Strategies for the Voltammetric Detection of Loop-Mediated Isothermal Amplification.

Loop-mediated isothermal amplification (LAMP) is rapidly developing into an important tool for the point-of-use detection of pathogens for both clinical and environmental samples, largely due to its sensitivity, rapidity, and adaptability to portable devices. Many methods are used to monitor LAMP, but not all are amenable to point-of-use applications. Common methods such as fluorescence often require bulky equipment, whereas colorimetric and turbidimetric methods can lack sensitivity. Electrochemical biosensors are becoming increasingly important for these applications due to their potential for low cost, high sensitivity, and capacity for miniaturization into integrated devices. This review provides an overview of the use of voltammetric sensors for monitoring LAMP, with a specific focus on how electroactive species are used to interface between the biochemical products of the LAMP reaction and the voltammetric sensor. Various strategies for the voltammetric detection of DNA amplicons as well as pyrophosphate and protons released during LAMP are presented, ranging from direct DNA binding by electroactive species to the creative use of pyrophosphate-detecting aptamers and pH-sensitive oligonucleotide structures. Hurdles for adapting these devices to point-of-use applications are also discussed.

Engineering a disulfide-gated switch in streptavidin enables reversible binding without sacrificing binding affinity.

Although high affinity binding between streptavidin and biotin is widely exploited, the accompanying low rate of dissociation prevents its use in many applications where rapid ligand release is also required. To combine extremely tight and reversible binding, we have introduced disulfide bonds into opposite sides of a flexible loop critical for biotin binding, creating streptavidin muteins (M88 and M112) with novel disulfide-switchable binding properties. Crystal structures reveal how each disulfide exerts opposing effects on structure and function. Whereas the disulfide in M112 disrupts the closed conformation to increase koff, the disulfide in M88 stabilizes the closed conformation, decreasing koff 260-fold relative to streptavidin. The simple and efficient reduction of this disulfide increases koff 19,000-fold, thus creating a reversible redox-dependent switch with 70-fold faster dissociation kinetics than streptavidin. The facile control of disulfide formation in M88 will enable the development of many new applications requiring high affinity and reversible binding.