The results of this study indicate that US may provide important prognostic information concerning fracture healing after unreamed tibial nailing, upon which subsequent treatment can be based.

Probing individual chemical reactions is key to mapping reaction pathways. Trace analysis of sub-kDa reactants and products is obfuscated by labels, however, as reaction kinetics are inevitably perturbed. The thiol-disulfide exchange reaction is of specific interest as it has many applications in nanotechnology and in nature. Redox cycling of single thiols and disulfides has been unresolvable due to a number of technological limitations, such as an inability to discriminate the leaving group. Here, we demonstrate detection of single-molecule thiol-disulfide exchange using a label-free optoplasmonic sensor. We quantify repeated reactions between sub-kDa thiolated species in real time and at concentrations down to 100’s of attomolar. A unique sensing modality is featured in our measurements, enabling the observation of single disulfide reaction kinetics and pathways on a plasmonic nanoparticle surface. Our technique paves the way towards characterising molecules in terms of their charge, oxidation state, and chirality via optoplasmonics.

Label-free optical sensor systems have emerged that exhibit extraordinary sensitivity for detecting physical, chemical, and biological entities at the micro/nanoscale. Particularly exciting is the detection and analysis of molecules, on miniature optical devices that have many possible applications in health, environment, and security. These micro- and nanosensors have now reached a sensitivity level that allows for the detection and analysis of even single molecules. Their small size enables an exceedingly high sensitivity, and the application of quantum optical measurement techniques can allow the classical limits of detection to be approached or surpassed. The new class of label-free micro- and nanosensors allows dynamic processes at the single-molecule level to be observed directly with light. By virtue of their small interaction length, these micro- and nanosensors probe light-matter interactions over a dynamic range often inaccessible by other optical techniques. For researchers entering this rapidly advancing field of single-molecule micro- and nanosensors, there is an urgent need for a timely review that covers the most recent developments and that identifies the most exciting opportunities. The focus here is to provide a summary of the recent techniques that have either demonstrated label-free single-molecule detection or claim single-molecule sensitivity.

Glycogen phosphorylase (GlyP) was the first allosteric enzyme to be described. Yet, the precise dynamic changes in solution phase structure and stability that underpin functional regulation have remained elusive. We have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. This enabled measurements of the solution phase local structural dynamics involved in allosteric regulation of GlyP. The sensitivity of these measurements discerned that the 250's loop is natively disordered in the apo T-state, adopting a more ordered conformation in the active state. The quantitative change in stability of the 280s loop is identified, providing the first direct evidence of the entropic switch that sterically regulates substrate access to the active site. Here, we quantify GlyP structural dynamics in solution, describing correlated changes in structure in the activated (pSer14) and inhibited (glucose-6-phosphate bound) forms of the enzyme. Significance StatementWe have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. Measurements of glycogen phosphorylase quantify the solution phase stability of local structure at near-amino acid structural resolution and with no appreciable lower limit of stability. This uncovered the highly-resolved local alterations in stability we provide direct evidence of the entropic mechanism by which access to the active site is gated by the 280s loop. Results Fully-automated and flexible pulse-labeling hydrogen/deuterium-exchange mass spectrometry with millisecond precisionOur goal was to quantify the structural switch in solution, along with other coincident dynamic changes in structure between activated (pSer14) and inhibited (glucose-6-phosphate bound) forms of GlyP. To enable this, we developed a fully automated broadband bottom-up HDX-MS approach capable of accurate quantitative assessment of peptide stability. The newly developed ms2min system design ( Figure 1A) offers certain significant advances over the other designs previously employed. Notably, it allows fully-automated, software-selection of mixing times over six orders of magnitude (ms to hours) with 1 ms time resolution, flexible labeling temperature control (0 -25 C), quench temperature control (0 C), on-line connection for 'bottom-up' workflows, two-way communication for reciprocal control of labeling, washing, digestion, desalting and chromatography, automated digestion column wash injection, intercalated blank injections and sample list scheduling of multiple runs. Precision of D-labeling.To evaluate the measurement variability and labeling precision of the system we determined the repeatability of the quench-flow labeling method. On three separate days we measured the D uptake of 50, 70 and 150 replicates of Bradykinin and Leucine enkephalin at 100 ms mixing time. The short labeling period close to the limit of quantification, wa...