Total analyte quantitation

Details Total analyte quantitation Total analyte quantitation

2001-04-12

2004-05-11

Nguyen, Nam (Department: 1753)

Chemistry: electrical and wave energy

Processes and products

Electrophoresis or electro-osmosis processes and electrolyte...

C204S604000, C422S070000, C702S022000

Reexamination Certificate

active

06733645

ABSTRACT:

COPYRIGHT NOTIFICATION
Pursuant to 37 C.F.R. § 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
BACKGROUND OF THE INVENTION
Biological molecules, such as proteins and nucleic acids are routinely fractionated and characterized, e.g., by capillary electrophoresis or using microfluidic separation technology. For example, U.S. Pat. No. 5,948,227 by Dubrow, entitled “Methods and Systems for Performing Electrophoretic Molecular Separations,” describes methods for electrophoretically separating molecular and macromolecular species in microfluidic devices.
Electrophoretic forces are typically used to separate materials in microfluidic devices, e.g., relying upon the electrophoretic mobility of charged species within an electric field applied to the material. Electrophoretic movement is used to separate mixtures of components as they move through a microfluidic channel. Signal peak area of separated components is typically used to assess the extent of reactions, reaction rate constants, concentration of reactants, products, separated components, and a variety of other chemical and biochemical parameters.
Just as in traditional capillary electrophoresis, electrokinetic sample introduction in a microfluidic device biases sample introduction. The electric fields can cause preferential movement of reagents due to differences in their mass to charge ratio, e.g., highly charged materials move to the front or back of a fluid plug. This effect is desirable when attempting to electrokinetically separate various compounds, but inhibits the ability to obtain measurements relating to entire samples, e.g., unseparated unbiased samples. For example, it is often desirable to identify the total concentration, e.g., of nucleic acids or proteins, in a sample in addition to the concentration of each nucleic acid fragment, e.g., after separation.
The calculation of kinetic constants in flowing systems has also been described. For example, published PCT application WO 98/56956, by Kopf-Sill et al., entitled “Apparatus and Methods for Correcting Variable Velocity in Microfluidic Systems,” describes methods of determining concentration, e.g., after an electrokinetically biased sample introduction, using variable velocities, e.g., of reactants and products. This reference also describes, e.g., the use of gated injections to achieve representative sample aliquots and other related phenomena.
The present invention provides methods and apparatus for obtaining representative or unbiased sample aliquots that are used to determine, e.g., total analyte concentrations. The methods and apparatus of the present invention provide these features and many others that will be apparent upon complete review of the following disclosure.
SUMMARY OF THE INVENTION
The present application provides methods for separating two or more analytes, e.g., in a microchannel, and determining a total analyte concentration for the two or more analytes. For example, a sample, e.g., a mixture of nucleic acid fragments, is optionally separated, the concentration or amount of each individual analyte in the sample determined, and the concentration of all analytes together determined. The methods typically involve summation of individual analyte concentrations using a representative sample aliquot, or alternatively, measurement of the total analyte concentration prior to separation.
In one aspect, a method for separating analytes and determining a total analyte concentration or amount is provided. The method comprises flowing at least two analytes through a first channel, e.g., electrokinetically or under pressure. The analytes typically flow into an intersection of the first channel with a separation channel. After a specified time, the analytes at the intersection are injected into the separation channel. The analytes are then separated, e.g., electrophoretically, resulting in two or more separated analytes. The analytes are then detected, resulting in, e.g., two or more signals. The two or more signals are used to determine the total analyte concentration, e.g., by summation of the two or more signals, e.g., summation of the signal peak areas or peak heights corresponding to individual analyte concentrations. The concentration or amount of each individual analyte is also optionally determined, e.g., to produce a ratio of the amount of at least one of the analytes to the total analyte amount or to a portion of the total analyte amount.
Typically, the analytes have different electrokinetic mobilities, wherein at least one of the analytes comprises a slowest analyte. Waiting the specified time to inject the analytes into the separation channel typically involves waiting until the slowest analyte reaches the intersection. By waiting until the slowest analyte has reached the intersection, a representative sample aliquot is obtained such that a total analyte concentration for the sample is optionally determined, e.g., after separation of the injected sample.
In another aspect, the method of separating two or more analytes and determining a total analyte concentration or amount comprises flowing, e.g., electrokinetically or under pressure, at least two analytes through a first channel region and through a measurement channel region. The analytes are detected, e.g., unseparated, in the measurement channel region, thereby determining the total analyte concentration or amount. Detection in the measurement channel typically leads to a signal that increases in value until it reaches a constant value, which constant value represents the total analyte concentration, e.g., after both slow and fast flowing analytes have reached a detection region. The analytes are then optionally injected into a separation channel, separated, e.g., electrophoretically, and detected. Concentrations or amounts of each individual analyte are optionally determined based on signals detected after separation of the analytes. Ratios of the amount of one or more of the separated analytes to the total analyte amount or to a portion of the total analyte amount are also optionally determined.
In another aspect, the invention provides systems for separating two or more analytes and determining a total analyte concentration or amount. Such a system typically comprises a microfluidic device comprising a body structure having a plurality of microscale channels disposed therein. The microscale channels typically comprise a first channel region for flowing one or more analytes, and a measurement channel region fluidly coupled to the first channel region. The measurement channel is used to obtain a total analyte concentration, e.g., after all analytes have had time to reach a detection channel region. Typically the devices include a separation channel fluidly coupled to the first channel region and two detection regions. A first detection region is typically positioned proximal to the measurement channel region and a second detection region is typically positioned proximal to the separation channel. The first detection region is typically used to detect the total analyte concentration or amount and the second detection region is typically used to detect individual analytes, e.g., after separation.
The system further comprises a fluid direction system fluidly coupled to the microfluidic device. The fluid direction system directs movement of the analytes through the first channel region; movement of the analytes from the first channel region into the measurement channel region; movement of the analytes through the first detection region; movement of the analytes from the first channel into the separation channel; and, movement of the separated analytes through the second detection region. The fluid direction system typically comprises one or more fluid control elements, such as pr

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