Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2002-08-28
2003-10-14
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S024000, C435S283100, C435S287100, C435S286100, C435S286500, C435S004000
Reexamination Certificate
active
06632629
ABSTRACT:
BACKGROUND OF THE INVENTION
There is considerable interest by biochemists, clinical chemists, pharmaceutical manufacturers, and other scientists in determining the concentration of amino acids, peptides, and other amine-functional compounds in complex biological samples. This analysis currently requires the detection of a plurality of compounds with a high degree of sensitivity and detection selectivity. Typical detection schemes involve, e.g., the use of fluorescent tags on the compounds of interest. A common method is to convert the compounds of interest into a derivative with a strong fluorescence signal. The derivatizing agent affects the ultimate sensitivity and accuracy of the analysis. Thus, a derivatizing reagent that maximizes sensitivity, yield, and stability of the derivatized amino acids is desired.
Many such derivatives have been studied, including heterocyclic aromatic carbamate compounds and aromatic dialdehydes. See, e.g., U.S. Pat. No. 5,296,599 issued Mar. 22, 1994 by Cohen and Michaud,
Analytical Biochemistry,
Cohen and Michaud, 211 (1993) 279-287, Jacobsen et al.,
Anal. Chem.
66 (1994) 3472-3476 and Krull et al.,
J. Chromatogr.
699 (1977) 173-208. Jacobsen, for example, describes the use of o-phthaldialdehyde for fluorescently tagging amino acids and Cohen describes the use of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate for labeling amino acids, peptides, and proteins.
The studies described above are typically performed in conjunction with chromatographic separation and detection. Improved methods for studying and detecting amino acids, peptides, and the like, are desired, e.g., improved methods for performing assays in microfluidic devices. Microfluidic devices useful in such methods have been described by the inventors and their coworkers in various publications, published PCT applications, and U.S. patent applications.
Improvements to such devices and methods would be desirable. For example, a method of labeling assay components in a microfluidic device would be useful. The methods and devices of the present invention provide these features and many others that will be apparent upon complete review of the following.
SUMMARY OF THE INVENTION
The present invention provides microfluidic methods, devices and systems for performing in-line labeling for continuous-flow assays, e.g., protease inhibition assays. For example, a protease assay is performed using unlabeled, e.g., non-fluorogenic substrates, and then the products are labeled using fast amino derivatization chemistry. Due to the fast derivatization, the labeling step is performed as the products are produced and/or flowed through the system. The method therefore provides a rapid, one step labeling procedure that forms stable derivatives that are readily amenable to separation, analysis, and detection. The products are optionally separated either before, or after, the labeling step.
In one aspect, the present invention provides a method of labeling a reaction product in a microfluidic system, e.g., when performing a protease assay, e.g., an inhibition assay. The method comprises flowing a protease through a microscale cavity and contacting the protease with a protease substrate. Other reagents are optionally mixed into the enzymatic reaction, e.g., modulators, inhibitors, activators, and the like. The protease acts on the protease substrate, e.g., in the presence of an inhibitor or modulator, to form products, e.g., amino acids, peptides, or proteins. The products are then labeled with a labeling reagent, thus producing labeled products. The labeled products are typically detected, e.g., fluorescently detected, and analyzed, e.g., quantitated.
The labeling step includes chemically reacting the products with a labeling reagent, which is typically an amine-derivatizing reagent, e.g., a heterocyclic aromatic carbamate compound or an aromatic dialdehyde.
Heterocyclic aromatic carbamate compounds include 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, 3-aminoquinolyl-N-hydroxysuccinimidyl carbamate, 5-aminoquinolyl-N-hydroxysuccinimidyl carbamate, 5-aminoisoquinolyl-N-hydroxysuccinimidyl carbamate, 6-amino-4-methylquinolyl-N-hydroxysuccinimidyl carbamate, 6-amino-2,4-dimethylquinolyl-N-hydroxysuccinimidylcarbamate, 6-amino-2-phenylquinolyl-N-hydroxysuccinimidyl-carbamate, 6-amino-2-methoxy-4-methylquinolyl-N-hydroxysuccinimidylcarbamate, 4-aminoquinaldine-N-hydroxysuccinimidyl carbamate, 9-aminoacridine-N-hydroxysuccinimidyl carbamate, 2-aminoacridine-N-hydroxysuccinimidylcarbamate, luminol-N-hydroxysuccinimidylcarbamate, isoluminol-N-hydroxysuccinimidylcarbamate, 7-amino-4-methylcoumarin-N-hydroxysuccinimidylcarbamate, 7-amino-4-(trifluoromethyl)coumarin-N-hydroxysuccinimidylcarbamate, 4′-(aminomethyl)fluorescein-N-hydroxysuccinimidylcarbamate, 5-(aminomethyl)fluorescein-N-hydroxysuccinimidylcarbamate, 5-aminoeosin-N-hydroxysuccinimidylcarbamate, Cascade Blue ethylenediamine-N-hydroxysuccinimidylcarbamate, and the like.
Aromatic dialdehydes include o-pthaldialdehyde, napthalene-2-3-dicarboxaldehyde, anthracene-2,3-dicarboxaldehyde, and the like. Other amine-derivatizing reagents useful in the present invention include 3-(4-carboxybenzoylquinoline-2-carboxaldehyde, 3-(2-furosyl)quinoline-2-carboxaldehyde, fluorescamine, 7-nitrobenz-2-oxa-1,3-diazole chloride, and the like.
In some embodiments, the methods of the present invention also involve separating the products before or after the labeling step. Separation comprises electrophoretically separating the various components in a polymer, a gel, a solution, or the like, e.g., polyacrylamide, polydimethylacrylamide/co-acrylic acid, or other separation matrices or polymers.
In other embodiments, the methods include flowing an inactivating reagent through the microscale cavity after the labeling step, thereby inactivating any labeling reagents that have not reacted with the products. For example, the inactivating reagent is optionally one that alters the pH of the materials or fluids in the microscale cavity.
In another aspect, the present invention provides devices and systems for performing the above assays and labeling reactions. The devices comprise a body structure having microscale channels disposed therein. The channels typically include a main channel, in which a protease and a protease substrate are combined to form one or more products, and a labeling channel region fluidly coupled to the main channel, in which the products are labeled. Also included are (a) detection channel region(s) fluidly coupled to the main channel, for detecting the labeled products, and sources for the components and reagents. Such sources include, but are not limited to, sources for labeling reagents, proteases, protease substrates, inactivating reagents, and the like. In addition, the device typically comprises a labeling reagent, e.g., an amine-derivatizing reagent such as those listed above. Separation channels, e.g., a channel comprising a polymer, gel, or solution, e.g., of polyacrylamide or polydimethylacrylamide/co-acrylic acid, are also optionally included in the above devices.
Systems incorporating the above devices are also provided. Such systems are used to perform assays or labeling reaction as described above. The systems typically include a fluid direction system operably coupled to the microfluidic device for transporting components, materials, enzymes, reagents, and the like through the plurality of microscale channels. Control systems are provided in the systems for instructing the fluid direction system to transport the reagents and a detection system is provided to detect the reagents. Computers and software are also optionally coupled to the system. Software for performing assays in the systems of the invention typically includes at least one instruction set to analyze signals produced from the detection system, quantitate signals produced from the detection system, and/or direct fluid movement in the system.
REFERENCES:
patent: 4390403 (1983-06-01), Batchelder
patent: 4908112 (1990-03-01), Pace
patent: 5126022 (1992-06-01), Soane et a
Sundberg Steven
Yang Hua
Caliper Technologies Corp.
Filler Andrew L.
Leary Louise N.
McKenna Donald R.
Quine Intellectual Property Law Group P.C.
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