Biology subjects

Newman, S. S.

Publications and source records attributed to Newman, S. S..

4 recordsLinked to original sources

bioRxiv2023

Theoretical framework and experimental validation of multiplexed analyte quantification using cross-reactive affinity reagents

Gold standard immunoassays depend on specific affinity reagents for accurate molecular quantification. Any cross-reactivity of affinity reagents, wherein the reagent non-specifically binds to unintended molecules, can create false positive binding signals and result in inaccurate quantification of analytes. Mitigating cross-reactivity represents one of the greatest challenges in molecular diagnostics, and remains an unsolved problem. To instead overcome the effects of cross-reactivity, we present a mathematical framework that uses generalized binding equations and noise estimation to enable the use of multiple cross-reactive reagents for multiplexed molecular quantification. As a proof-of-concept, we experimentally demonstrate accurate quantification of a small molecule for which no specific affinity reagents are available, even at high concentrations of a cross-reactive molecule. Furthermore, this robust schema yields well-defined bounds of quantification that make it easier to assess the quality of assay results and predicts under which conditions assay performance is likely to break down. This work turns cross-reactive affinity reagents, which were previously a liability, into an asset for achieving accurate quantification of analytes.

bioengineering↗

bioRxiv2023

Real-Time Spatiotemporal Measurement of Extracellular Signaling Molecules Using an Aptamer Switch-Conjugated Hydrogel Matrix

Cells rely on secreted signaling molecules to coordinate essential biological functions including development, metabolism, and immunity. Unfortunately, such signaling processes remain difficult to measure with sufficient chemical specificity and temporal resolution. To address this need, we have developed an aptamer-conjugated hydrogel matrix that enables continuous fluorescent measurement of specific secreted analytes - in two dimensions, in real-time. As a proof of concept, we performed real-time imaging of Dictyostelium discoideum cells, a well-studied amoeba model wherein inter-cellular communication is performed though cAMP signaling. We engineered a set of aptamer switches that generate a rapid and reversible change in fluorescence in response to cAMP signals. By combining multiple switches with different dynamic ranges, we can measure cAMP concentrations spanning three orders of magnitude in a single experiment. These sensors are embedded within a biocompatible hydrogel on which cells are cultured and their cAMP secretions can be imaged using fluorescent microscopy. Using this aptamer-hydrogel material system, we achieved the first direct measurements of oscillatory cAMP signaling that correlate closely with previous indirect measurements. Using different aptamer switches, this approach could be generalized for measuring other secreted molecules to directly visualize diverse extracellular signaling processes and the biological effects that they trigger in recipient cells.

bioengineering↗

bioRxiv2023

Extending the dynamic range of biomarker quantification through molecular equalization

Precision medicine requires highly scalable methods of multiplexed biomarker quantification that can accurately describe patient physiology. Unfortunately, contemporary molecular detection methods are generally limited to a dynamic range of sensitivity spanning just 3-4 orders of magnitude, whereas the actual physiological dynamic range of the human plasma proteome spans more than 10 orders of magnitude. Current methods rely on sample splitting and differential dilution to compensate for this mismatch, but such measures greatly limit the reproducibility and scalability that can be achieved--in particular, the effects of non-linear dilution can greatly confound the analysis of multiplexed assays. We describe here a two- pronged strategy for equalizing the signal generated by each analyte in a multiplexed panel, thereby enabling simultaneous quantification of targets spanning a wide range of concentrations. We apply our EVROS strategy to a proximity ligation assay and demonstrate simultaneous quantification of four analytes present at concentrations spanning from low femtomolar to mid-nanomolar levels. In this initial demonstration, we achieve a dynamic range spanning seven orders of magnitude in a single 5 {micro}l sample of undiluted human serum, highlighting the opportunity to achieve sensitive, accurate detection of diverse analytes in a highly multiplexed fashion.

bioengineering↗

bioRxiv2021

Improved immunoassay sensitivity and specificity using single-molecule colocalization

Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still suffers some notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background arising from the interaction of detection antibodies with the assay substrate or interferents in the sample matrix. Single-Molecule Colocalization Assay (SiMCA) overcomes this problem by employing total internal reflection fluorescence (TIRF) microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized fluorescent signals, we can essentially eliminate the confounding effects of background produced by non-specific binding of detection antibodies. We further employed a normalization strategy to account for the heterogeneous distribution of the capture antibodies, greatly improving the reproducibility of our measurements. In a series of experiments with TNF-, we show that SiMCA can achieve a three-fold lower limit of detection compared to conventional single-color assays using the same antibodies and exhibits consistent performance for assays performed in complex specimens such as chicken serum and human blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects.

biophysics↗