Biology subjects

Feagin, T.

Publications and source records attributed to Feagin, T..

3 recordsLinked to original sources

bioRxiv2023

Continuous optical detection of small-molecule analytes in complex biomatrices

Current technology for measuring specific biomarkers - continuously in complex samples, without sample preparation - is limited to just handful of molecules such as glucose and blood oxygen. In this work, we present the first optical biosensor system that enables continuous detection of a wide range of biomarkers in complex samples, such as human plasma. Our system employs a modular duplex-bubble switch (DBS) architecture that converts aptamers into structure-switching fluorescence probes whose affinity and kinetics can be readily tuned. These DBS constructs are coupled to a fiber-optic detector that measures the fluorescence change only within an evanescent field, thereby minimizing the impact of background autofluorescence and enabling direct detection of analytes at physiologically relevant concentrations even in interferent-rich sample matrices. Using our system, we achieved continuous detection of dopamine in artificial cerebrospinal fluid for >24 hours with sub-second resolution and a limit of detection (LOD) of 1 {micro}M. We subsequently demonstrated the systems generalizability by configuring it to detect cortisol with nanomolar sensitivity in undiluted human plasma. Both sensors achieved LODs orders of magnitude lower than the KD of the DBS element, highlighting the potential to achieve sensitive detection even when using aptamers with modest affinity.

biophysics↗

bioRxiv2021

Discovery of indole-modified aptamers for highly specific recognition of protein glycoforms

Glycosylation is one of the most abundant forms of post-translational modification, and can have a profound impact on a wide range of biological processes and diseases. Unfortunately, efforts to characterize such modifications in the context of basic and clinical research are severely hampered by the lack of affinity reagents that can differentiate protein glycoforms. This lack of reagents is largely due to the challenges associated with generating affinity reagents that can bind to particular glycan epitopes with robust affinity and specificity. In this work, we use a fluorescence-activated cell sorting (FACS)-based approach to generate and screen aptamers with indole-modified bases in an effort to isolate reagents that can differentiate between protein glycoforms. Using this approach, we were able to select multiple aptamers that exhibit strong selectivity for specific glycoforms of two different proteins, with the capacity to discriminate between molecules with identical tertiary structures that differ only in terms of their glycan modifications.

bioengineering↗

bioRxiv2020

Automated platform for high-throughput screening of base-modified aptamers for affinity and specificity

Aptamers incorporating chemically modified bases can achieve superior affinity and specificity compared to natural aptamers, but their characterization remains a labor-intensive, low-throughput task. Here we describe the non-natural aptamer array (N2A2) system, in which a minimally modified Illumina MiSeq instrument is used for the high-throughput generation and characterization of large libraries ([~]106) of base-modified DNA aptamer candidates on the basis of both target affinity and specificity. We first demonstrate the capability to screen multiple different base modifications to identify the optimal chemistry for high-affinity target binding. We next use N2A2 to generate aptamers that can maintain excellent specificity even in complex samples, with equally strong target affinity in both buffer and diluted human serum. Given that N2A2 requires only minor mechanical modifications to the MiSeq, we believe N2A2 offers a broadly accessible tool for generating high-quality affinity reagents for diverse applications.

bioengineering↗