Biology subjects

Maganzini, N.

Publications and source records attributed to Maganzini, N..

6 recordsLinked to original sources

bioRxiv2023

Aptamer-antibody chimera sensors for sensitive, rapid and reversible molecular detection in complex samples

The development of receptors suitable for the continuous detection of analytes in complex, interferent-rich samples remains challenging. Antibodies are highly sensitive but difficult to engineer in order to introduce signaling functionality, while aptamer switches are easy to construct but often yield only modest target sensitivity. We present here the programmable antibody and DNA aptamer switch (PANDAS), which combines the best features of both systems by using a nucleic acid tether to link an analyte-specific antibody to an internal strand-displacement (ISD)-based aptamer switch that recognizes the same target. The monoclonal antibody mediates initial analyte binding due to its higher affinity; the resulting increase in local analyte concentration then leads to cooperative binding and signaling by the ISD switch. We developed a PANDAS sensor for the clotting protein thrombin and show that this design achieves 100-fold enhanced sensitivity compared to using an aptamer alone. This design also exhibits reversible binding, enabling repeated measurements with temporal resolution of [~]10 minutes, and retains excellent sensitivity even in interferent-rich samples. With future development, this PANDAS approach could enable the adaptation of existing protein-binding aptamers with modest affinity into sensors that deliver excellent sensitivity and minute-scale resolution in minimally prepared biological specimens.

bioengineering↗

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↗

bioRxiv2022

Pre-equilibrium biosensors: A new approach towards rapid and continuous molecular measurements

Almost all biosensors that use ligand-receptor binding operate under equilibrium conditions. However, at low ligand concentrations, the equilibration with the receptor (e.g., antibodies and aptamers) become slow and thus equilibrium-based biosensors are inherently limited in making measurements that are both rapid and sensitive. In this work, we provide a theoretical foundation for a novel method through which biosensors can quantitatively measure ligand concentration before reaching equilibrium. Rather than only measuring receptor binding at a single time-point, the pre-equilibrium approach leverages the receptors kinetic response to instantaneously quantify the changing ligand concentration. Importantly, analyzing the biosensor output in frequency domain, rather than in the time domain, we show the degree to which noise in the biosensor affects the accuracy of the pre-equilibrium approach. Through this analysis, we provide the conditions under which the signal-to-noise ratio of the biosensor can be maximized for a given target concentration range and rate of change. As a model, we apply our theoretical analysis to continuous insulin measurement and show that with a properly selected antibody, the pre-equilibrium approach could make the continuous tracking of physiological insulin fluctuations possible.

bioengineering↗

bioRxiv2021

Accelerated electron transfer in nanostructured electrodes improves the sensitivity of electrochemical biosensors

Electrochemical biosensors hold the exciting potential to integrate molecular detection with signal processing and wireless communication in a miniaturized, low-cost system. However, as electrochemical biosensors are miniaturized to the micron scale, their detection sensitivity degrades precipitously, thereby greatly reducing their utility in the context of molecular diagnostic applications. Studies have reported that nanostructured electrodes can greatly improve electrochemical biosensor sensitivity, but the underlying mechanism remains poorly understood, thus making it difficult to fully exploit this phenomenon to improve biosensor performance. In this work, we propose and experimentally validate a novel mechanism in which electron transfer is physically accelerated within nanostructured electrodes due to reduced charge screening, resulting in enhanced sensitivity. We show that this mechanism can be exploited to achieve up to 24-fold increase in signal and nearly four-fold lower limit-of-detection relative conventional planar electrodes. This accelerated electron transfer mechanism should prove broadly applicable for improving the performance of electrochemical biosensors.

bioengineering↗