Method and apparatus for performing peptide digestion on a microfluidic device
Inventors
Fathollahi, Bahram • Farinas, Javier A. • Chow, Andrea W. • Mouradian, Stephane
Assignees
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Publication Number
US-9097723-B2
Publication Date
2015-08-04
Expiration Date
Abstract
The invention provides methods and apparatuses that allow a protein sample to undergo reduction, alkylation, and digestion in a continuous flow process carried out within a microfluidic device. Methods and apparatuses in accordance with the invention can be employed as part of an automated proteomics analysis carried out in an integrated proteomics system.
Core Innovation
The invention is a microfluidic lab-on-a-chip system for continuous-flow protein processing in which a protein sample is driven through a microfluidic device under a single negative pressure driving force. The microfluidic device includes a sample reservoir and a waste reservoir connected by a plurality of parallel channels, with channel regions arranged proximate to the sample reservoir, adjacent to an intermediate region, and proximate to the waste reservoir. Reagents are introduced at various points along the parallel channels via a plurality of reagent reservoirs in fluid communication with at least two of the parallel channels.
In the first region proximate the sample reservoir, the protein sample is sequentially exposed to a reduction reagent; in the second region adjacent the first region, the protein sample is sequentially exposed to alkylation and quenching reagents. The device further places immobilized trypsin in a portion of each channel in the third region proximate to the waste reservoir, so that the protein sample flows through the immobilized trypsin after the reduction, alkylation, and quenching exposures. The continuous flow sequentially performs the reduction, alkylation, quenching, and digestion steps before collecting the processed protein-containing fluid in the waste reservoir.
The microfluidic device employs region-specific temperature control, maintaining the first region at 50 °C and the third region below 50 °C but above room temperature, or maintaining the third region at 37 °C and the first region above 37 °C. The system is configured for continuous-flow operation in parallel channels so multiple samples can be processed using the device/interface architecture described.
Claims Coverage
The partial content provides two independent method claims. Each independent claim centers on parallel microfluidic channels with reagent reservoirs for sequential reduction, alkylation, quenching, and digestion using immobilized trypsin, driven by a single negative-pressure draw and collected in a waste reservoir; the primary differences are the region-specific temperature conditions.
Parallel microfluidic regions for sequential reduction, alkylation, quenching, and immobilized-trypsin digestion
A microfluidic device includes a sample reservoir, a waste reservoir, and a plurality of parallel channels defining a first region proximate the sample reservoir, a second region adjacent the first region, and a third region proximate the waste reservoir, and includes a plurality of reagent reservoirs introducing reagents at various points along each of the plurality of parallel channels; immobilized trypsin is placed in a portion of each of the plurality of channels in the third region; the flow sequentially exposes the protein sample to the reduction reagent in the first region, and the alkylation and quenching reagents in the second region before the protein sample flows through the immobilized trypsin in the third region; and the fluid including the protein sample is collected in the waste reservoir.
Single negative pressure continuous-flow driving force with sequential reagent exposure and collection in waste reservoir
Applying a single negative pressure driving force to the waste reservoir generates a continuous flow of fluid from the sample reservoir, through the plurality of channels, to the waste reservoir, such that the flow sequentially exposes the protein sample to the reduction reagent in the first region, and the alkylation and quenching reagents in the second region before the protein sample flows through the immobilized trypsin in the third region; and collecting the fluid including the protein sample in the waste reservoir.
Region-specific temperature control for reduction/alkylation/quenching and immobilized trypsin
Maintaining the first region at 50 °C and the third region at a temperature below 50 °C but above room temperature, or maintaining the third region at 37 °C and the first region at a temperature above 37 °C, while applying the continuous flow so that immobilized trypsin in the third region performs digestion after reduction, alkylation, and quenching in the upstream regions.
Across the independent claims, the inventive coverage is directed to a microfluidic, continuous-flow, parallel-channel process that uses reagent reservoirs to sequentially perform reduction, alkylation, and quenching upstream of a region containing immobilized trypsin, where the overall fluid movement is generated by a single negative pressure and the processed protein-containing fluid is collected in a waste reservoir; the claims further specify different region-specific temperature relationships for the first and third regions.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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