A high-dimensional microfluidic approach for selection of aptamers with programmable binding affinities.

Aptamers are being applied as affinity reagents in analytical applications owing to their high stability, compact size and amenability to chemical modification. Generating aptamers with different binding affinities is desirable, but systematic evolution of ligands by exponential enrichment (SELEX), the standard for aptamer generation, is unable to quantitatively produce aptamers with desired binding affinities and requires multiple rounds of selection to eliminate false-positive hits. Here we introduce Pro-SELEX, an approach for the rapid discovery of aptamers with precisely defined binding affinities that combines efficient particle display, high-performance microfluidic sorting and high-content bioinformatics. Using the Pro-SELEX workflow, we were able to investigate the binding performance of individual aptamer candidates under different selective pressures in a single round of selection. Using human myeloperoxidase as a target, we demonstrate that aptamers with dissociation constants spanning a 20-fold range of affinities can be identified within one round of Pro-SELEX.

Biomolecular sensors for advanced physiological monitoring.

Body-based biomolecular sensing systems, including wearable, implantable and consumable sensors allow comprehensive health-related monitoring. Glucose sensors have long dominated wearable bioanalysis applications owing to their robust continuous detection of glucose, which has not yet been achieved for other biomarkers. However, access to diverse biological fluids and the development of reagentless sensing approaches may enable the design of body-based sensing systems for various analytes. Importantly, enhancing the selectivity and sensitivity of biomolecular sensors is essential for biomarker detection in complex physiological conditions. In this Review, we discuss approaches for the signal amplification of biomolecular sensors, including techniques to overcome Debye and mass transport limitations, and selectivity improvement, such as the integration of artificial affinity recognition elements. We highlight reagentless sensing approaches that can enable sequential real-time measurements, for example, the implementation of thin-film transistors in wearable devices. In addition to sensor construction, careful consideration of physical, psychological and security concerns related to body-based sensor integration is required to ensure that the transition from the laboratory to the human body is as seamless as possible.

Electrochemical Detection of Borrelia burgdorferi Using a Biomimetic Flow Cell System.

Lyme disease, caused by infection with pathogenic Borrelia bacteria, has emerged as a pervasive illness throughout North America and many other regions of the world in recent years, owing in part to climate-mediated habitat expansion of the tick vectors. Standard diagnostic testing has remained largely unchanged over the past several decades and is indirect, relying on detection of antibodies against the Borrelia pathogen, rather than detection of the pathogen itself. The development of new rapid, point-of-care tests for Lyme disease that directly detects the pathogen could drastically improve patient health by enabling faster and more frequent testing that could better inform patient treatment. Here, we describe a proof-of-concept electrochemical sensing approach to the detection of the Lyme disease-causing bacteria, which utilizes a biomimetic electrode to interact with the Borrelia bacteria that induce impedance alterations. In addition, the catch-bond mechanism between bacterial BBK32 protein and human fibronectin protein, which exhibits improved bond strength with increased tensile force, is tested within an electrochemical injection flow-cell to achieve Borrelia detection under shear stress.

Monitoring Cardiac Biomarkers with Aptamer-Based Molecular Pendulum Sensors.

Reagent-free electronic biosensors capable of analyzing disease markers directly in unprocessed body fluids will enable the development of simple & affordable devices for personalized healthcare monitoring. Here we report a powerful and versatile nucleic acid-based reagent-free electronic sensing system. The signal transduction is based on the kinetics of an electrode-tethered molecular pendulum-a rigid double stranded DNA with one of the strands displaying an analyte-binding aptamer and the other featuring a redox probe-that exhibits field-induced transport modulated by receptor occupancy. Using chronoamperometry, which enables the sensor to circumvent the conventional Debye length limitation, the binding of an analyte can be monitored as these species increase the hydrodynamic drag. The sensing platform demonstrates a low femtomolar quantification limit and minimal cross-reactivity in analyzing cardiac biomarkers in whole blood collected from patients with chronic heart failure.

Capillary-Assisted Molecular Pendulum Bioanalysis.

The development of robust biosensing strategies that can be easily implemented in everyday life remains a challenge for the future of modern biosensor research. While several reagentless approaches have attempted to address this challenge, they often achieve user-friendliness through sacrificing sensitivity or universality. While acceptable for certain applications, these trade-offs hinder the widespread adoption of reagentless biosensing technologies. Here, we report a novel approach to reagentless biosensing that achieves high sensitivity, rapid detection, and universality using the SARS-CoV-2 virus as a model target. Universality is achieved by using nanoscale molecular pendulums, which enables reagentless electrochemical biosensing through a variable antibody recognition element. Enhanced sensitivity and rapid detection are accomplished by incorporating the coffee-ring phenomenon into the sensing scheme, allowing for target preconcentration on a ring-shaped electrode. Using this approach, we obtained limits of detection of 1 fg/mL and 20 copies/mL for the SARS-CoV-2 nucleoproteins and viral particles, respectively. In addition, clinical sample analysis showed excellent agreement with Ct values from PCR-positive SARS-CoV-2 patients.

Nanostructured Cu2O Synthesized via Bipolar Electrochemistry.

Cuprous oxide (Cu2O) was synthesized for the first time via an open bipolar electrochemistry (BPE) approach and characterized in parallel with the commercially available material. As compared to the reference, Cu2O formed through a BPE reaction demonstrated a decrease in particle size; an increase in photocurrent; more efficient light scavenging; and structure-correlated changes in the flat band potential and charge carrier concentration. More importantly, as-synthesized oxides were all phase-pure, defect-free, and had an average crystallite size of 20 nm. Ultimately, this study demonstrates the impact of reaction conditions (e.g., applied potential, reaction time) on structure, morphology, surface chemistry, and photo-electrochemical activity of semiconducting oxides, and at the same time, the ability to maintain a green synthetic protocol and potentially create a scalable product. In the proposed BPE synthesis, we introduced a common food supplement (potassium gluconate) as a reducing and complexing agent, and as an electrolyte, allowing us to replace the more harmful reactants that are conventionally used in Cu2O production. In addition, in the BPE process very corrosive reactants, such as hydroxides and metal precursors (required for synthesis of oxides), are generated in situ in stoichiometric quantity, providing an alternative methodology to generate various nanostructured materials in high yields under mild conditions.