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Label-Free Single Peptide Detection and Identification using Nanoscale Electrochromatography and Resistive Pulse Sensing in Thermoplastic Nanofluidic Devices for Peptide Fingerprinting
Chibuike, Maximillian Ifeanyi
Chibuike, Maximillian Ifeanyi
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Abstract
Proteins perform pivotal functions in various biological processes, including metabolism and cellular communication, with errors in protein expression or molecular composition giving rise to different disease states. Thus, identification and quantification of proteins carried by both normal and diseased cells is of high importance. However, current protein determinations can be fraught with challenges, such as the inability to deal with mass-limited samples, for example, those encountered in liquid biopsy samples.We are developing a method for protein identification through solid-phase enzymatic protein digestion that will generate peptide (protein peptide fingerprints, PPF) using nanoscale electrochromatography and resistive pulse sensing (RPS) in a label-free fashion. In order to realize this technology, we reported in the dissertation nanoscale electrochromatography (nEC) of model peptides and proteins using dual in-plane nanosensors fabricated in thermoplastic with surfaces engineered for optimal identification via molecular-dependent electrophoretic mobility of the analytes evaluated from the Time-of-Flight (ToF). The ToFs were obtained using the nanosensor, which consists of two in-plane nanopores separated by a nanoelectrochromatography column. The nanosensor is fabricated either in poly (methyl methacrylate) (PMMA), or cyclic olefin polymer (COP), with the surfaces activated with O2 plasma for PMMA and UV/O3 for COP to create hydrophilic carboxylic acid groups (COOH) and aiding in the differential electrokinetic transport of the peptide or protein molecules. We performed microchip and nanochannel electrochromatography of the peptides labeled with an ATTO-532 dye to permit electrokinetic tracking of single molecules and understand scaling effect phenomena. Our results demonstrated a clear difference in terms of apparent electrophoretic mobilities between the two columns. Next, we carried out RPS of label-free peptides and proteins using the dual in-plane nanosensor that contained two pores spaced by 5 µm that were ~10 nm in apparent diameter with a 50 nm column (width and depth). As a single peptide molecule moved through the nanopore, the sensor read a resistive pulse pair from which the ToF could be deduced. Peptides were identified via their apparent electrophoretic mobilities evaluation in terms of time-of-flight (ToF), which is the time it takes for a single peptide molecule to travel between the two in-plane nanopores, current transient amplitude, and dwell time (peak shape). With the aid of machine learning algorithm, we were able to classify these single molecules to over 99.5%. Changes in peptide or proteins apparent electrophoretic mobilities and other RPS parameters may be utilized to identify disease biomolecules.
Next, we carried out surface modification of COP/COC activated with UV/O3 by appending Tris on a dual in-plane nanosensor after thermal fusion bonding using EDC/NHS coupling chemistry to reduce surface charge density and increase capture rate of biomolecules during electrokinetic transport. Our results showed that ToF, dwell time, and normalized peak amplitude (?I/Io) were affected. Finally, we investigated the use of a two-dimensional (2D) nanoscale electrophoresis device to increase peptide peak capacity. The 2D nEP device consists of two columns (first and second dimensions) with three nanopores —two in the first dimension and one in the second dimension. The two columns have different cross-sectional areas to affect orthogonality and increase electrokinetic transport efficiency. We showed that the average peak capacity of the peptides with the 2D nEP device is above 74,000 and the migration of the peptides in the two columns is different, indicating some level of device orthogonality. The 2D device is a multipurpose device that could be used for solid-phase enzymatic digestion of proteins that will provide peptide fingerprint information from single protein targets and potentially transform single-molecule protein sequencing.
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Date
2025-05-31
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University of Kansas
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This item contains archived web content.
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Available after December 31, 2027
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Keywords
Electrochromatography, Identification, Label-Free, Nanoscale, Single-Peptide, Thermoplastics