|Publication number||USRE43122 E1|
|Application number||US 12/820,390|
|Publication date||24 Jan 2012|
|Filing date||22 Jun 2010|
|Priority date||26 Nov 1999|
|Also published as||US6432290|
|Publication number||12820390, 820390, US RE43122 E1, US RE43122E1, US-E1-RE43122, USRE43122 E1, USRE43122E1|
|Inventors||D. Jed Harrison, Richard Oleschuk, Loranelle L. Lockyear, Cameron Skinner, Paul Li|
|Original Assignee||The Governors Of The University Of Alberta|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (100), Non-Patent Citations (181), Referenced by (2), Classifications (38), Legal Events (1)|
|External Links: USPTO, USPTO Assignment, Espacenet|
The present invention relates generally to microfluidic analysis systems, and more specifically to micro-Total Analysis Systems μ-TAS), for performing liquid phase analysis at a miniaturized level.
Recent developments in the field of micro-Total Analysis Systems (μ-TAS) have led to systems that perform chemical reactions, separation and detection at a miniaturized level on a single microchip [see, for example Harrison, D. J. ; Fluri, K.; Fan, Z.; Effenhauser, C.S.; and Manz, A., Science 1993, 261, 895-897. Harrison, D. J.; and van den Berg, E.; Eds., Micro Total Analysis Systems '98, Proceedings of the μTAS '98 Workshop (Kluwer: Dordrecht, 1998), Coyler, C. L.; Tang, T.; Chiem, N.; and Harrison, D. J., Electrophoresis 1997, 18 1733-1741].
Most prior art microfluidic devices are based on conventional open tubular flow designs and solution phase reagents. While the functionality of these devices has continued to increase, one key feature that is presently lacking in these prior art devices is the ability to effectively incorporate on-chip packed reactor beds, for introduction of packing materials with immobilized reagents or stationary phases. While a few attempts have been made to employ packed reactor beds in some prior art designs, the difficulty of packing portions of a complex microfluidic manifold with packing material (such as microscopic beads) has so far hindered the effective utilization of these reagent deliver vehicles within microfluidic devices. (The difficulty of packing has been well recognized by practitioners in the field [see, for example, Ericson, C; Holm, J.; Ericson, T.; and Hjertén, S., Analytical Chemistry.)
In one prior art example, a packed bed chromatographic device with a bead trapping frit was fabricated in a silicon substrate [Oevirk, G., Verpoorte, E., Manz, A., Grasserbauer, M., and Widmer, H. M. Analytical Methods and Instrumentation 1995, 2, 74-82]. However, the packing material in this prior art design could not be readily packed or exchanged, thus limiting its utility.
Several authors have also described the difficulties associated with reproducibly fabricating frits for retaining packing material in conventional capillaries [Boughtflower, R. J.; Underwood, T.; Patterson, C. J. Chromatographia 1995, 40, 329-335. Van den Bosch, S. E.; Heernstra, S.; Kraak, J. C.; Poppe, H. J. Chromatogr. A 1996,755, 165-177. Colon, L. A.; Reynolds, K. J.; Alicea-Maldonado, K.; Fermier, A. M. Electrophoresis 1997, 18, 2162-2174. Majors, R. E. LC CC 1998, 16, 96-110.]. The frits used in conventional systems are prepared using time and labor intensive procedures, the most commonly used method involving the use of pure silica gel, wetted down with aqueous sodium silicate. The frit is made by first tapping a capillary end into a paste made from silica and aqueous sodium silicate. The resulting plug of silica is then heated to make a frit.
Furthermore, using frits produced by prior art methods of construction often leads to the formation of undesirable bubbles. Altria, K. D.; Smith, N. W.; and Turnbull, C. H., Chromatographia, 46 (1997) 644. Majors, R. E., LC-GC, 16 (1998) 96. ]Bubbles cause discontinuity within a column, hindering solution flow and ultimately preventing separation from occurring. The bubbles are thought to arise from a change in electroosmotic flow (EOF) velocity caused by moving from a bead trapping frit into an open capillary. The formation of bubbles, which have been observed to increase at higher voltages, also limits the amount of voltage that can be applied across the capillary, thereby limiting column lengthy, separation efficiency, and speed of analysis.
Developing a functional on-chip packed reactor bed design which overcomes the limitations in the prior art would significantly enhance the range of the microfluidic toolbox and extend the number of applications of such devices.
Generally, the present invention provides an on-chip packed reactor bed design using one or more weir structures that allow for an effective exchange of packing materials (beads for example) at a miniaturized level. The present invention extends the function of microfluidic analysis systems to new applications. For example, the packed reactor bed formed according to the present invention allows on-chip solid phase extraction (SPE) and on-chip capillary electrochromatography (CEC), as explained in detail further below. The design can be further extended to include, for example, integrated packed bed immuni- or enzyme reactors.
More specifically, the present invention provides:
A microfluidic analysis system, comprising:
The microfluidic analysis system may further comprise at least one side channel formed into said planar substrate, said side channel being connected at a first end to said main channel at a location upstream from said first weir, and at a second end to a reservoir, said side channel providing a higher flow resistance than said main channel.
The microfluidic analysis system may further comprise a second weir located upstream from said connected firs end of said side channel, said first and second weirs forming a chamber therebetween, said second weir providing at least one flow gap to allow, in use, at least some fluid to flow past said second weir while trapping said packing material within said chamber.
Each side channel connection to said main channel may be provided with a hook structure whereby, in use, packing material is sprayed into said chamber to facilitate even packing.
Said hook structure preferably at least partially obstructs direct line-of-sight entry of packing material from said side channel into said chamber and forms a chamber mouth to one side of said hook structure.
The present invention also provides a method of packing the chamber in the microfluidic analysis system as claimed above, said method comprising, providing a non-conductive substrate and effecting an electrokinetic flow by applying a relatively high voltage at said reservoir, said reservoir containing packing material, and providing relatively low voltages at said first and second ends of said main channel, whereby, packing material flows from said reservoir into said chamber and is trapped by said first and second weirs.
For a better understanding of the present invention, and by way of example, reference will now be made to the accompanying drawings, which show preferred embodiments of the present invention in which:
As explained above, the present invention is designed to provide a convenient system and method of trapping packing materials (such as beads) on-chip, and of effectively packing and unpacking the trapping zones, to provide a functional on-chip packed reactor bed which significantly extends the number of applications of microfluidic analysis devices.
One such extended application facilitated by the present invention is on-chip sample preconcentration by solid phase extraction (SPE). In microfluidic analysis, SPE is often required to overcome detection limit problems, or to eliminate a potential interferent. To date, preconcentration within microchips has been performed by sample stacking using “isoelectric focusing” [Jacobson, S. C. and Ramsey, M. Electrophoresis 1995, 16, 481-486]. Advantageously, unlike sample stacking, SPE can be made selective for a particular analyte and does not require precise control of buffer concentrations. For SPE the amount of preconcentration is limited by the preconcentration time, which makes it more flexible than sample stacking. The SPE of an analyte can be beneficial not only for analyte preconcentration, but also for removing other impurities or changing solvent conditions. While the coupling of SPE with microfluidic devices has been accomplished [Figeys, D. and Aebersold, R. Anal. Chem. 1998, 70, 3721-3727], the SPE component in these prior art devices have been made in a capillary or similar cartridge external to the chip, thus resulting in a more complex and more expensive system. The present invention is designed to overcome this prior art limitation by facilitating an on-chip SPE component.
As realized by the present inventors, an integrated, on-chip SPE component is ultimately easier to manufacture, does not require low dead volume coupling to the chip, and eliminates sample handling looses or contamination problems arising from the off-chip sample manipulation required in the prior art. It is anticipated that routine incorporation of SPE onto a chip, as facilitated by the present invention, will reduce problems with on-chip detection limits and will improve the range of sample preparation steps which can be integrated.
Another extended application facilitated by the present invention is on-chip capillary electrochromatography (CEC). CEC has recently received significant attention due to the fact that it combines the separation power of both liquid chromatography and capillary electrophoresis. To date the difficulty associated with packing chromatographic material within devices has focused most previous chromatographic efforts upon prior art open channel methods [Manz, A., Miyahara, U., Miura, J., Watanabe, Y., MIyagi and H. Sato, K. Sens Actuators 1990, B1, 249-255; Jacobson, S. C., Hergenröder, R., Koutny, L. B. and Ramsey, J. M. Anal. Chem. 1994, 66, 2369-2373; Kutter, J. P., Jacobson, S. C., Matsubara, N. and Ramsey, J. M. Anal. Chem. 1998, 70, 3291-3297; He, B., Tait, N. and Regnier, F. Anal. Chem. 1998, 70, 3790-3797]. In the prior art, open channel method devices with channel widths of 2 μm or less were required to improve mobile-phase transfer in open columns leading to other practical considerations such as clogging and a short path length for detection. There were also problems with the reproducibility and the cost of stationary phase coating in such structures.
As realized by the inventors, on-chip packed bed chromatography according to the present invention has the benefit of providing low mobile-phase mass transfer, and makes available a wide variety of stationary phases. In this case, the use of an off-chip prepared stationary phase offers the advantage that it eliminates the need for coating the chip and allows for optimization of the stationary phase preparation.
Yet another extended application facilitated by the present invention is providing on-chip bead-based immunoassay and enzyme based assays. These applications are described further below.
To illustrate the present invention by way of example, the inventors conducted a series of experiments, which are described here.
Solutions and Reagents
Various solutions and reagents were used in these experiments. Acetonitrile (BDH, Toronto, ON) was filtered through a 0.45 μm Nylon-6,6 filter (Altech, Deerfield, Ill.) prior to use. Otherwise, the acetonitrile was used as received, with no added electrolyte. Also, 50 mM potassium phosphate (pH 7.0) and ammonium acetate (pH 8.5) buffers were prepared in ultra-pure water (Millipore Canada, Mississauga, ON). A 1:1 (v/v) mixture of acetonitrile and buffer was prepared. A stock solution of 0.10 mM, 4,4-difluoro1,3,5,67,8 penta methyl -4-bora-3a4a-diaza-s-indacene, BODIPY 493/503 (Molecular Probes, Eugene, Ore.) was prepared in HPLC grade methanol (Fisher, Fair Lawn, N.J.) A 1 mM stock solution of fluorescein di-sodium salt (Sigma) was prepared in phosphate buffer. Both stock solutions were then diluted in the 50 mM phosphate and 50 mM ammonium acetate buffers to give 1.0 μM solutions, which were then diluted to 1.0 nM. This 1.0 nM solution served as the sample for preconcentration and electrochromatography. All aqueous (buffer and sample) solutions were filtered through a cellulose acetate syringe filter (0.2 μm pore size) (Nalgene, Rochester, N.Y.) prior to use.
One suitable packing material used in these experiments comprises a revers-phase chromatographic stationary resin. The resin was Spherisorb ODS1 (Phase Separations, Flintshire, UK), a porous C-18 resin whose particles ranged from 1.5 to 4.0 μm in diameter, as determined by scanning electron microscopy (ODS beads 12). A slurry of approximately 0.003 g/mL of ODS1 was prepared in acetonitrile. This slurry was used to supply the packing material reservoir 3, to subsequently pack the chamber 4.
Certain solvent and additive combinations were found to help the packing material stay in the packed chambers. For example, if ODS beads are introduced in acetonitrile they flow readily, while subsequently switching to an aqueous or predominantly aqueous solvent causes the beads to aggregate and become trapped within the chamber. With ODS heads up to 30% acetonitrile could be present in the aqueous solution without disrupting the aggregation observed to the point of de-stabilizing the packed bed. Up to 50% acetonitrile could be present with only modest loss in aggregation and weak demobilization of the bed. As another example protein G or protein A coated beads formed aggregates in aqueous solution, which made it hard to introduce them into the trapping zone. However, the addition of a neutral surfactant such as Tween 20 or Brij 35 (both are trademarks) prevented such aggregation and allowed the beads to be introduced. Conversely, subsequent removal of the surfactants form the aqueous solvent resulted in aggregation and enhanced stability of the trapped bed. The following trend was observed: when using non-polar or partially non-polar bead phases (for example, ODS and protein coated beads) lowering the surface tension of the solvent from that of water or buffered water, by the addition of organic additives such as organic solvents or surfactants, reduced the tendency to aggregate. Conversely reducing or eliminating materials with lower surface tension from aqueous solution increased the tendency to lock the beads in place on the bed, creating a “solvent lock” method to enhance bead trapping within these devices. Other organic solvents other than acetonitrile, miscible with water may also be used for these purposes, such as methanol, ethanol, dimethylsulfoxide, propylene carbonate, etc. Charged surfactants may also be used instead of neutral surfactants, so long as they are compatible with the proteins that may be present on the beads or in the sample.
Magnetic beads used for magnetic packing may comprise AbeBaw—protein “A” coated beads: composition 36-40% magnetite dispersed within a copolymer matrix consisting of styrene and divinyl benzene (Prozyme, Calif.) Also, Guifeng-oligo (dT)25 coated beads may be used for the isolation of mRNA. The beads have an even dispersion of magnetic material (Fe2 O3 and Fe3O4) through out the bead. The beads are coated with a polystyrene which encases the magnetic material (Dynal, Oslo, Norway).
Various instruments were used in conducting the present experiments. As these instruments and their operation are well known to those skilled in the art, only a brief description is provided, and the instruments are not shown in the figures.
A power supply and relay system used to control the electrophoretic voltages necessary for bead packing and all liquid handling on-chip has been described previously [Fluir, K., Fitzpatrick, G., Chiem, N. and Harrison, D. J. Anal. Chem. 1996, 68, 4285-4290]. LabVIEW programs (National Instruments, Austin, Tex.), were written for computer control of the voltage system and for data acquisition.
A laser-induced fluorescence detection system used in this experiment consisted of a 488 nm argon ion laser (Uniphase, San Jose, Calif.), operated at 4.0 mW, and associated focusing optics [Manz. A., Miyahara, Y., Miura, J., Watanabe, Y., Miyagi, H. and Sato, K. Sens. Actuators 1990, B1, 249-255] (Melles Griot, Irvine, Calif.). Fluorescence emitted from the BODIPY sample (as described above) was collected by a 25 X, 0.35 NA microscope objective (Leitz Wetzlar, Germany). The images were observed with a SONY CCD-IRIS camera. Alternatively a 530 nm emission filter and a photo multiplier tube (PMT) (R1477, Hamamatus, Bridgewater, N.J.) were used as a detector positioned so that the narrow channel 5 between the chamber 4 and packing material reservoir 3 could be monitored. Data were collected from the section of main channel 11 just next to the chamber 4. The weir 6 was just out of the field of view. The PMT was biased at 530 V while the PMT signal was amplified, filtered (25 Hz Butterworth) and sampled at a frequency of 50 Hz.
The fluorescence of the buffer, acetonitrile, and 1.0 nM BODIPY in both buffer and acetonitrile was measured using a Shimadzu RF-5301PC Spectrofluorophotometer.
While specific models and manufacturers have been provided for various instrumentation described above, it will be understood by those skilled in the art that any suitable, functional equivalent may be used.
Referring back to
The device 10 was not conditioned with any aqueous solutions prior to use. The chamber 4, channels 5, 11, and reservoirs 1, 2, 3 were first filled with acetonitrile. The chamber 4 was packed with ODS treads 12 (
Once the chamber 4 was packed, a step gradient was performed to introduce aqueous solution to the main channel 11 and the ODS beads 12 in the chamber 4. A 1:1 (v/v) mixture of acetonitrile and buffer was placed in reservoirs 1 and 2. Acetonitrile replaced the slurry in packing material reservoir 3. A voltage was then applied to main reservoir 1 and was ramped from 200 V to 800 V, with packing material reservoir 3 biased at 400 V and main reservoir 2 grounded. After 2 to 5 min at 800 V, the acetonitrile/buffer mixture in reservoirs 1 and 2 was replaced with buffer, and the same voltage program repeated. The chamber 4 was monitored visually to ensure that the acetonitrile was completely replaced by buffer and that the packing material (beads 12) did not shift or unpack during this procedure. (The beads 12 could be seen to agglomerate as the acetonitrile was expelled, and the index of refraction change at the water/acetonitrile interface was clearly visible.) The experiments conducted are described in further detail below.
Experimental Results and Discussion
In order to conduct the experiments, it was necessary to pack the chamber 4 with packing material (beads 12), as shown in
The narrow side channel 5 shown in
Reverse phase ODS beads 12 (as described previously) were used in the SPE device because of their extensive use for the chromatography of proteins, peptides and tryptic digests [Seifar, R. M.; Kok, W. T.; Kraak, J. C.; and Poppe, II. Chromatographia, 1997, 46, 131-136. Yan, C.; Dadoo, R.; Zhao, H.; Zare, R. N.; and Rakestraw, D. J., Anal. Chem. 1995, 67, 2026-2029. ] as well as other applications of SPE and CEC [Nielsen, R. G.; Riggin, R. M.; Rickard, E. C. J. Chromatogr. 1989, 480, 393-401. Hancock, W. S.; Chloupek, R. C.; Kirkland, J. J.; Snyder, L. R. J. Chromatogr. A 1994, 686, 31-43.] Electrokinetic packing of conventional capillaries has been described previously, [Yan, C.; U.S. Pat. No. 5,453,163, 1995. Knox, J. H.; Grant, I. H. Chromatographia 1991, 32, 317-328.], and the inventors have adapted the method for the present invention.
As briefly explained earlier, the packing procedure involved applying a positive voltage (ramped from 200-*00 V) to the packing material reservoir 3, while grounding main reservoirs 1 and 2. The applied voltage induced EOF to flow down the bead channel, carrying the beads into the cavity. An organic solvent was required to suspend the chromatographic beads 12 to prevent them from aggregating and plugging the narrow side channel 5. Studies have shown that capillaries filled with acetonitrile exhibit substantial electgroosmostic flow [Wright, P. B.; Lister, A. S.; Dorsey, J. G. Anal. Chem. 1997, 69 3251-3259, Lister, A. S.; Dorsey, J. G.; Burton, D. E. J. High Resol. Chromatogr. 1997, 20, 523-528. Schwer, C.; Kenndler, E. Anal. Chem. 1991, 63, 1801—14 1807. Salimi-Moosavi, H.; Cassidy, R. M. Anal. Chem. 1995, 67, 1067-1073. ].
As shown in
As shown in
The weir design of the present invention allows electric fields to be applied across the trapping zone formed by two weirs, when filled with beads, in a range as high as 20,000 to 80,000 V/cm without bubble formation at the weir. Separations performed in devices with these weirs can use electric fields at least as high as 15,000 V/cm. The power dissipated across a weir can be as high as 3-7 V/m without the formation of bubbles. In contrast firts formed in conventional columns have at the best been reported to form bubbles at power dissipations above 0.6 W/m, and electric fields in the range of 150-600 V/cm are the best that have been reported without bubble formation.
It is possible to couple an external capillary to a chip and allow the weir to be used as the trapping element for the beads packed within the external electrochromatography capillary. This can be accomplished using a low dead volume coupling, such as described by bings et al. ( N. H. Bings, C. Wang, C. D. Skinner, C. L. Colyer, P. Thibeault, D. J. Harrison, Anal. Chem. 71 (1999) 3292-3296.) In this way the chip based weir can replace the frits normally formed within external capillaries, and allow higher electric fields to be used, improving speed and separation efficiency.
(It is noted here that it was also possible to pack the cavity by applying a vacuum at main reservoirs 1 and 2, although this was less convenient when electrokinetic flow was used for sample loading and elution).
If for some reason the beads 12 did not pack as tightly as was desirable (as shown in
Significantly, a design utilizing a hook structure 13 at the chamber entrance (
As explained earlier, during the packing step, the packing material reservoir 3 has a positive bias applied with reservoirs 1 and 2 grounded. The inventors believe that the hooked structure 13 causes electric field lines to follow a curved pathway into the cavity. Consequently, as the chromatographic beads 12 follow the electric field lines into the chamber mouth 4A they appear to be “sprayed” as if from a snow blower (
During the packing procedure the chamber 4 filled only to the beginning of the hook structure 13 (see
A key aspect of the hooked structure as shown is the asymmetric entrance into the traping zone, which allows for better packing. A symmetric entrance means the entering beads can go to both weir equally, which tends to lead to uneven or difficult packing. An asymmetric structure allows the beads to pack preferentially at one end of the trapping zone firt and then build up in one direction from that location. The key role of the hook structure is to prevent line-of sight outflow form the trapping zone during use of the packed bed.
Chambers constructed without an asymmetry in the entrance were not observed to pack as well as asymmetric entry designs. In these cases, packing material tended to fill the corners furthest from the entrance, but no additional material would enter the chamber. The inventors believe that, due to its symmetric design, this type of chamber exhibits solvent back flow, after it has filled to a certain extent. That is, the partially filled chamber may resemble a closed or restricted system. Such an occurrence would preclude the filling of the symmetric chamber with beads and is consistent with previously observed behavior, as explained by Shaw. Such behavior may account for the ability to fill symmetric structures on some occasions but less readily on others. In contrast, an asymmetric design, with or without a hook structure 13 guarding the entrance is less likely to experience back flow directly into the narrow bead introduction channel 5.
Solid Phase Extraction (SPE) On-Chip
As explained earlier, the present invention allows applications of microfluidic analysis systems to be extended. One such extension is facilitating SPE directly on-chip. Preconcentration is a valuable tool that can be used to enhance the sensitivity of microfluidic devices. To determine the ability of a packed SPE bed constructed on a microchip to preconcentrate an analyte, we concentrated a 1.0 nM solution of BODIPY reagent from 50 mM phosphate buffer. Solution conditions utilized were similar to those used for protein and peptide analysis in HPLC-CE systems. [Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62, 978-984. Castagnola, M.; Cassiano, L.; Rabino, R.; Rossetti, D. V. J. Chromatogr. 1991, 572, 51-58.] The BODIPY reagent, when diluted in aqueous buffer, exhibits a high affinity for ODS material and is an excellent fluorophore. The preconcentration and elution of the BODIPY reagent was carried out in four steps: equalization of the SPE bed with buffer; sample introduction; buffer flush; and elution of analyte.
Following rinsing of the packed bed with phosphate buffer, a solution of 1.0 nM BODIPY was placed in main reservoir 1, and +200 V was applied for 2 minutes, with main reservoir 2 grounded. The EOF (0.2 mm/sec, 1.2×10−9 L/sec) flowed towards reservoir 2, carrying the BODIPY onto the SPE bed during the loading step.
As shown in
A buffer wash step was used after loading to wash sample remaining within the channel 11 onto the bed (in chamber 4). The solutions in reservoirs 1 and 2 were then replaced with acetonitrile, and the dye was eluted with solvent moving in the same direction as the initial loading step (or by reversal of the potential gradient during the elution step, it could be directed back towards the original sample reservoir). Both procedures work well, but the latter was more convenient for our testing.
where Vi is the volume of buffer containing analyte and Vf is the volume of acetonitrile containing analyte. The volume Vi is the product of the preconcentration time (tpre, sec.) and the electroosmotic flow of the sample being concentrated (fbuff, L/sec.) while Vf is the product of width of the eluted analyte peak (telute, sec.) and the flow rate of the eluting solvent felute (1./sec). For this case, the analyte was preconcentrated by a factor of at least 100 times. After sufficient concentration the BODIPY is easily observed visually on the SPE bed.
Different sample loading times were utilized to increase the amount of preconcentration. In the experiments, reconsecration times ranging from 120-532 seconds were studied yielding preconcentration factors of 80-500. Peak area (rsd 3-11%) plotted versus preconcentration time yielded a linear relationship (r2=0.9993) over the studied conditions.
Capillary Electrochromatography (CEC) On-Chip
As explained earlier, another application facilitated by the present invention is on-chip capillary electrochromatography (CEC). Reversed phase mode CEC was performed on a chamber 4 packed with octadecyl silane beads 12 equilibrated with buffer. Due to the lack of an injector within the chip design, the samples were loaded onto the front of the chromatographic bed in 50 mM ammonium acetate buffer, pH 8.5 (see “Solutions and Reagents,” above). Both compounds were totally retained under these conditions, as indicated by a lack of analyte signal in the loading and flush steps. The loading step functioned to both introduce the sample and preconcentrate the retained analyzes at the front of the bed [Swartz, M. E. ; Merion, M.; J. Chromatogr, 1993, 632, 209-213.]
Our present results are comparable to that reported for open tubular CEC on a chip [Jacobson, S. C., Hergenröder, R., Koutny, L. B., Ramsey, J. M. Anal. Chem. 1994, 66, 2369-2373. Kutter, J. P.; Jacobson, S. C.; Matsubara, N.; Ramsey, J. M. Anal. Chem. 1998, 70, 3291-3297. Ho, B., Tait, N., Regnier, F. Anal. Chem. 1998, 70, 3790-3797.]
Immunoassay Using Bead-based Reagents
Immunoassay on beads, or immunosorbent assays involves placing either an antibody or antigen on the surface of the bead. As a solution containing an antigen passes over the beads, the antigen specifically binds the antibody. In this way the specificity of the antigen for the antibody is utilized to separate it from other species in solution. Later the solution conditions are changed so that the antibody or antigen is eluted from the beads and is detected as either complex or the free antibody. The development of immunosorbent assays on chip is attractive because of the small amounts of reagents that are consumed. In addition microchips offer very fast analysis times compared to conventional methods performed in micro titer plates or in syringes packed with immuno-beads. Immunosorbent assays on-chip also provide lower concentration detection limits than solution phase immunoassays on-chip. Making the development of bead based immunoassay on-chip important.
Beads that have specific enzymes linked to them are packed into the chamber created by the two weirs. The use of beads is preferential because of the increased surface area of the beads as opposed surface area of the channel walls. The higher surface area leads to a greater capacity and more efficient trapping of the analyte. The weirs form a well-defined chamber for the immunoassay beads to pack. We have demonstrated bead-based immunoassay on chip for the enzyme theoplylline. In the experiment magnetic beads coated with protein A are packed within the chamber of the chip. Later the antibody (antitheophylline) is flowed across the bed in a 1 mM tricine buffer pH 8.0. When the antitheophylline flows through the packed bed the antibody binds to the protein A. The antitheophylline was passed over the bed for several minutes to ensure that the bed is saturated with antibody. A buffer washing step was then utilized to remove the remaining unbound antibody from the chamber and channels. The bed was then saturated with fluorescently labeled theophylline (diluted from a kit) by flowing it through the bed where it binds to the antitheophylline. The point at which the bed was saturated was determined by monitoring fluorescence below the bed and determining the point where the breakthrough curve plateaus. Following breakthrough the theophylline solution is washed from the device using a buffer flush step. A chaotropic agent is then added to elute the theophylline from the bed as either free protein or theophylline/antibody complex. Chaotropic agents can be of various types, however in this example a mixture of 90% acetone/10% tricine buffer was used. Once the chaotropic agent reaches the packed bed the theophylline is eluted in a relatively narrow band.
Although normally under these circumstances a competitive assay would be performed the direct assay demonstrates the ability of the chamber on the weir device to act as an immunoassay bed.
Enzyme Reactor Beds
There have been several methods developed for immobilizing enzymes onto solid supports like beads. Once immobilized the enzyme beads can be packed into beds to perform chemical reactions on solutions as they are flowed through them. Normally a solution containing a substrate is passed through the bed. When the substrate comes in contact with the enzyme reacts with the substrate to yield a product. The product resulting from the reaction of the immobilized enzyme and substrate can be later used as a method of detection or in other synthetic processes. This example illustrates the use of the immobilized enzyme horse radish peroxidase(HRP) and xanthine oxidase(XO) on porous silica beads (5 μm diameter). These results show that enzymes, once immobilized onto beads, can be trapped/packed into the weir device, where they are still active and can be used as an enzyme reactor bed.
XOD and HRP were immobilized onto Nucleosil 1000-5 silica beads (Machrey-Nagel, Germany) that has been silanized with 3-aminopropyltriethoxysilane, by crosslinking with gluteraldehyde (Sigma). The immobilization of enzymes on glass beads has been described previously and is known by practitioners of the art. All studies were performed using 50 mM boric acid adjusted with 1 M NaOH to pH 9.
The immobilization of HRP and XOD was performed to demonstrate two principals. First was the ability to pack the enzyme immobilized beads within the weir device and then second was to demonstrate that the enzyme was still active and could be utilized to catalyze reactions once packed. To show each of these principals a chemiluminscent reaction was performed using the weir device.
The ability to pack immobilized enzymes allows different methods of detection to be used for certain analytes. For example the luminol chemiluminescence (CL) reaction can be used for very sensitive determinations when only small amounts of analyte are available or when labeling reactions are otherwise difficult to perform. CL reactions are unique in that they do not require a light source simplifying the detection scheme. The chemiluminescence reaction catalyzed by HRP is shown below.
Luminol+H2O2+HRP (Light(425 nm)+other products
Beads immobilized with HRP were packed into the weir device and a solution containing the reagents for the reaction passed through the bed. The immobilized HRP was found to catalyze the chemiluminescent reaction when a solution of H2O2 (100(M) and luminol (10 mM) was flowed over a bed that had been packed with beads containing immobilized HRP. Light generated from the reaction was detected downstream from the enzyme bed.
However, it was noticed that with each successive trial the light generated from the CL reaction was lower than in the previous trial. This is probably caused by a decrease in the activity of the enzyme with each successive run. These results evidence the advantage of a method of removing the exhausted beads and replacing them with fresh ones, such as discussed for the replacement of ODS beads within the weir device.
While a two weir embodiment of the design according to the present invention has been described above, other embodiments are also possible. For example, it is possible to implement a single weir design to form an on-chip reactor bead (i.e. not having a second weir 6 located upstream in the main channel 11). Specifically, by providing a downstream weir 7 formed across the main channel and providing pressure only in a downstream direction (i.e. from main reservoir 2 and side channel 5 to main reservoir 1), it has been observed that packing can be achieved against the downstream weir 7. However, it is noted that a single weir design may result in the formation of a ragged leading edge for the packed bed that reduces separation efficiency when used for SPE or CEC. Additionally, the high back pressure associated with a long bed of small beads limited the length of the pack to about 4-6 mm. A high pressure fitting for the microchip would allow high pressure pumping and allowing somewhat greater lengths.
Other types of forces may also be used to create a packed bed using a single weir design. For example, it was also possible to achieve a limited degree of packing (to a length of a few millimeters) using electrokinetic forces, directed only in a downstream direction (i.e. from main reservoir 2 and packing material reservoir 3, to main reservoir 1).
In addition to varying the numbers of weirs, it is also possible to provide more than one inlet or outlet to a chamber, as shown in alternative embodiments of the present invention in
As shown in
Another embodiment is shown in
In all three embodiments shown in
Loading of beads with more than one side channel, as shown in
When using pressure driven flow to load beads, a back pressure must be applied to the additional side channels during loading, or else the reservoirs attached to the additional side channels may be temporarily sealed. When flushing the beads from the chamber 4, a pressure may be applied to the bead supply channel 5a to flush beads out of one or more additional side channels.
When performing SPE or CEC using a multiple side channel design and electrokinetic forces, a voltage may be applied to the additional side channels to prevent leakage of sample or beads out of the trapping zone and into the side channels, substantially in the same manner as described for a single side channel in the trapping zone. When using pressure drive pumping, the side channels may a have enough positive pressure applied to eliminate flow into the side channel, or else the reservoirs attached to the respective side channels can be temporarily sealed.
While the theoretical limits of various dimensions of a microfluidic device designed according to the present invention are not known, the inventors have adopted some general guidelines for practical purposes, which are discussed below.
It is thought that the length of the trapping zone may range anywhere from about 10 μm up to about 200 cm (using a coiled or serpentine path if necessary to allow for incorporation of such a length within the confines of a single device wafer). The trapping zone length required will be dependent upon the application and will also be limited by the forces which may be applied to achieve packing and unpacking. For example, on-chip CEC would require relatively long trapping zones, with a preferred upper limit of about 5 cm.
As to the depth of the trapping zone, sample and waste channels, a practical range is estimated to be about 400 μm to 0.25 μm. More preferably, the upper limit should be about 100 μm and the lower limit should be about 10% larger than the particle depth at a minimum.
Also, in order to reduce the likelihood of clogging, the bead delivery and bead waste channels (side channels 5, 5a 5d) preferably should be at least about 3 times deeper and three times wider than the bead diameter.
The maximum dimensions of the side channels 5, 5a-5d are also dependent upon the relative flow resistances required (i.e. the flow resistance of the side channel versus the main channel and the weirs, so as to minimize side channel backflow during use). Generally, speaking, the flow resistance of the side channels should be higher than the flow resistance of weirs to minimize the backflow problem.
The accompanying tables provide information on the calculated effect of channel and weir dimensions on the volumetric flow rates out of the trapping zone, as a function of flow channel depth, weir depth and side channel length using pressure driven flow.
In the tables below, what is called channel W is element 1 in
Correlation to FIG. 1A Width Length 20 μm Deep Element 1 Channel W 600 6,500 Weir 280 variable Element 5 Channel C 50 variable Element 3 Channel C′ 600 3,500 10 μm Deep Element 1 Channel W 580 6,500 Weir 280 variable
The volumetric flow rates were estimated using the Navier-Stokes equation for a rectangular channel cross section and Perry's tabulated values of the effect of channel shape. The flow resistance of a channel with half width a and half depth b is given by equation 2:
where ΔP is the pressure drop along a channel segment of length L, U is the average linear flow velocity, h is the viscosity, and N is a form factor dependent upon the cross sectional ratio b/a (b<a). The factor N may be estimated from solutions to the Navier-Stokes equation for pressure driven, parabolic flow, and was tabulated by Perry in Chemical Engineer's Handbook, (3rd edition, 1950) pp 387. The goal in device design is to make the resistance of the side channel, C in the Tables, higher than the resistance of the weir and the following flow channel W, so that flow across the weir is favoured. When flow elements are in series the fluid resistance given by the right hand side of equation 1 for each segment can be added in the manner that the resistance of series electrical impedances can be added. When fluid elements are in parallel the inverse of their fluid resistance can be added to obtain the inverse of the total impedance, as is done for parallel electrical resistances. The volumetric flow rate Q, through a channel or a combination of channels is then given by equation 3.
Where Rf is the resistance to fluid flow defined by the right hand side of equation 1, combined to gether for all channel segments as discussed above. The ratio, r, of volumetric flow rate across the weir, QW versus into the side channel, QC, r−QW/QC, should be large to ensure the percent of solution flowing across the weir, % QW=1(1+r), is high. This can be accomplished by using a long narrow side channel compared to a wide main channel, by increasing the depth of the weir relative to the depth of the other channels, by decreasing the depth of the side channel relative to the main channel, etc, as indicated by several calculations presented in the Tables.
Volumetric Flow Ratios for 10 and 20 μm Deep Designs
TABLE Fixed Device Dimensions for Calculations with a Given Etch Depth Width Length 20 μm Deep Channel W 600 6,500 Weir 280 variable Channel C 50 variable Channel C′ 600 3,500 10 μm Deep Channel W 580 6,500 Weir 280 variable Channel C 30 variable Channel C′ 580 3,500
Integrated Analytical Procedures
It will be appreciated that the various features of the present invention as described above may be utilized in a more complex microfluidic design.
As an illustrative example, in a first trapping zone 25, formed between weirs 6a and 6b, beads loaded with an antibody to a specific protein are introduced via side channel 24 (and exit via side channel 26). A cell lysate or serum sample or other protein source is directed from a sample reservoir (not shown) and loaded into the chip via sample inlet 21 and entrance channel 38 (the sample is removed at sample outlet 22 and an eluent inlet 23 is also provided at the entrance channel. The sample is then passed into the antibody bead bed in trapping zone 25 to isolate a specific protein, while the effluent is directed towards waste outlet 27.
A chaotropic elution agent, such as an acetonitrile, water mix, is then introduced (eluent inlet 23) to elute the protein from the column and deliver it to the next trapping zone 30 (formed between weirs 6c and 6d) where it is designed by a protease enzyme immobilized on beads loaded into the zone 30 (via side channels 29, 31). The effluent at this stage would be directed towards waste outlet 32. After sufficient reaction time, a buffer is delivered (elution inlet 28, running buffer 28a, waste from bed 25) to flush the protein digest from the bed and into the next trapping zone 35 (formed between weirs 6e and 6f) with effluent delivered to waste outlet 39.
The third trapping zone 35 contains a solid phase extraction material (packed and unpacked via side channels 34, 36), allowing concentration of the digest peptides onto the bed in zone 35. An elution solvent, such as a methanol/aqueous mixture or acetonitrile/aqueous mixture is then introduced (elution inlet 33, running buffer 33a) to deliver (exit channel 37, waste 39, or collection 40) a concentrated protein digest to another location on the chip for final analysis.
Packed Bed Chip to Electrospray Mass Spectrometry Interface
Packed bed flow channels may according to the present invention may be interfaced to a mass spectrometer via an electrospray coupler 41, as illustrated in
While the present invention has been described by reference to various preferred embodiments, it will be understood that obvious changes may be made and equivalents substituted without departing from the true spirit and scope of the invention which is set out in the following claims.
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US3352643||2 Sep 1964||14 Nov 1967||Hitachi Ltd||Liquid chromatography and chromatographs|
|US3433257||1 Feb 1966||18 Mar 1969||Ibm||Diaphragm type fluid logic latch|
|US3568692||27 Nov 1967||9 Mar 1971||Bowles Eng Corp||Optical machining process|
|US4113665||3 Feb 1977||12 Sep 1978||Ameron, Inc.||Coatings prepared from trialkoxysilanes|
|US4847120||7 Nov 1988||11 Jul 1989||Cogent Limited||Compositions and processes producing scratch resistant polysiloxane coatings|
|US4963498||15 Jan 1988||16 Oct 1990||Biotrack||Capillary flow device|
|US5275645||24 Nov 1992||4 Jan 1994||Ameron, Inc.||Polysiloxane coating|
|US5376252||10 Nov 1992||27 Dec 1994||Pharmacia Biosensor Ab||Microfluidic structure and process for its manufacture|
|US5387505||4 May 1990||7 Feb 1995||Eastman Kodak Company||Preparation and isolation of single-stranded biotinylated nucleic acids by heat avidin-biotin cleavage|
|US5453163||29 Oct 1993||26 Sep 1995||Yan; Chao||Electrokinetic packing of capillary columns|
|US5482836||14 Jan 1993||9 Jan 1996||The Regents Of The University Of California||DNA purification by triplex-affinity capture and affinity capture electrophoresis|
|US5571410||7 Jun 1995||5 Nov 1996||Hewlett Packard Company||Fully integrated miniaturized planar liquid sample handling and analysis device|
|US5587128||14 Nov 1994||24 Dec 1996||The Trustees Of The University Of Pennsylvania||Mesoscale polynucleotide amplification devices|
|US5635358||14 Feb 1994||3 Jun 1997||Trustees Of The University Of Pennsylvania||Fluid handling methods for use in mesoscale analytical devices|
|US5639428||19 Jul 1994||17 Jun 1997||Becton Dickinson And Company||Method and apparatus for fully automated nucleic acid amplification, nucleic acid assay and immunoassay|
|US5705813||1 Nov 1995||6 Jan 1998||Hewlett-Packard Company||Integrated planar liquid handling system for maldi-TOF MS|
|US5726026||14 Nov 1994||10 Mar 1998||Trustees Of The University Of Pennsylvania||Mesoscale sample preparation device and systems for determination and processing of analytes|
|US5741462||25 Apr 1995||21 Apr 1998||Irori||Remotely programmable matrices with memories|
|US5750015||13 Mar 1996||12 May 1998||Soane Biosciences||Method and device for moving molecules by the application of a plurality of electrical fields|
|US5770029||30 Jul 1996||23 Jun 1998||Soane Biosciences||Integrated electrophoretic microdevices|
|US5775371||8 Mar 1995||7 Jul 1998||Abbott Laboratories||Valve control|
|US5776748||6 Jun 1996||7 Jul 1998||President And Fellows Of Harvard College||Method of formation of microstamped patterns on plates for adhesion of cells and other biological materials, devices and uses therefor|
|US5830662||23 Sep 1994||3 Nov 1998||The Trustees Of Columbia University In The City Of New York||Method for construction of normalized cDNA libraries|
|US5842787||9 Oct 1997||1 Dec 1998||Caliper Technologies Corporation||Microfluidic systems incorporating varied channel dimensions|
|US5856174||19 Jan 1996||5 Jan 1999||Affymetrix, Inc.||Integrated nucleic acid diagnostic device|
|US5863502||23 Jan 1997||26 Jan 1999||Sarnoff Corporation||Parallel reaction cassette and associated devices|
|US5872010||3 Jul 1996||16 Feb 1999||Northeastern University||Microscale fluid handling system|
|US5885470||14 Apr 1997||23 Mar 1999||Caliper Technologies Corporation||Controlled fluid transport in microfabricated polymeric substrates|
|US5908552||10 Oct 1997||1 Jun 1999||Hewlett-Packard Company||Column for capillary chromatographic separations|
|US5922591||27 Jun 1996||13 Jul 1999||Affymetrix, Inc.||Integrated nucleic acid diagnostic device|
|US5942443||28 Jun 1996||24 Aug 1999||Caliper Technologies Corporation||High throughput screening assay systems in microscale fluidic devices|
|US5948684||25 Jul 1997||7 Sep 1999||University Of Washington||Simultaneous analyte determination and reference balancing in reference T-sensor devices|
|US5971158||13 Jun 1997||26 Oct 1999||University Of Washington||Absorption-enhanced differential extraction device|
|US6001229||1 Aug 1994||14 Dec 1999||Lockheed Martin Energy Systems, Inc.||Apparatus and method for performing microfluidic manipulations for chemical analysis|
|US6007690||30 Jul 1997||28 Dec 1999||Aclara Biosciences, Inc.||Integrated microfluidic devices|
|US6007775||26 Sep 1997||28 Dec 1999||University Of Washington||Multiple analyte diffusion based chemical sensor|
|US6010607||16 Sep 1998||4 Jan 2000||Lockheed Martin Energy Research Corporation||Apparatus and method for performing microfluidic manipulations for chemical analysis and synthesis|
|US6048100||10 Mar 1999||11 Apr 2000||Industrial Label Corp.||Resealable closure for a bag|
|US6056860||18 Sep 1997||2 May 2000||Aclara Biosciences, Inc.||Surface modified electrophoretic chambers|
|US6073482||20 Jan 1999||13 Jun 2000||Ysi Incorporated||Fluid flow module|
|US6074827||5 Feb 1998||13 Jun 2000||Aclara Biosciences, Inc.||Microfluidic method for nucleic acid purification and processing|
|US6103199||15 Sep 1998||15 Aug 2000||Aclara Biosciences, Inc.||Capillary electroflow apparatus and method|
|US6120184||17 Nov 1999||19 Sep 2000||Stone Container Corporation||Bag apparatus with reclosable pour spout|
|US6136212||6 Aug 1997||24 Oct 2000||The Regents Of The University Of Michigan||Polymer-based micromachining for microfluidic devices|
|US6168948||12 Jan 1998||2 Jan 2001||Affymetrix, Inc.||Miniaturized genetic analysis systems and methods|
|US6176962||18 Jun 1997||23 Jan 2001||Aclara Biosciences, Inc.||Methods for fabricating enclosed microchannel structures|
|US6190616||11 Sep 1997||20 Feb 2001||Molecular Dynamics, Inc.||Capillary valve, connector, and router|
|US6197595||19 Apr 1999||6 Mar 2001||Affymetrix, Inc.||Integrated nucleic acid diagnostic device|
|US6207031||14 Sep 1998||27 Mar 2001||Whitehead Institute For Biomedical Research||Methods and apparatus for processing a sample of biomolecular analyte using a microfabricated device|
|US6235471||3 Apr 1998||22 May 2001||Caliper Technologies Corp.||Closed-loop biochemical analyzers|
|US6238538||6 Apr 1999||29 May 2001||Caliper Technologies, Corp.||Controlled fluid transport in microfabricated polymeric substrates|
|US6251343||24 Feb 1998||26 Jun 2001||Caliper Technologies Corp.||Microfluidic devices and systems incorporating cover layers|
|US6280589||12 Apr 1994||28 Aug 2001||Zeptosens Ag||Method for controlling sample introduction in microcolumn separation techniques and sampling device|
|US6319476||2 Mar 1999||20 Nov 2001||Perseptive Biosystems, Inc.||Microfluidic connector|
|US6321791||4 Oct 2000||27 Nov 2001||Caliper Technologies Corp.||Multi-layer microfluidic devices|
|US6322683||14 Apr 1999||27 Nov 2001||Caliper Technologies Corp.||Alignment of multicomponent microfabricated structures|
|US6348318||3 Apr 1998||19 Feb 2002||Biosite Diagnostics||Methods for concentrating ligands using magnetic particles|
|US6379929||19 Nov 1997||30 Apr 2002||The Regents Of The University Of Michigan||Chip-based isothermal amplification devices and methods|
|US6387707||28 May 1999||14 May 2002||Bioarray Solutions||Array Cytometry|
|US6403338||27 Jun 2000||11 Jun 2002||Mountain View||Microfluidic systems and methods of genotyping|
|US6408878||28 Feb 2001||25 Jun 2002||California Institute Of Technology||Microfabricated elastomeric valve and pump systems|
|US6423536||23 May 2000||23 Jul 2002||Molecular Dynamics, Inc.||Low volume chemical and biochemical reaction system|
|US6429025||24 Jun 1997||6 Aug 2002||Caliper Technologies Corp.||High-throughput screening assay systems in microscale fluidic devices|
|US6432191||8 Feb 2001||13 Aug 2002||John B. Schutt||Silane-based, coating compositions, coated articles obtained therefrom and methods of using same|
|US6432290||26 Nov 1999||13 Aug 2002||The Governors Of The University Of Alberta||Apparatus and method for trapping bead based reagents within microfluidic analysis systems|
|US6454924||23 Feb 2001||24 Sep 2002||Zyomyx, Inc.||Microfluidic devices and methods|
|US6489112||2 Aug 2000||3 Dec 2002||Molecular Dynamics, Inc.||Methods and apparatus for template capture and normalization for submicroliter reaction|
|US6521188||22 Nov 2000||18 Feb 2003||Industrial Technology Research Institute||Microfluidic actuator|
|US6524456||29 Sep 1999||25 Feb 2003||Ut-Battelle, Llc||Microfluidic devices for the controlled manipulation of small volumes|
|US6532997||28 Dec 2001||18 Mar 2003||3M Innovative Properties Company||Sample processing device with integral electrophoresis channels|
|US6533914||27 Jun 2000||18 Mar 2003||Shaorong Liu||Microfabricated injector and capillary array assembly for high-resolution and high throughput separation|
|US6537757||8 Aug 2000||25 Mar 2003||The Regents Of The University Of Michigan||Nucleic acid sequencing and mapping|
|US6544734||9 Dec 1999||8 Apr 2003||Cynthia G. Briscoe||Multilayered microfluidic DNA analysis system and method|
|US6551839||25 Jan 2001||22 Apr 2003||Amersham Biosciences Corp.||Method of merging chemical reactants in capillary tubes|
|US6581441||1 Feb 2002||24 Jun 2003||Perseptive Biosystems, Inc.||Capillary column chromatography process and system|
|US6605454||22 Mar 2001||12 Aug 2003||Motorola, Inc.||Microfluidic devices with monolithic microwave integrated circuits|
|US6613525||17 Jan 2002||2 Sep 2003||Aclara Biosciences, Inc.||Microfluidic apparatus and method for purification and processing|
|US6614228||24 Oct 2001||2 Sep 2003||Bruker Analytik Gmbh||Device for trapping at least one component of a chromatography flow in an LC/NMR environment|
|US6618679||27 Jan 2001||9 Sep 2003||Althea Technologies, Inc.||Methods for analysis of gene expression|
|US6623613||2 Oct 2000||23 Sep 2003||The Regents Of The University Of California||Microfabricated liquid sample loading system|
|US6627446||2 Jul 1998||30 Sep 2003||Amersham Biosciences (Sv) Corp||Robotic microchannel bioanalytical instrument|
|US6629820||26 Jun 2001||7 Oct 2003||Micralyne Inc.||Microfluidic flow control device|
|US6632619||16 May 1997||14 Oct 2003||The Governors Of The University Of Alberta||Microfluidic system and methods of use|
|US6632655||22 Feb 2000||14 Oct 2003||Caliper Technologies Corp.||Manipulation of microparticles in microfluidic systems|
|US6663833||5 Mar 1999||16 Dec 2003||Strategic Diagnostics Inc.||Integrated assay device and methods of production and use|
|US6685442||20 Feb 2002||3 Feb 2004||Sandia National Laboratories||Actuator device utilizing a conductive polymer gel|
|US6752922||5 Apr 2002||22 Jun 2004||Fluidigm Corporation||Microfluidic chromatography|
|US6764648||20 Apr 2000||20 Jul 2004||Amersham Biosciences (Sv) Corp.||Robotic microchannel bioanalytical instrument|
|US6782746||24 Oct 2000||31 Aug 2004||Sandia National Laboratories||Mobile monolithic polymer elements for flow control in microfluidic devices|
|US6786708||18 Jul 2002||7 Sep 2004||The Regents Of The University Of Michigan||Laminated devices and methods of making same|
|US6787111||13 Dec 2000||7 Sep 2004||Amersham Biosciences (Sv) Corp.||Apparatus and method for filling and cleaning channels and inlet ports in microchips used for biological analysis|
|US6793753||28 Feb 2001||21 Sep 2004||California Institute Of Technology||Method of making a microfabricated elastomeric valve|
|US6802342||26 Nov 2001||12 Oct 2004||Fluidigm Corporation||Microfabricated fluidic circuit elements and applications|
|US6803019||15 Oct 1998||12 Oct 2004||Aclara Biosciences, Inc.||Laminate microstructure device and method for making same|
|US6824663||25 Aug 2000||30 Nov 2004||Aclara Biosciences, Inc.||Efficient compound distribution in microfluidic devices|
|US6829753||27 Jun 2001||7 Dec 2004||Fluidigm Corporation||Microfluidic design automation method and system|
|US6833246||29 Mar 2002||21 Dec 2004||Solexa, Ltd.||Polynucleotide sequencing|
|US6852287||12 Sep 2001||8 Feb 2005||Handylab, Inc.||Microfluidic devices having a reduced number of input and output connections|
|US6870185||1 Aug 2003||22 Mar 2005||Amersham Biosciences (Sv) Corp||Integrated microchip design|
|JPH08327594A *||Title not available|
|1||Amendment and Request for Correction of Inventorship mailed Jan. 10, 2008 in U.S. Appl. No. 10/750,533.|
|2||Anderson, et al. A miniature integrated device for automated multistep genetic assays. Nucleic Acids Research. 2000;28:e60.|
|3||Armani, et al. Re-configurable fluid circuits by PDMS elastomer micromachining. Proceedings of IEEE Micro Electro Mechanical Systems: MEMS. 1999; 222-227.|
|4||Bennett, et al. Toward the 1,000 dollars human genome. Pharmacogenomics, 6 (4) 373-382. (Jun. 2005).|
|5||Bings, et al. Microfluidic Devices Connected to Fused-Silica Capillaries with Minimal Dead Dead Volume. Analytical Chemistry. 1999;71(15):3292-3296.|
|6||Bings, Nicolas H. et al., "Microfluidic Devices Connected to Fused-Silica Capillaries with Minimal Dead Volume," Analytical Chemistry, vol. 71, No. 15, Aug. 1999, pp. 3292-3296.|
|7||Blazej, et al. Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing. Proc. Natl. Acad. Sci. USA 2006;103:7240-7245.|
|8||Blazej, et al. Polymorphism Ratio Sequencing: A New Approach for Single Nucleotide Polymorphism Discovery and Genotyping. Genome Research. 2003;13:287-293.|
|9||Brenner, et al. Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nature Biotechnology. 2000;18(6):630-634.|
|10||Buchholz, et al. The use of light scattering for precise characterization of polymers for DNA sequencing by capillary electrophoresis. Electrophoresis. 2001;22:4118-4128.|
|11||Caplus abstract of Krohkin et al. Modified silica as a stationary phase for ion chromatography. Journal of Chromatography A. 1995;706:93-8.|
|12||*||CAPLUS abstract of Krokhin et al. ("Modified silica as a stationary phase for ion chromatography", J. Chromatogr., A (1995), 706(1+2), 93-8).|
|13||Chan, et al. Microfabricated Polymer Devices for Automated Sample Delivery of Peptides for Analysis by Electrospray Ionization Tandem Mass Spectrometry. Analytical Chemistry. 1999;71(20):4437-4444.|
|14||Chan, Jason H. et al., "Microfabricated Polymer Devices for Automated Sample Delivery of Peptides for Analysis by Electrospray Ionization Tandem Mass Spectrometry," Analytical Chemistry, vol. 71, No. 20, Oct. 1999, pp. 4437-4444.|
|15||Chiem, et al. Microchip systems for immunoassay: an integrated immunoreactor with electrophoretic separation for serum theophylline determination. Clinical Chemistry.1998;44(3):591-598.|
|16||Chiem, et al. Room temperature bonding of micromachined glass devices for capillary electrophoresis. Sensors and Actuators. 2000;B63(3):147-152.|
|17||Chinese office action dated Jan. 31, 2011 for CN 200580035911.7. (In Chinese with English translation).|
|18||Coleman, et al. A sequential injection microfluidic mixing strategy. Microfluidics and Nanofluidics. 2005;319-327.|
|19||Curcio, et al. Continuous Segmented-Flow Polymerase Chain Reaction for High-Throughput Miniaturized DNA Amplification. Analytical Chemistry. 2003;75(1):1-7.|
|20||Datasheet Cycle Sequencing, Retrieved from the internet, URL:http//answers.com/topic/cycle sequencing. Printed Sep. 3, 2010, pp. 1-2.|
|21||Diehl, et al. BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions. Nature Methods. 2006;3(7):551-9.|
|22||Doherty, et al. Sparsely Cross-linked "Nanogel" Matrices as Fluid, Mechanically Stablized Polymer Networks for High-Throughput Microchannel DNA Sequencing. Analytical Chemistry. 2004;76:5249-5256.|
|23||Doherty, et al. Sparsely cross-linked "nanogels" for microchannel DNA sequencing. Electrophoresis. 2003;24(24):4170-4180.|
|24||Dorfman, et al. Contamination-Free Continuous Flow Microfluidic Polymerase Chain Reaction for Quantitative and Clinical Applications. Analytical Chemistry. 2005;77(11):3700-3704.|
|25||Doyle, et al. Self-Assembled Magnetic Matrices for DNA Separation Chips. Science. 2000;295:2237.|
|26||Dressman, et al. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc Natl Acad Sci USA. 2003;100(15):8817-8822.|
|27||Emrich, et al. Microfabricated 384-Lane Capillary Array Electrophoresis Bioanalyzer for Ultrahigh-Throughput Genetic Analysis. Analytical Chemistry. 2002;74(19):5076-5083.|
|28||Ericson, et al. Electroosmosis- and Pressure-Driven Chromatography in Chips Using Continuous Beds. Analytical Chemistry. 2000;72(1):81-87.|
|29||Erratum for Margulies, et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature. 2005;437(7057):376-80.: Margulies, et al. Nature. 441(7089):120. (May 4, 2006).|
|30||European search report and search opinion dated Jun. 6, 2011 for Application No. 10011511.2.|
|31||European search report dated Dec. 18, 2009 for Application No. 03808583.3.|
|32||European search report dated Sep. 1, 2010 for Application No. 5804847.1.|
|33||Ewing, et al. Base-Calling of Automated Sequencer Traces Using Phred. I. Accuracy Assessment. Genome Research. 1998;8:175-185.|
|34||Ewing, et al. Base-Calling of Automated Sequencer Traces Using Phred. II. Error probabilities. Genome Research. 1998;8:186-194.|
|35||Figeys, Daniel et al., "A Microfabricated Device for Rapid Protein Identification by Microelectrospray Ion Trap Spectrometry," Analytical Chemistry, vol. 69, No. 16, Aug. 1997, pp. 3153-3160.|
|36||Figeys, Daniel et al., "An Integrated Microfluidics-Tandem Mass Spectrometry System for Automated Protein Analysis," Analytical Chemistry, vol. 70, No. 18, Sep. 1998, pp. 3728-3734.|
|37||Figeys, Daniel et al., "Microfabricated Device Coupled with an Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometer: Protein Identifications Based on Enhanced-Reslution Mass Spectrometry and Tandem Mass Spectrometry Data," Rapid Communications In Mass Spectrometry, vol. 12, Aug. 1998, pp. 1435-1444.|
|38||Figeys, Daniel et al., "Nanoflow Solvent Gradient Delivery from a Microfabricated Device for Protein Identifications by Electrospray Ionization Mass Spectrometry," Analytical Chemistry, vol. 70, No. 18, Sep. 1998, pp. 3721-3727.|
|39||Figeys, et al. A Microfabricated Device for Rapid Protein Identification by Microelectrospray Ion Trap Mass Spectrometry. Analytical Chemistry. 1997;69(16):3153-3160.|
|40||Figeys, et al. An Integrated Microfluidics-Tandem Mass Spectrometry System for Automated Protein Analysis. Analytical Chemistry. 1998;70(18):3728-3734.|
|41||Figeys, et al. Microfabricated Device Coupled with an Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometer: Protein Identifications Based on Enhanced-Resolution Mass Spectrometry and Tandem Mass Spectrometry Data. Rapid Communications in Mass Spectrometry. 1998;12:1435-1444.|
|42||Figeys, et al. Nanoflow Solvent Gradient Delivery from a Microfabricated Device for Protein Identifications by Electrospray Ionization Mass Spectrometry. Analytical Chemistry. 1998;70(18):3721-3727.|
|43||Francis, et al. Flow analysis based on a pulsed flow of solution: theory, instrumentation and applications. Talanta. 2002;58(6):1029-1042.|
|44||Ghadessy, et al. Directed evolution of polymerase function by compartmentalized self-replication. Proc Natl Acad Sci USA. 2001;98:4552-4557.|
|45||Giddings, et al. A software system for data analysis in automated DNA sequencing. Genome Research. 1998;8:644-665.|
|46||Goll, et al. Microvalves with bistable buckled polymer diaphragms. Journal of Micromechanics and Microengineering. 1996;6:77-79.|
|47||Grover, et al. An integrated microfluidic processor for single nucleotide polymorphism-based DNA computing. Lab on a Chip. 2005;5(10):1033-1040.|
|48||Grover, et al. Development and multiplexed control of latching pneumatic valves using microfluidic logical structures. Lab on a chip. 2006;6:623-631.|
|49||Grover, et al. Monolithic membrane valves and diaphragm pumps for practical large-scale integration into glass microfluidic devices. Sensors and Actuators. 2003;B89:315-323.|
|50||Grover, et al. Practical Valves and Pumps for Large-Scale Integration into Microfludic Analysis Devices. Micro Total Analysis Systems. 2002;1:136-138.|
|51||Hansen, et al. A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion. Proc Natl Acad Sci USA. 2002;99(26): 16531-16536.|
|52||Harrison, et al. Micromachining a Miniaturized Capillary Electrophoresis-Based Chemical Analysis System on a Chip. Science. 1993;261(5123):895-897.|
|53||Hayes, et al. EDGE: A Centralized Resource for the Comparison, Analysis, and Distribution of Toxicogenomic Information. Molecular Pharmacology. 2005;67(4):1360-1368.|
|54||Hultman, et al. Bidirectional Solid-Phase Sequencing of In Vitro-Amplified Plasmid DNA. BioTechniques. 1991;10(1):84-93.|
|55||International Preliminary Report for corresponding PCT Application No. PCT/CA2000/01421 dated Feb. 14, 2002.|
|56||International Preliminary Report for corresponding PCT Application No. PCT/US2005/018678 dated Nov. 13, 2007.|
|57||International Preliminary Report for corresponding PCT Application No. PCT/US2005/033347 dated Mar. 20, 2007.|
|58||International Preliminary Report for corresponding PCT Application No. PCT/US2007/007381 dated Sep. 23, 2008.|
|59||International Preliminary Report for corresponding PCT Application No. PCT/US2007/02721 dated Aug. 5, 2008.|
|60||International Preliminary Report for corresponding PCT Application No. PCT/US2007/061573 dated Aug. 26, 2008.|
|61||International search report and written opinion dated Jun. 9, 2011 for PCT Application No. US2011/30973.|
|62||International search report and written opinion dated Mar. 24, 2011 for PCT Application No. US2010/58227.|
|63||International search report dated Apr. 5, 2001 for PCT Application No. CA2000/01421.|
|64||International search report dated Aug. 18, 2009 for PCT Application No. US09/00419.|
|65||International search report dated Aug. 23, 2006 for PCT Application No. US2005/033347.|
|66||International search report dated Aug. 26, 2004 PCT Application No. US2003/41466.|
|67||International search report dated Jul. 11, 2008 for PCT Application No. US07/61573.|
|68||International search report dated Jul. 30, 2010 for PCT Application No. US2010/36464.|
|69||International search report dated May 14, 2010 for PCT Application No. US2009/06640.|
|70||International search report dated Oct. 6, 2010 for PCT Application No. US10/37545.|
|71||International search report dated Sep. 1, 2010 for PCT/US2010/040490.|
|72||International search report dated Sep. 25, 2007 for PCT Application No. US2007/02721.|
|73||International Search Report for PCT/US2005/033347.|
|74||Jacobson, et al. Electrokinetic Focusing in Microfabricated Channel Structures. Anal. Chem., 1997, 69 (16), pp. 3212-3217.|
|75||Japanese Office Action dated Apr. 27, 2010 for Application No. JP2001-540363 (in Japanese with English translation).|
|76||Japanese Office Action dated Dec. 21, 2010 for Application No. JP2001-540363 (in Japanese with English translation).|
|77||*||JAPIO Abstract of Arai (JP408327594A).|
|78||*||JPO computer generated English language translation of JP 08-327594 A, patent published on Dec. 13, 1996.|
|79||Ju, et al. Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis. Proc. Natl. Acad. Sci. USA. 1995;92;4347-4351.|
|80||Kan, et al. A novel thermogelling matrix for microchannel DNA sequencing based on poly-N-alkoxyalkylaclylamide copolymers. Electrophoresis. 2003;24(24):4161-4169.|
|81||Koh, et al. Integrating Polymerase Chain Reaction, Valving, and Electrophoresis in a Plastic Device for Bacterial Detection. Analytical Chemistry. 2003;75(17):4591-4598.|
|82||Kopp, et al. Chemical Amplification Continuous-Flow PCR on a Chip. Science. 1998;280:1046-1048.|
|83||Kopp, Martin U. et al., "Chemical Amplification: Continuous-Flow PCR on a Chip," Science, vol. 280, May 1998, pp. 1046-1048.|
|84||Lagally, et al. Fully integrated PCR-capillary electrophoresis microsystem for DNA analysis. Lab on a Chip. 2001;1(2):102-107.|
|85||Lagally, et al. Integrated Portable Genetic Analysis Microsystem for Pathogen/Infectious Disease Detection. Analytical Chemistry. 2004;76:3162-3170.|
|86||Lagally, et al. Monolithic integrated microfluidic DNA amplification and capillary electrophoresis analysis system. Sensors and Actuators. 2000;B63(3):138-146.|
|87||Lagally, et al. Single-Molecule DNA Amplification and Analysis in an Integrated Microfluidic Device. Analytical Chemistry. 2001;73(3): 565-570.|
|88||Lazar, et al. Subattomole-Sensitivity Microchip Nanoelectrospray Source with Time-of-Flight Mass Spectrometry Detection. Analytical Chemistry. 1999;71(17):3627-3631.|
|89||Lazar, Julia M. et al., "Subattomole-Sensitivity Microchip-Nanoelectrospray Source with Time-of-Flight Mass Spectrometry Detection," Analytical Chemistry, vol. 71, No. 17, Sep. 1999, p. 3627-3631.|
|90||Li, et al. Integration of Microfabricated Devices to Capillary Electrophoresis-Electrospray Mass Spectrometry Using a Low Dead Volume Connection: Application to Rapid Analyses of Proteolytic Digests. Analytical Chemistry. 1999;71(15):3036-3045.|
|91||Li, et al. Rapid and sensitive separation of trace level protein digests using microfabricated devices coupled to a quadrupole-time-of-flight mass spectrometer. Electrophoresis. 2000;21:198-210.|
|92||Li, et al. Rapid and sensitive separation of trace level protein digests using microfabricated devices coupled to a quadrupole—time-of-flight mass spectrometer. Electrophoresis. 2000;21:198-210.|
|93||Li, et al. Separation and Identification of Peptides from Gel-Isolated Membrane Proteins Using a Microfabricated Device for Combined Capillary Electrophoresis/Nanoelectrospray Mass Spectrometry. Analytical Chemistry. 2000;72(3):599-609.|
|94||Li, Jianjun et al., "Integration of Microfabricated Devices to Capillary Electrophoresis-Electrospray Mass Spectrometry Using a Low Dead Volume Connection: Application to Rapid Analyses of Proteolytic Digests," Analytical Chemistry, vol. 71, No. 15, Aug. 1999, pp. 3036-3045.|
|95||Li, Jianjun et al., "Rapid and Sensitive Separation of Trace Level Protein Digests Using Microfabricated Devices Coupled to a Quadrupole—Time-of-Flight Mass Spectrometer," Electrophoresis, 2000, pp. 198-210.|
|96||Li, Jianjun et al., "Separation and Identification of Peptides from Gel-Isolated Membrane Proteins Using a Microfabricated Device for Combined Capillary Electrophoresis/Nanoelectrospray Mass Spectrometry," Analytical Chemistry, vol. 72, No. 3, Feb. 1999, pp. 599-609.|
|97||Licklider, et al. A Micromachined Chip-Based Electrospray Source for Mass Spectrometry. Analytical Chemistry. 2000;72(2):367-375.|
|98||Licklider, Larry et al., "A Micromachined Chip-Based Electrospray Source for Mass Spectrometry," Analytical Chemistry, vol. 72, No. 2, Jan. 2000, pp. 367-375.|
|99||Lisec, et al. A bistable pneumatic microswitch for driving fluidic components. Sensors and Actuators. 1996;A54:746-749.|
|100||Liu, et al. Automated parallel DNA sequencing on multiple channel microchips. Proc. Natl. Acad. Sci. USA. 2000;97(10):5369-5374.|
|101||Liu, et al. Optimization of High-Speed DNA Sequencing on Microfabricated Capillary Electrophoresis Channels. Analytical Chemistry. 1999;71:566-573.|
|102||Margulies, et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature. 2005;437(7057):376-80. (Abstact only).|
|103||Melin, et al. A Passive 2-Dimensional Liquid Sample Micromixer. 7th International Conference on Miniaturized Chemical and Biochemical Analysis Systems. 2003;167170.|
|104||MillGat pump user manual, version 2.12, published 2005, pp. 1-28.|
|105||Mitra, et al. Digital genotyping and haplotyping with polymerase colonies. Proc Nati Acad Sci USA. 2003.100(10):15926-5931.|
|106||Norris, et al. Fully-integrated, multiplexed STR-based human identification using a single microfluidic chip and automated instrument. Available at http://www.promega.com/geneticidproc/ussymp20proc/oralpresentations/landersbienvenue.pdf. Accessed Jun. 2, 2010.|
|107||Notice of allowance dated Jun. 9, 2011 for U.S. Appl. No. 12/831,949.|
|108||Obeid, et al. Microfabricated Device for DNA and RNA Amplification by Continuous-Flow Polymerase Chain Reaction and Reverse Transcription-Polymerase Chain Reaction with Cycle Number Selection. Analytical Chemistry. 2003;75(2): 288-295.|
|109||Ocvirk, et al. High Performance Liquid Chromatography Partially Integrated onto a Silicon Chip. Analytical Methods and Instrumentation. 1995;2:74-82.|
|110||Ocvirk, et al. Optimization of confocal epifluorescence microscopy for microchip-based miniaturized total analysis systems. The Analyst. 1998;123:1429-1434.|
|111||Ocvirk, Gregor et al., "High Performance Liquid Chromatography Partially Integrated onto a Silicon Chip," Research Articles, Nov. 1994, 9 pp.|
|112||Ocvirk, Gregor et al., "Optimization of Confocal Epifluorescence Microscopy for Microchip-Based Miniaturized Total Analysis Systems," The Analyst, vol. 123, Jun. 1998, pp. 1429-1434.|
|113||Office Action Final dated Feb. 19, 2008 issued in U.S. Appl. No. 10/540,658.|
|114||Office Action Final dated Feb. 6, 2008 issued in U.S. Appl. No. 11/139,018.|
|115||Office Action mailed Apr. 27, 2007 in U.S. Appl. No. 11/139,018, filed May 25, 2005.|
|116||Office Action mailed Jul. 12, 2007 in U.S. Appl. No. 10/750,533, filed Dec. 29, 2003.|
|117||Office Action mailed Jul. 2, 2007 in U.S. Appl. No. 10/540,658, filed Jun. 23, 2005.|
|118||Ohori, et al. Partly disposable three-way mirovalve for a medical micro total analysis system (muTAS). Sensors and Actuators. 1998;A64(1): 57-62.|
|119||Oleschuk, et al. Trapping of Bead-Based Reagents within Microfluidic Systems: On-Chip Solid-Phase Extraction and Electrochromatography. Analytical Chemistry. 2000;72:585-590.|
|120||Olsen, et al. Immobilization of DNA Hydrogel Plugs in Microfluidic Channels. Analytical Chemistry. 2002;74:1436-1441.|
|121||Paegel, et al. High-throughput DNA sequencing with a 96-lane capillary array electrophoresis bioprocessor. Proc Natl Acad Sci USA. 2002;99:574-579.|
|122||Paegel, et al. Microchip Bioprocessor for Integrated Nanovolume Sample Purification and DNA Sequencing. Analytical Chemistry. 2002;74(19):5092-5098.|
|123||Paegel, et al. Microfluidic devices for DNA sequencing: sample preparation and electrophoretic analysis. Current Opinion in Biotechnology. 2003;14(1):42-50.|
|124||Paegel, et al. Turn Geometry for Minimizing Band Broadening in Microfabricated Capillary Electrophoresis Channels. Analytical Chemistry. 2000;72:3030-3037.|
|125||PCT Notification of Transmittal of the International Search Report and The Written Opinion of the International Searching Authority, or the Declaration, mailed Jun. 17, 2008, Application No. PCT/US2007/082568 (UCALP067WO).|
|126||Peterson, et al. Enzymatic Microreactor-on-a-Chip: Protein Mapping Using Trypsin Immobilized on Porous Polymer Monoliths Molded in Channels of Microfluidic Devices. Analytical Chemistry. 2002;74:4081-4088.|
|127||Ramsey, et al. Generating Electrospray from Microchip Devices Using Electroosmotic Pumping. Analytical Chemistry. 1997;69(6):1174-1178.|
|128||Ramsey, R.S. et al., "Generating Electrospray from Microchip Devices Using Electroosmotic Pumping," Analytical Chemistry, vol. 69, No. 6, Mar. 1997, pp. 1174-1178.|
|129||Rohr, et al. Porous polymer monoliths: Simple and efficient mixers prepared by direct polymerization in the channels of microfluidic chips. Electrophoresis. 2001;22:3959-3967.|
|130||Rye, et al. High-sensitivity two-color detection of double-stranded DNA with a confocal fluorescence gel scanner using ethidium homodimer and thiazole orange. Nucleic Acids Research. 1991;19(2):327-333.|
|131||Scherer, et al. High-Pressure Gel Loader for Capillary Array Electrophoresis Microchannel Plates. Biotechniques. 2001;31(5):1150-1154.|
|132||Schomburg, et al. Design Optimization of Bistable Microdiaphragm Valves. Sensors and Actuators. 1998;A64:259-264.|
|133||Seifar, et al. Capillary electrochromatography with 1.8-mum ODS-modified porous silica particles. Journal of Chromatography. 1998; A808:71-77.|
|134||Simpson, et al. High-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates. Proc Natl Acad Sci USA. 1998;95:2256-2261.|
|135||Simpson, et al. Microfabrication Technology for the Production of Capillary Array Electrophoresis Chips. Biomedical Microdevices. 1998;1:7-26.|
|136||Soper, et al. Sanger DNA Sequencing Reactions Performed in a Solid-Phase Nanoreactor Directly Coupled to Capillary Gel Electrophoresis. Analytical Chemistry. 1998;70:4036-4043.|
|137||Spiering, et al. Novel microstructures and technologies applied in chemical analysis techniques. 1997 International Conference on Solid-State Sensors and Actuators. 1997;1:511-514.|
|138||Spiering, Vincent L., et al., "Novel Microstructures and Technologies Applied in Chemical Analysis Techniques," Transducers '97, Jun. 1997, pp. 511-514.|
|139||Takao, et al. A Pneumatically Actuated Full In-Channel Microvalve With MOSFET-Like Function in Fluid Channel Networks. Journal of Microelectromechanical Systems. 2002;11(5):421-426.|
|140||Takao, et al. Microfluidic Integrated Circuits for Signal Processing Using Analogous Relationship Betweeen Pneumatic Microvalve and MOSFET. Journal of Microelectromechanical Systems. 2003;12(4):497-505.|
|141||Thomas, et al. Application of Genomics to Toxicology Research. Environmental Health Perspectives. 2002;110(6):919-923.|
|142||Thorsen, et al. Microfluidic Large-Scale Integration. Science. 2002;298(5593):580-584.|
|143||Tice, et al. Formation of Droplets and Mixing in Multiphase Microfluidics at Low Values of the Reynolds and the Capillary Numbers. Langmuir. 2003;19:9127-9133.|
|144||U.S. Appl. No. 12/026,510, filed Feb. 5, 2008, Jovanovich et al.|
|145||U.S. Appl. No. 12/526,015, filed Nov. 3, 2010, Jovanovich et al.|
|146||U.S. Appl. No. 12/815,685, filed Jun. 15, 2010, Jovanovich et al.|
|147||U.S. Appl. No. 12/845,650, filed Jul. 28, 2010, Jovanovich et al.|
|148||U.S. Appl. No. 12/852,370, filed Aug. 6, 2010, Harrison et al.|
|149||U.S. Appl. No. 12/949,623, filed Nov. 18, 2010, Kobrin et al.|
|150||U.S. Appl. No. 13/075,165, filed Mar. 29, 2011, Eberhart et al.|
|151||U.S. Appl. No. 13/113,968, filed May 23, 2011, Majlof et al.|
|152||U.S. Appl. No. 13/202,877, filed Aug. 23, 2011, Vangbo et al.|
|153||U.S. Appl. No. 13/202,884, filed Aug. 23, 2011, Jovanovich et al.|
|154||Unger, et al. Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography. Science. 2000;288:113-116.|
|155||Van Der Moolen, et al. A Micromachined Injection Device for CZE: Application to Correlation CZE. Analytical Chemistry. 1997;69(20):4220-4225.|
|156||Van Der Moolen, et al. Correlation Capillary Zone Electrophoresis, a Novel Technique to Decrease Detection Limits. Chromatographia. 1995;40(7/8):368-374.|
|157||van der Moolen, J.N. et al., "Correlation Capillary Zone Electrophoresis, a Novel Technique to Decrease Detection Limits," Chromatographia, vol. 40, No. 7/8, Apr. 1995, pp. 368-374.|
|158||van der Moolen, Johannes N. et al., "A Micromachined Injection Device for CZE: Application to Correlation CZE," Analytical Chemistry, vol. 69, No. 20, Oct. 1997, pp. 4220-4225.|
|159||Vazquez, et al. Electrophoretic Injection within Microdevices. Analytical Chemistry. 2002;74:1952-1961.|
|160||Veenstra, et al. The design of an in-plane compliance structure for microfluidical systems. Sensors and Actuators. 2002;B81:377-383.|
|161||Waller, et al. Quantitative Immunocapture PCR Assay for Detection of Campylobacter jejuni in Foods. Applied Environmental Microbiology. 2000; 66(9):4115-4118.|
|162||Weimer, et al. Solid-Phase Capture of Proteins, Spores, and Bacteria. Applied Environmental Microbiology. 2001;67(3):1300-1307.|
|163||Wen, et al. Microfabricated isoelectric focusing device for direct electrospray ionization-mass spectrometry. Electrophoresis. 2000;21:191-197.|
|164||Wen, Jenny et al., "Microfabricated Isoelectric Focusing Device for Direct Electrospray Ionization-Mass Spectrometry," Electrophoresis, vol. 21, 2000, pp. 191-197.|
|165||Wikipedia brochure for defining stocahstic process. Sep. 2, 2009.|
|166||Williams, et al. Amplification of complex gene libraries by emulsion PCR. Nature Methods. 2006;3(7):545-50.|
|167||Woolley, et al. Functional Integration of PCR Amplification and Capillary Electrophoresis in a Microfabricated DNA Analysis Device. Analytical Chemistry. 1996;68(23):4081-4086.|
|168||Wright, et al. Behavior and Use of Nonaqueous Media without Supporting Electrolyte in Capillary Electrophoresis and Capillary Electrochromatography. Analytical Chemistry. 1997;69(16):3251-3259.|
|169||Xiang, et al. An Integrated Microfabricated Device for Dual Microdialysis and On-Line ESI-Ion Trap Mass Spectrometry for Analysis of Complex Biological Samples. Analytical Chemistry. 1999;71(8):1485-1490.|
|170||Xiang, Fan et al., "An Integrated Microfabricated Device for Dual Microdialysis and On-Line ESI-Ion Trap Mass Spectrometry for Analysis of Complex Biological Samples," Analytical Chemistry, vol. 71, No. 8, Apr. 1999, pp. 1485-1490.|
|171||Xue Qifeng et al., "Multichannel Microchip Electrospray Mass Spectrometry," Analytical Chemistry, vol. 69, No. 3, Feb. 1997, pp. 426-430.|
|172||Xue, et al. Integrated Multichannel Microchip Electrospray Ionization Mass Spectrometry: Analysis of Peptides from On-Chip Tryptic Digestion of Melittin. Rapid Communications in Mass Spectrometry. 1997;11:1253-1256.|
|173||Xue, et al. Multichannel Microchip Electrospray Mass Spectrometry. Analytical Chemistry. 1997;69(3):426-430.|
|174||Xue, Qifeng et al., "Integrated Multichannel Microchip Electrospray Ionization Mass Spectrometry: Analysis of Peptides from On Chip Tryptic Digestion of Melittin," Rapid Communications in Mass Spectrometry, vol. 11, Jun. 1997, pp. 1253-1256.|
|175||Yang, et al. A MEMS thermopneumatic silicone rubber membrane valve. Sensors and Actuators. 1998;A64(1):101-108.|
|176||Yu, et al. Preparation of Monolithic Polymers with Controlled Porous Properties for Microfluidic Chip Applications Using Photoinitiated Free Radial Polymerization. Journal of Polymer Science. 2002;40:755-769.|
|177||Yu, et al. Towards stationary phases for chromatography on a microchip: Molded porous polymer monoliths prepared in capillaries by photoinitiated in situ polymerization as separation media for electrochromatography. Electrophoresis. 2000;21:120-127.|
|178||Zhang, B. et al., "Microfabricated Devices for Capillary Electrophoresis-Electrospray Mass Spectrometry," Analytical Chemistry, vol. 71, No. 15, Aug. 1999, pp. 3258-3264.|
|179||Zhang, Bailin et al., "A Microdevice with Integrated Liquid Junction for Facile Peptide and Protein Analysis in Capillary Electrophoresis/Electrospray Mass Spectrometry," Analytical Chemistry, vol. 72, No. 5, Mar. 2000, pp. 1015-1022.|
|180||Zhang, et al. A Microdevice with Integrated Liquid Junction for Facile Peptide and Protein Analysis by Capillary Electrophoresis/Electrospray Mass Spectrometry. Analytical Chemistry. 2000;72(5):1015-1022.|
|181||Zhang, et al. Microfabricated Devices for Capillary Electrophoresis-Electrospray Mass Spectrometry. Analytical Chemistry. 1999;71(15):3258-3264.|
|Citing Patent||Filing date||Publication date||Applicant||Title|
|US8748165||21 Aug 2012||10 Jun 2014||Integenx Inc.||Methods for generating short tandem repeat (STR) profiles|
|WO2015013210A1 *||21 Jul 2014||29 Jan 2015||The Regents Of The University Of California||Microfluidics cell culture device|
|U.S. Classification||204/453, 422/503, 204/605, 204/455, 204/604, 422/513, 422/502, 204/601, 204/451|
|International Classification||G01N30/28, G01N30/56, G01N27/26, G01N33/00, B81B1/00, G01N30/60, B01L3/00, B01L3/02, G01N30/00, G01N27/447|
|Cooperative Classification||B01L2300/0816, B01L3/5027, B01L2400/0487, G01N27/44704, B01L3/502753, G01N30/56, B01L2300/087, B01L2400/0415, G01N27/44791, B01L2200/0668, G01N2030/565, G01N30/6095, G01N2030/009, B01L3/0241, G01N2030/285|
|European Classification||G01N27/447C7, G01N27/447B, G01N30/56, B01L3/5027|