Chemistry: analytical and immunological testing – Including sample preparation – Volumetric liquid transfer
Reexamination Certificate
2000-06-30
2003-09-02
Warden, Jill (Department: 1641)
Chemistry: analytical and immunological testing
Including sample preparation
Volumetric liquid transfer
C436S167000, C436S172000, C436S174000, C436S179000, C422S068100, C422S082050, C422S091000
Reexamination Certificate
active
06613580
ABSTRACT:
BACKGROUND OF THE INVENTION
The determination of kinetic relationships for reactants and other components in microfluidic systems can be a relatively complex process. In this regard, the inventors and their co-workers have determined a variety of useful methods of determining kinetic information for reactions and other phenomena in such microfluidic systems. For example, “APPARATUS & METHODS FOR CORRECTING FOR VARIABLE VELOCITY IN MICROFLUIDIC SYSTEMS,” W098/56956 by Kopf-Sill et al. provides pioneering methods of obtaining kinetic information for moving reactants, based, e.g., upon the conservation of flux in microscale systems which use electrokinetic forces to move fluids.
The difficulties of determining kinetic information are compounded in high-throughput systems where thousands of test compounds per day can be screened in a single microscale system for activity on one or more selected targets. Pioneering high-throughput screening methods and relevant apparatus are described by the inventors and their co-workers in Knapp et al. “CLOSED LOOP BIOCHEMICAL ANALYZERS” (WO 98/45481; PCT/US98/06723); Parce et al. “HIGH THROUGHPUT SCREENING ASSAY SYSTEMS IN MICROSCALE FLUIDIC DEVICES” WO 98/00231 and in, e.g., No. 60/128,643, filed Apr. 4, 1999, entitled “MANIPULATION OF MICROPARTICLES IN MICROFLUIDIC SYSTEMS,” by Mehta et al.
One of the rate limiting aspects of high-throughput screening in general is the need to sample and re-sample the effect of a given test reagent at a variety of concentrations to provide kinetic or other activity data, such as dose-response information. Irrelevant samples (i.e., those with little or no activity in the system of interest) are often unnecessarily re-sampled to determine whether they provide an activity of interest. The need for multiple sampling strategies effectively increases the number of sampling events that a system performs, decreasing the overall throughput of the system. Furthermore, this resampling can involve multiple xyz spatial translations of a microfluidic sample loader or sample library, or both, to provide access to samples of interest, further reducing system throughput.
Accordingly, it would be extremely useful to be able to acquire kinetic information from one or a few sampling events in a microfluidic system. The present invention provides methods, apparatus and systems for generating and deconvoluting signal information and for reduced resampling in high throughput systems (including both pressure-based and electrokinetic systems), as well as a variety of other features which will become apparent upon complete review of the following.
SUMMARY OF THE INVENTION
The present invention provides for the use of signal profile information from one or a few sampling events to produce information regarding kinetics and/or reactant concentrations in a microfluidic system. In particular, the shape of the signal profile can be deconvoluted to provide kinetic information and to provide directed re-sampling of a source of sample materials.
In one set of methods, the dwell time for a system sampler is varied to modulate the profile of a signal profile, thereby providing a basis for activity determination and for limiting the need for re-sampling by the system. The system sampler will often utilize pressure-based sampling elements (e.g., pressure-based pipettor channels), although the methods herein can also be adapted to electrokinetic and other fluid movement systems.
In other methods, the dwell time is not necessarily varied, with kinetic information resulting from the deconvolution of signal profile information. Although not necessary, microfluidic channel geometry can be used to facilitate signal deconvolution, e.g., by first dispersing the sample and then reacting the dispersed sample to produce concentration gradient dependent signal information.
For example, the present invention provides high-throughput methods of sampling fluidic materials in which a plurality of different aliquots of the fluidic material are introduced into a microfluidic cavity (typically a channel, well, chamber, reservoir or other structure of microscale dimensions). These aliquots (which typically include a test material to be assayed for activity on a selected target) are produced by systematically varying dwell time at a source of the fluidic material for a microfluidic sample loader which is fluidly coupled to the microfluidic cavity, or by systematically varying a volume of the material in each aliquot. For example, the percent modulation versus target reagent concentration time can be determined by systematically varying the dwell time for a microfluidic sample loader loading material from a source of a test reagent.
The microfluidic sample loader can be configured in a variety of ways, e.g., as a fluid pressure modulatory channel, an electrokinetic modulatory channel, an electrokinetic controller, a fluid pressure controller (e.g., a vacuum source), or the like. Following loading of a test material into the system by the microfluidic sample loader, the material can be flowed into contact with target compositions in the microfluidic cavity for typical mixing reactions, e.g., with a target material.
The system is particularly useful for examining potential activity modulators (i.e., compounds or compositions which facilitate (activate) or inhibit (repress) a reaction of interest, or between an interaction between two or more moieties which interact to produce a detectable result. Typically, the modulator modulates an activity between one or more reactant and one or more reactant substrate. For example, the target composition or the test composition can include an enzyme or other catalyst, a substrate and/or an activity modulator (it will be appreciated that, e.g., kinetic activity can be determined by varying any one or all of these components, depending on the format of the reaction at issue). For example, the modulator can inhibit (e.g., as a competitive inhibitor) or enhance essentially any reaction conducted in the microfluidic system. In the methods herein, it is typical to measure a signal produced by at least one test composition, a target composition, a reaction modulator, and, an interaction between any of these components. Signals are generally produced by one or more labels in the system, and can be a component of, or released by, a reaction.
Label is typically initially confined in a region −h<x<h, as a function of time (t) and spatial position (x) with respect to the peak center (x=0) and the concentration (C) of the label, or of a component corresponding to the label, is equal to ½ C
0
{erf[(h−x)/(2Dt)
½
)]}, where C
0
is the initial concentration at time t=0, erf is an error function, and D is a coefficient of overall dispersion. D is equal to the sum of thermal diffusion and Taylor dispersion (D
T
) in the system. In turn, the Taylor dispersion (D
T
) is dependent on the dimensions and shape of the microfluidic cavity through which the label is flowed, the flow velocity (u) and the thermal diffusivity (D). Typically, D=K(d
2
u
2
)/D, where K is a proportionality factor which is a function of the microfluidic cavity through which the label is flowed and d is a characteristic microfluidic cavity length. For example, where the microfluidic cavity is a circular channel and K=1/192, d is the diameter of the circular channel and D=D+D
T
.
In the methods herein, a kinetic rate constant is typically determined for a reaction between at least one test reagent and a target reagent. For example, the kinetic rate constant can be determined by establishing a calibration curve relating dwell time to dilution factor using one or more dye with a molecular weight which is similar to the molecular weight of the reaction modulator. For example, where the target composition is an enzyme and the test composition is an enzyme substrate, or a potential enzyme substrate, the method can include contacting the at least one test composition or the target composition with
Chow Andrea W.
Kopf-Sill Anne R.
Parce J. Wallace
Caliper Technologies Corp.
Filler Andrew L.
Handy Dwayne K
Murphy Matthew B.
Quine Intellectual Property Law Group P.C.
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