Biology subjects

Thompson, I. A. P.

Publications and source records attributed to Thompson, I. A. P..

9 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

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

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

A modular intramolecular triplex photo-switching motif that enables rapid and reversible control of aptamer binding activity

DNA switches that can change conformation in response to certain wavelengths of light could enable rapid and non-invasive control of chemical processes for a wide range of applications. However, most current photo-responsive DNA switches are limited either by irreversible switching or reversible switching with impractically slow kinetics. Here, we report the design of an intramolecular triplex photoswitch (TPS) design based on single-stranded DNA that undergoes rapid and reversible photoswitching between folded and unfolded states through isomerization of internal azobenzene modifications. After optimizing the performance of our photoswitch design, we used molecular dynamics (MD) simulations to reveal how individual azobenzenes contribute to the stabilization or destabilization of the triplex depending on their photoisomerization state. By coupling our TPS to an existing aptamer, we can reversibly modulate its binding affinity with less than 15 seconds of UV light exposure. We further demonstrate reproducible shifting in affinity over multiple cycles of UV and blue light irradiation without substantial photobleaching. Given that our TPS can introduce switching functionality to aptamers without manipulating the aptamer sequence itself, we believe our design methodology should offer a versatile means for integrating photo-responsive properties into DNA nanostructures.

bioengineering↗

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↗

bioRxiv2020

Rational Design of Aptamer Switches with Programmable pH Response

Aptamer switches that respond sensitively to local variations in pH could enable the development of molecular devices that greatly enhance diagnostic accuracy and therapeutic efficacy. Previous design strategies relied on inserting a known pH-sensitive DNA motif into the aptamer structure. Unfortunately, their performance was ultimately governed by the intrinsic pH-response of the inserted motif and could not be readily tuned to operate across arbitrary pH ranges. In this work, we present a general methodology for converting virtually any aptamer into a pH-responsive switch that can be modulated to undergo a strong change in affinity - in acidic, neutral, or alkaline conditions. This was achieved by inserting two orthogonal motifs that can be manipulated in parallel to tune sensitivity to different pH conditions without altering the core sequence of the aptamer itself. Using a single ATP aptamer, we achieve pH-dependent target binding and release under diverse pH conditions, producing as much as 1,000-fold pH-induced change in affinity. Importantly, we also demonstrate the novel capability to predictably engineer aptamer switches that only exhibit strong target affinity within a narrowly defined pH range. Our approach should offer a highly generalizable strategy for integrating pH-responsiveness into molecular devices.

bioengineering↗

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↗