Aptamer biosensor that can switch its structure upon target binding offers a powerful strategy for molecular detection. However, the process of converting an aptamer into a "structure-switching" biosensor is challenging and often relies on trial-and-error without established design principles. In this Sensor Issues, we examine a variety of design approaches for incorporating structure-switching functionality into existing aptamers, and provide thermodynamic analyses to highlight the variables that most strongly influence their performance. Finally, we also describe emerging efforts for incorporating the structure-switching functionality directly into the aptamer selection process.
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 micrometer scale, their signal-to-noise ratio degrades and reduces their utility for molecular diagnostics. Studies have reported that nanostructured electrodes can improve electrochemical biosensor signals, but since the underlying mechanism remains poorly understood, it remains difficult to fully exploit this phenomenon to improve biosensor performance. In this work, electrochemical aptamer biosensors on nanoporous electrode are optimized to achieve improved sensitivity by tuning pore size, probe density, and electrochemical measurement parameters. Further, a novel mechanism in which electron transfer is physically accelerated within nanostructured electrodes due to reduced charge screening, resulting in enhanced sensitivity is proposed and experimentally validated. In concert with the increased surface areas achieved with this platform, this newly identified effect can yield an up to 24-fold increase in signal level and nearly fourfold lower limit of detection relative to planar electrodes with the same footprint. Importantly, this strategy can be generalized to virtually any electrochemical aptamer sensor, enabling sensitive detection in applications where miniaturization is a necessity, and should likewise prove broadly applicable for improving electrochemical biosensor performance in general.
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 µM. We subsequently demonstrated the system’s generalizability by configuring it to detect cortisol with nanomolar sensitivity in undiluted human plasma. Both sensors achieved LODs orders of magnitude lower than theKDof the DBS element, highlighting the potential to achieve sensitive detection even when using aptamers with modest affinity.
Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still faces notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background. Here, we developed a Single-Molecule Colocalization Assay (SiMCA) that overcomes this problem by employing total internal reflection fluorescence microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized signals, we can eliminate the effects of background produced by non-specific binding of detection antibodies. Using TNF-α, we show that SiMCA achieves a three-fold lower limit of detection compared to conventional single-color assays and exhibits consistent performance for assays performed in complex specimens such as serum and 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.
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) becomes 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 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 receptor’s kinetic response to instantaneously quantify the changing ligand concentration. Importantly, by 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.
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