Biology subjects

Hariri, A. A.

Publications and source records attributed to Hariri, A. A..

5 recordsLinked to original sources

bioRxiv2023

A rapid ELISA platform with no sample preparation requirement

Since its invention in the 1970s, the enzyme-linked immunosorbent assay (ELISA) has served as the "gold-standard" for blood and plasma protein biomarker quantification. However, ELISAs require significant amounts of sample preparation entailing multiple reagent additions, incubations, and washing steps, limiting their clinical usefulness in the context of diagnosis and prognosis of rapidly evolving medical conditions. In this work, we describe the instant ELISA biosensor platform, a probe that can be exposed directly to blood or other biological samples and quantifies protein biomarkers within 15 minutes. The sensor leverages a novel affinity reagent termed monolithic dual-antibody clamp (MDAC) which preserves the specificity, sensitivity, and generalizability of ELISA while also enabling rapid analysis of unprocessed blood and other complex matrices. Using MDAC in chicken media, we demonstrate picomolar quantification of the inflammatory marker tumor necrosis factor alpha (TNF), as well as monocyte chemotactic protein (MCP)-1, a useful prognostic indicator of cytokine release syndrome (CRS) during chimeric antigen receptor (CAR) T-cell immunotherapy. Finally, we demonstrate MCP-1 quantification in plasma samples from patients who had undergone CAR T-cell treatment.

bioengineering↗

bioRxiv2023

An Antibody-Based Molecular Switch for Continuous Biosensing

We present a generalizable approach for designing biosensors that can continuously detect specific biomarkers in real time and without sample preparation. This is achieved by converting existing antibodies into target-responsive "antibody-switches" that enable continuous optical biosensing. To engineer these switches, antibodies are linked to a molecular competitor through a DNA scaffold, such that competitive target binding induces scaffold switching and fluorescent signaling of changing target concentrations. As a demonstration, we designed antibody-switches that achieve rapid, sample-preparation-free sensing of digoxigenin and cortisol in undiluted plasma. We showed that, by substituting the molecular competitor, we can further modulate the sensitivity of our cortisol switch to achieve detection at concentrations spanning 3.3 nM to 3.3 mM. Finally, we integrated this switch with a fiber-optic sensor to achieve hours-long continuous sensing of cortisol in buffer with <5-minute time resolution. We believe this modular sensor design can enable continuous biosensor development for many biomarkers.

bioengineering↗

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

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↗

bioRxiv2019

Independent Control of the Thermodynamic and Kinetic Properties of Aptamer Switches

Molecular switches that change their conformation upon target binding offer powerful capabilities for biotechnology and synthetic biology. In particular, aptamers have proven useful as molecular switches because they offer excellent binding properties, undergo reversible folding, and can be readily engineered into a wide range of nanostructures. Unfortunately, the thermodynamic and kinetic properties of the aptamer switches developed to date are intrinsically coupled, such that high temporal resolution (i.e., switching time) can only be achieved at the cost of lower sensitivity or high background. Here, we describe a general design strategy that decouples the thermodynamic and kinetic behavior of aptamer switches to achieve independent control of sensitivity and temporal resolution. We used this strategy to generate an array of aptamer switches with effective dissociation constants (KD) ranging from 10 M to 40 mM and binding kinetics ranging from 170 ms to 3 s--all generated from the same parent ATP aptamer. Our strategy is broadly applicable to other aptamers, enabling the efficient development of switches with characteristics suitable for diverse range of biotechnology applications.

bioengineering↗