pumps were made on silicon, glass , and quartz substrates using aluminum as the sacrificial material .
  • Figure HA shows a top view photo of the critical section of an electroosmotic pump taken with an optical microscope .
  • the width of the small channels 100 on the left of the photograph was approximately 3 ⁇ m, while the width of the larger channel 102 shown on the right was 25 ⁇ m .
  • Figure HB shows a cross section of the channels 100 taken using an SEM .
  • a pump fabricated on an SiO 2 substrate with 100 channels (1 /xm in width and depth each) feeding into a single 40 ⁇ m wide channel was evaluated. The pump was initially filled with a pH 9.5 carbonate buffer solution.
  • a reservoir surrounding the small channels was filled with carbonate buffer containing 9.1 ppm rhodamine B .
  • Figure 12 shows a plot of liquid linear velocity and flow rate in the large channel as a function of voltage applied.
  • the characterized pump represents a minimum attainable geometry for the narrow channels .
  • Flow rates will increase with increase in number of total channels and/or channel diameter .
  • Waveguides were mentioned previously as being an important element of the microfluidic components .
  • optical detection on microfluidic platforms will be played by ARROW waveguides .
  • These structures enable light to be routed through liquid channels on the surface of a chip from optical sources to points of detection, and from points of detection to on-chip and off-chip optical detectors .
  • ARROW construction requires the deposition of several alternating layers of silicon dioxide and silicon nitride of thicknesses specific to the wavelength of light to be guided. These layers surround the sacrificial core material in all dimensions .
  • FIG. 13A A cross sectional view of an ARROW is shown in Figure 13A.
  • Figure 13B Optical tests on these waveguides are shown in Figure 13B in which the structure has been filled with ethylene glycol and illuminated with a 785 nm wavelength laser on one side of a cleaved facet while the other side was imaged as shown.
  • the geometry of the waveguide is outlined to better indicate the location of the propagating optical signal in relation to the top and side walls .
  • Ethylene glycol was used as the liquid in this test to reduce the amount of evaporation and to allow time to perform measurements .
  • Figure 13C shows the extent of the measured optical mode profile compared to theoretical computer models . The excellent agreement indicates that we can accurately design and build ARROWs given any liquid core and any light wavelength . Optical loss for these structures , the most important figure of merit for waveguides , was measured to be close to 0.1 cm "1 . This is well within the required performance range for on-chip devices .
  • ARROW waveguides were also filled with fluorophore containing liquids as illustrated in Figure 14A.
  • the structures were specifically designed to guide the fluorescence signal from a fluorophore pumped at 632 nm .
  • the fluorescence spectrum from the waveguide is shown in Figure 14B .
  • This same setup was used to measure detection limits for fluorescence signals .
  • the results are shown in Figure 15 , indicating detection down to several pmol/L (corresponding to 500 dye molecules in the waveguide) . These limits of detection will be improved significantly by using a better detector and filter setup .
  • liquid waveguides with solid-core waveguides for routing optical pump or measurement signals .
  • One application would be to illuminate only a very small volume of liquid inside a waveguide ( femtoliters , fL) for detecting single molecules by intersecting a solid core waveguide with a liquid one as illustrated in Figure 16A.
  • the integrated structure is created as shown in Figure 16B .
  • the hollow waveguide was filled with an ethylene glycol solution containing Alexa 647 , the same dye used in the previous experiments , and a 632 nm laser illuminated the solid-core ARROW.
  • the total amount of liquid illuminated in the hollow core was 59 fL.
  • Microscale impedance measurements are another application of the present invention . The most sensitive detection methods for biological molecules and agents are currently based on fluorescent tags .
  • This mechanism requires the necessary optical sources and detectors , and the introduction of a relevant fluorophore that can attach to a molecule of interest . It would be desirable for many reasons to have a sensitive method of detection based upon electric measurements , including ease of integration .
  • One potential electrical characteristic to measure would be impedance or, inversely, conductivity .
  • microfluidic components based on planar, thin film technology are almost limitless , and each depends on the application of interest . In fact , most fluidic manipulations can be addressed by this technology .
  • the previous sections have addressed the development of a variety of components that could comprise a microfluidic chip .
  • This next discussion describes the design of a microfluidic device that integrates the components necessary to address a very complex application.
  • the area of proteomics is extremely challenging, requiring complex multidimensional approaches to separate and identify the vast number of proteins in biological samples , especially those that are present at trace levels .
  • the planar microfluidic technology taught by the present invention allows the integration of many protein manipulation steps in a microdevice for separation and identification of complex protein samples at resolution and speed never before achieved .
  • the present invention makes possible the fabrication of a microfluidic chip that integrates the steps of extraction, concentration, separation, and identification of complex protein samples .
  • Each microfluidic system is designed to fit on a 3 cm x 3 cm chip .
  • Each analytical scheme begins by moving a sample from reservoir l_through a multichannel electroosmotic pump 2_ and through a porous monolith 3_ that has bonded affinity groups selective for the abundant proteins , such as albumin. This step removes high concentration proteins so that the less abundant ones can be concentrated and detected more easily . These non-bound proteins are introduced into a second porous monolith 4_ containing a different selective affinity ligand or a nonselective solid- phase extraction (SPE) binder for proteins , where all of the remaining proteins are concentrated. Sampling and trapping can continue until sufficient protein has accumulated in monolith 4 ⁇ for further separation and detection.
  • SPE solid- phase extraction
  • This two-step sample clean-up and concentration process is effected by applying voltage between sample reservoir 1 and a waste reservoir !5. Termination of sample loading and further clean-up of the bound protein sample can be accomplished by switching the voltage from sample reservoir 1 to a rinse reservoir 6_ so that current flows from rinse reservoir (5 (activating electroosmotic pump 1 ) through monolith 4_, rinsing off non-bound species to waste reservoir _5.
  • the protein sample is desorbed from monolith 4_ by switching the voltage from rinse reservoir £ to a desorber reservoir j3 and from waste reservoir 15 to another waste reservoir 10_, allowing electroosmotic pump £ to move desorber buffer through monolith ⁇ , displacing the bound proteins from the monolith.
  • the desorber solution will flow into waste reservoir 5_ by pressure flow because channel Vl_ between reservoir j5 and waste reservoir l_0 is filled with isoelectric focusing gel .
  • As the desorbed proteins enter the T-junction of waste reservoir 5_ they are drawn into the isoelectric focusing channel 11 by electrophoresis .
  • the isoelectric focusing channel VL contains gel bonded immobilines that create a pH gradient along the channel to focus and concentrate proteins according to their pi values .
  • the proteins are driven into numerous orthogonal gel electrophoresis channels 12 by switching the voltage from desorber reservoir £5 to buffer reservoir 1_5 and from waste reservoir IJD to buffer reservoir 2JK Proteins will be separated according to size and charge by gel electrophoresis in channels V2_.
  • buffer lf> will be continuously pumped by electroosmotic pump jL£ through intersection point 33 into waste reservoir 17.
  • the proteins will be introduced into a monolith 14_ containing a bonded protein digestion enzyme .
  • the peptides that are formed in the monolith will move from the monolith immediately into a peptide concentrating area IjB (separated merely by a conductive membrane from flowing buffer in contact with the voltage source at reservoir 20) before being released for CZE separation in channels 3JK
  • Buffer 2_0 will be continuously pumped by electroosmotic pump 2JL through intersection points IL4_ into common waste reservoir Tl ⁇ in order to maintain constant pH in the intersection points 14.
  • voltage will be momentarily switched from reservoir 3J5 to reservoir 2_3 to release the concentrated peptides and initiate fast CZE separation in channels 1_9.
  • migration in the CGE channels will be stopped.
  • proteins that migrate into the digestion monolith will be fragmented, trapped, and subsequently separated by CZE to produce peptide profiles that are characteristic of each of the proteins in the sample .
  • These peptide digest profiles will be used in a similar way that mass spectra are used to identify compounds .
  • Different bonded digestion enzymes can be used in different microfluidic systems to provide complementary fragmentation profiles for more definitive identification of the proteins .
  • Electrodes 2jt_ will be located just before waste reservoir 2_3.
  • fluorescence detection a fluorescent tag reagent in reservoir Z5 will be added to the separated peptides at the end of the CZE channels 19_ using electroosmotic pump 2£ just before the laser 27_ illuminated ARROW waveguide excitation junctions . Fluorescence will be detected at the ends of the ARROW/CZE channels 19_ using off- chip solid state detectors .
  • the main purpose of this subsystem is to isolate the primary proteins of interest away from highly concentrated proteins such as albumin and IgG, and other interfering compound types that might be present in biological samples .
  • highly concentrated proteins such as albumin and IgG, and other interfering compound types that might be present in biological samples .
  • a few abundant proteins can occupy over 80% of the sample, so some pretreatment is necessary to remove these abundant components before introducing the sample onto the microfluidic system.
  • FIG. 20 Another subsystem that can be created using the teachings of the present invention is an integrated 2-D separation, consisting of IEF followed by CGE . Development of this package will be critical to achieving high peak capacity separations .
  • a device layout for this subsystem is illustrated schematically in Figure 20.
  • Protein mixtures are loaded into a sample reservoir, and IEF occurs when a potential is applied between the sample and buffer reservoirs , across the gel-filled IEF channel containing immobilines to supply a pH gradient .
  • proteins will be focused into ⁇ concentrated bands within the IEF channel according to pi values .
  • bands will be inj ected into the array of CGE channels by applying a potential between the buffer and waste reservoirs addressing the ends of the CGE columns .
  • an EO pump can be designed to flush fresh buffer through the microchannel loops that address the ends of the CGE channels .
  • FIG. 21 A zoom view of the intersection of the IEF channel with a few CGE channels is shown in Figure 21. This figure shows that the looped buffer channels that allow sample inj ection are offset from the CGE channels to enable more efficient loading of analyte bands into the second separation dimension. Moreover, the interfaces between the different types of photopolymerized gels in the various channels are shown. Two different approaches can be used for filling the microdevices with the appropriate gels , using masked UV photopolymerization.
  • the array of CGE channels will be filled through the common waste reservoir at the end of the CGE columns with pre-polymer solution (e . g . buffer solution having 4% acrylamide with a photoinitiator) .
  • pre-polymer solution e . g . buffer solution having 4% acrylamide with a photoinitiator
  • an optical mask will be placed on top of the CGE channel array, which will allow UV radiation to polymerize the gel only in regions in the CGE channels ( Figure 21) up to the intersection with the IEF channel .
  • residual pre-polymer solution will be flushed from the fluidic system by flowing buffer solution between the sample and buffer reservoirs , and the buffer and waste/buffer reservoirs ( Figure 20) .
  • the IEF gel pre-polymer solution will be loaded into the IEF channel from the sample reservoir, acidic and basic immobilines will be added to the sample and buffer reservoirs , respectively, and a potential will be applied along the IEF channel to cause the immobilines to migrate to their appropriate positions and generate a pH gradient in the channel . 135
  • spatially defined, UV-masked photopolymerization will create a pH gradient gel in the desired regions in the IEF channel ( Figure 23 ) .
  • Unpolymerized material will be removed by flushing buffer solution ( Figure 23 ) through the looped buffer channel .
  • An alternate , potentially simpler approach involves filling the entire device with pre-polymer solution, adding acidic and basic immobilines to the sample and buffer reservoirs , respectively, and then migrating the immobilines into the IEF channel in an applied field. Masked UV polymerization will form gel in both the CGE and. IEF channels , and then unpolymerized materials in the unexposed buffer channels will be flushed out .
  • immobiline migration for either approach it will be critical to minimize heat generation to prevent premature polymerization; therefore, a combination of low current and active device cooling will be utilized to avoid this issue .
  • the next step is to integrate CGE, protein digestion, peptide separation and fluorescent labeling .
  • CGE protein digestion
  • peptide separation and fluorescent labeling To obtain a "peptide fingerprint" of each of the separated proteins , we will integrate CGE columns with monolithic beds for protein digestion, followed by an additional separation dimension having on-column fluorescent labeling . Appropriate design of this subsystem will enable separation- based identification of each protein analyzed, providing information similar to MS detection . A layout of a device designed for optimization of this operation is depicted in Figure 22. This subsystem has a single injector and CGE column, which is followed by a set up for fragmenting proteins and analyzing the resultant peptides . Separate devices having different digestion enzymes will provide multiple peptide fragmentation patterns , which will allow definitive identification of the proteins being separated.
  • On-column fluorescent labeling will be performed at the end of the separation system, enabling detection with a confocal laser- induced fluorescence setup . If greater peak capacity is desired, we can omit the enzymatic digestion monolith in the columns , and create a third separation dimension; if a conservative estimate of 20 protein bands can be separated in the CE channels , then the overall peak capacity of such a 3 -D separation system would be 50 , 000 , a substantial improvement over existing approaches .
  • Critical to the success of a complex planar microfluidic device is a detection system that fits into the fabrication mold outlined in previous sections .
  • Figure 23 illustrates a capillary electrophoresis separation module created from liquid waveguides . Fluorescently tagged proteins will be introduced into the module and then separated along a channel . Intersecting the liquid waveguide will be a solid core waveguide carrying an optical pumping signal . Proteins flowing through the illuminated region will emit fluorescence that will be collected and transmitted by the liquid waveguide to a downstream detector . Thin film detectors created using PECVD deposited amorphous silicon could ultimately be grown on the chip and interfaced directly with individual waveguides .
  • Figure 24 shows a schematic diagram of this simplified but fully integrated analyzer .
  • the principal difference lies in the number of CGE channels and, hence, the number of CZE channels, fluorescent tag channels , and optical waveguide channels required.

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Abstract

L'invention concerne un système et un procédé destinés à mettre en oeuvre des séparations de capacité de crête élevée et automatisée, rapide, de mélanges de protéines complexes par combinaison d'éléments électriques et fluidiques sur un circuit intégré, par micro-usinage d'un film mince planaire à la fois pour les composants électriques et fluidiques.
PCT/US2006/002157 2005-01-20 2006-01-20 Systemes bioanalytiques microfluidiques planaires integres WO2006078968A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103055973A (zh) * 2012-12-31 2013-04-24 苏州汶颢芯片科技有限公司 一种新型微流控分离芯片及其制备方法
CN103055971A (zh) * 2012-12-31 2013-04-24 苏州汶颢芯片科技有限公司 一种微流体流动可控的微流控芯片及其制备方法
FR3008690A1 (fr) * 2013-07-22 2015-01-23 Commissariat Energie Atomique Dispositif comportant un canal fluidique muni d'au moins un systeme micro ou nanoelectronique et procede de realisation d'un tel dispositif

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020108097A1 (en) * 2000-06-27 2002-08-08 Fluidigm Corporation Object oriented microfluidic design method and system
US20020127736A1 (en) * 2000-10-03 2002-09-12 California Institute Of Technology Microfluidic devices and methods of use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020108097A1 (en) * 2000-06-27 2002-08-08 Fluidigm Corporation Object oriented microfluidic design method and system
US20020127736A1 (en) * 2000-10-03 2002-09-12 California Institute Of Technology Microfluidic devices and methods of use

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103055973A (zh) * 2012-12-31 2013-04-24 苏州汶颢芯片科技有限公司 一种新型微流控分离芯片及其制备方法
CN103055971A (zh) * 2012-12-31 2013-04-24 苏州汶颢芯片科技有限公司 一种微流体流动可控的微流控芯片及其制备方法
FR3008690A1 (fr) * 2013-07-22 2015-01-23 Commissariat Energie Atomique Dispositif comportant un canal fluidique muni d'au moins un systeme micro ou nanoelectronique et procede de realisation d'un tel dispositif
EP2829511A3 (fr) * 2013-07-22 2015-04-08 Commissariat à l'Énergie Atomique et aux Énergies Alternatives Dispositif comportant un canal fluidique muni d'au moins un systeme micro ou nanoélectronique et procédé de réalisation d'un tel dispositif
US9234879B2 (en) 2013-07-22 2016-01-12 Commissariat à l'énergie atomique et aux énergies alternatives Device comprising a fluid channel provided with at least one micro or nanoelectronic system and method for carrying out such a device

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