The application of single molecule nanopore sensing for quantitative analysis

Nanopore-based sensors typically work by monitoring transient pulses in conductance via current-time traces as molecules translocate through the nanopore. The unique property of being able to monitor single molecules gives nanopore sensors the potential as quantitative sensors based on the counting of single molecules. This review provides an overview of the concepts and fabrication of nanopore sensors as well as nanopore sensing with a view toward using nanopore sensors for quantitative analysis. We first introduce the classification of nanopores and highlight their applications in molecular identification with some pioneering studies. The review then shifts focus to recent strategies to extend nanopore sensors to devices that can rapidly and accurately quantify the amount of an analyte of interest. Finally, future prospects are provided and briefly discussed. The aim of this review is to aid in understanding recent advances, challenges, and prospects for nanopore sensors for quantitative analysis

The application of personal glucose meters as universal point-of-care diagnostic tools

Personal glucose meters (PGMs) have been used for the measurement of blood glucose for decades now such that they have become the most used analytical method in the world. They are also well placed to be repurposed for point-of-care testing of other analytes as they are inexpensive, portable and quantitative. Efforts to repurpose PGMs for the detection of any analyte at the point-of-care have been one focus of biosensor research for several years now with a number of successful efforts in the detection of a wide range of analytes. This article reviews the published methods to repurpose a PGM to detect analytes other than glucose, and analyses the potential and the challenges to be overcome in developing a PGM-based biosensor and bring it to market

FRET theoretical predictions concerning freely diffusive dyes inside spherical container: how to choose the best pair?

FRET has been massively used to see if biomolecules were bounded or not by labelling both biomolecules by one dye of a FRET pair. This should give a digital answer to the question (fluorescence of the acceptor: high FRET efficency: molecules associated, fluorescence of the donor: low FRET efficency: molecules dissociated). This has been used, inter alia, at the single-molecule scale in containers, such as liposomes. One perspective of the field is to reduce the container’s size to study the effect of confinement on binding. The problem is that if the two dyes are encapsulated inside a small liposome, they could have a significant probability to be close one from the other one (and thus to undergo a high FRET efficiency event without binding). This is why we suggest here a theoretical model which gives mean FRET efficiency as a function of liposome radius (the model applies to any spherical container) and Förster radius to help the experimentalist to choose their experimental set-up. Besides, the influence of side effect on mean FRET efficiency has been studied as well. We show here that if this “background FRET” is most of the time non-quantitative, it can remain significant and which makes data analysis trickier. We could show as well that if this background FRET obviously increases when liposome radius decreases, this variation was lower than the one which could be expected because of side effect. We show as well the FRET efficiency function distribution which let the experimentalist know the probability to get one FRET efficiency value

Electrochemical cellular biosensor combined with fluorescence microscopy: An investigation of subtle changes in response of cells to a drug

This study presents the combination of electrochemical cell based biosensor and fluorescence microscopy on a single platform to track biomolecular processes contributing to the morphology changes of the cells. Initial experiments demonstrate that using interdigitated electrodes where the gold microfingers were comparable in width and spacing to a single cell have the optimal sensitivity in the final electrochemical cell based-sensing device. This was determined by measuring the cell index, based on the impedance analysis of bare and cell-covered microelectrodes. The fabrication of the electrodes on a glass substrate enabled the capture of high-resolution fluorescence microscopy images of single cells and related intracellular calcium release inside the HeLa cells via a window incorporated into the gold microelectrode design. As an illustration of the enhanced capability of the combined approach over traditional impedance cellular assay, the opto-electric assay was utilized as a functional readout for G protein couple receptor activation. The simultaneous examinations of cells stimulated with histamine demonstrated an association between time courses of changes in cytosolic calcium concentration and reductions in cell-cell adhesions

Optical Nanopore Sensors for Quantitative Analysis

Nanopore sensors have received significant interest for the detection of clinically important biomarkers with single-molecule resolution. These sensors typically operate by detecting changes in the ionic current through a nanopore due to the translocation of an analyte. Recently, there has been interest in developing optical readout strategies for nanopore sensors for quantitative analysis. This is because they can utilize wide-field microscopy to independently monitor many nanopores within a high-density array. This significantly increases the amount of statistics that can be obtained, thus enabling the analysis of analytes present at ultralow concentrations. Here, we review the use of optical nanopore sensing strategies for quantitative analysis. We discuss optical nanopore sensing assays that have been developed to detect clinically relevant biomarkers, the potential for multiplexing such measurements, and techniques to fabricate high density arrays of nanopores with a view toward the use of these devices for clinical applications

Spiers Memorial Lecture. Next generation nanoelectrochemistry: the fundamental advances needed for applications

Nanoelectrochemistry, where electrochemical processes are controlled and investigated with nanoscale resolution, is gaining more and more attention because of the many potential applications in energy and sensing and the fact that there is much to learn about fundamental electrochemical processes when we explore them at the nanoscale. The development of instrumental methods that can explore the heterogeneity of electrochemistry occurring across an electrode surface, monitoring single molecules or many single nanoparticles on a surface simultaneously, have been pivotal in giving us new insights into nanoscale electrochemistry. Equally important has been the ability to synthesise or fabricate nanoscale entities with a high degree of control that allows us to develop nanoscale devices. Central to the latter has been the incredible advances in nanomaterial synthesis where electrode materials with atomic control over electrochemically active sites can be achieved. After introducing nanoelectrochemistry, this paper focuses on recent developments in two major application areas of nanoelectrochemistry; electrocatalysis and using single entities in sensing. Discussion of the developments in these two application fields highlights some of the advances in the fundamental understanding of nanoelectrochemical systems really driving these applications forward. Looking into our nanocrystal ball, this paper then highlights: the need to understand the impact of nanoconfinement on electrochemical processes, the need to measure many single entities, the need to develop more sophisticated ways of treating the potentially large data sets from measuring such many single entities, the need for more new methods for characterising nanoelectrochemical systems as they operate and the need for material synthesis to become more reproducible as well as possess more nanoscale control

Understanding and modelling the magnitude of the change in current of nanopore sensors

Nanopores are promising sensing devices that can be used for the detection of analytes at the single molecule level. It is of importance to understand and model the current response of a nanopore sensor for improving the sensitivity of the sensor, a better interpretation of the behaviours of different analytes in confined nanoscale spaces, and quantitative analysis of the properties of the targets. The current response of a nanopore sensor, usually called a resistive pulse, results from the change in nanopore resistance when an analyte translocates through the nanopore. This article reviews the theoretical models used for the calculation of the resistance of the nanopore, and the corresponding change in nanopore resistance due to a translocation event. Models focus on the resistance of the pore cavity region and the access region of the nanopore. The influence of the sizes, shapes and surface charges of the translocating species and the nanopore, as well as the trajectory that the analyte follows are also discussed. This review aims to give a general guidance to the audience for understanding the current response of a nanopore sensor and the application of this class of sensor to a broad range of species with the theoretical models

Building a total internal reflection microscope (TIRF) with active stabilization (feedback SMLM)

The data quality of high-resolution imaging can be markedly improved with active stabilization, which is based on feedback loops within the microscope that maintain the sample in the same location throughout the experiment. The purpose is to provide a highly accurate focus lock, therefore eliminating drift and improving localization precision. Here, we describe a step-by-step protocol for building a total internal reflection microscope combined with the feedback loops necessary for sample and detection stabilization, which we routinely use in single-molecule localization microscopy (SMLM). The performance of the final microscope with feedback loops, called feedback SMLM, has previously been described. We demonstrate how to build a replica of our system and include a list of the necessary optical components, tips, and an alignment strategy

A modular design strategy to integrate mechanotransduction concepts in scaffold-based bone tissue engineering

Repair or regeneration of load-bearing bones has long been an incentive for the tissue engineering community to develop a plethora of synthetic bone scaffolds. Despite the key role of physical forces and the mechanical environment in bone regeneration, the mechanotransduction concept has rarely been incorporated in structural design of bone tissue scaffolds, particularly those made of bioactive materials such as hydrogels and bioceramics. Herein, we introduce a modular design strategy to fabricate a load bearing device that can support a wide range of hydrogel- and ceramic-based scaffolds against complex in-vivo loading conditions to induce desirable mechanical strains for bone regeneration within the scaffolds. The device is comprised of a fenestrated polymeric shell and ceramic structural pillars arranged in a sophisticated configuration to provide ample internal space for the scaffold, also enabling it to purposely regulate the levels of strains and stresses within the scaffolds. Utilizing this top-down design approach, we demonstrate that the failure load of alginate hydrogels increases 3200-fold in compression, 300-fold in shear and 75-fold in impact, achieving the values that enable them to withstand physiological loads in weight-bearing sites, while allowing generation of osteoinductive strains (i.e., 0.2-0.4%) in the hydrogel. This modular design approach opens a broad range of opportunities to utilize various bioactive but mechanically weak scaffolds for the treatment of load-bearing defects and exploiting mechanobiology strategies to improve bone regeneration