Rachel A. McKendry

Multiple label-free biodetection and quantitative DNA-binding assays on a nanomechanical cantilever array

We report a microarray of cantilevers to detect multiple unlabeled biomolecules simultaneously at nanomolar concentrations within minutes. Ligand-receptor binding interactions such as DNA hybridization or protein recognition occurring on microfabricated silicon cantilevers generate nanomechanical bending, which is detected optically in situ. Differential measurements including reference cantilevers on an array of eight sensors can sequence-specifically detect unlabeled DNA targets in 80-fold excess of nonmatching DNA as a background and discriminate 3′ and 5′ overhangs. Our experiments suggest that the nanomechanical motion originates from predominantly steric hindrance effects and depends on the concentration of DNA molecules in solution. We show that cantilever arrays can be used to investigate the thermodynamics of biomolecular interactions mechanically, and we have found that the specificity of the reaction on a cantilever is consistent with solution data. Hence cantilever arrays permit multiple binding assays in parallel and can detect femtomoles of DNA on the cantilever at a DNA concentration in solution of 75 nM.

Rapid and label-free nanomechanical detection of biomarker transcripts in human RNA

The availability of entire genome sequences has triggered the development of microarrays for clinical diagnostics that measure the expression levels of specific genes. Methods that involve labelling can achieve picomolar detection sensitivity, but they are costly, labour-intensive and time-consuming. Moreover, target amplification or biochemical labelling can influence the original signal. We have improved the biosensitivity of label-free cantilever-array sensors by orders of magnitude to detect mRNA biomarker candidates in total cellular RNA. Differential gene expression of the gene 1-8U, a potential marker for cancer progression or viral infections, has been observed in a complex background. The measurements provide results within minutes at the picomolar level without target amplification, and are sensitive to base mismatches. This qualifies the technology as a rapid method to validate biomarkers that reveal disease risk, disease progression or therapy response. We foreseee cantilever arrays being used as a tool to evaluate treatment response efficacy for personalized medical diagnostics.

Nanomechanical detection of antibiotic mucopeptide binding in a model for superbug drug resistance

The alarming growth of the antibiotic-resistant superbugs methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) is driving the development of new technologies to investigate antibiotics and their modes of action. We report the label-free detection of vancomycin binding to bacterial cell wall precursor analogues (mucopeptides) on cantilever arrays, with 10 nM sensitivity and at clinically relevant concentrations in blood serum. Differential measurements have quantified binding constants for vancomycin-sensitive and vancomycin-resistant mucopeptide analogues. Moreover, by systematically modifying the mucopeptide density we gain new insights into the origin of surface stress. We propose that stress is a product of a local chemical binding factor and a geometrical factor describing the mechanical connectivity of regions activated by local binding in terms of a percolation process. Our findings place BioMEMS devices in a new class of percolative systems. The percolation concept will underpin the design of devices and coatings to significantly lower the drug detection limit and may also have an impact on our understanding of antibiotic drug action in bacteria.

Chiral discrimination by chemical force microscopy

Chirality is a fundamental aspect of chemical biology, and is of central importance in pharmacology. Consequently there is great interest in techniques for distinguishing between different chiral forms of a compound. Chemical force microscopy is a technique that combines chemical discrimination with atomic force microscopy by chemical derivatization of the scanning probe tip. It has been applied to the study of hydrophobic and hydrophilic interactions, the binding between biotin and streptavidin, and between DNA bases. Here we report on the use of chemical force microscopy to discriminate between chiral molecules. Using chiral molecules attached to the probe tip, we can distinguish the two enantiomers of mandelic acid arrayed on a surface, through differences in both the adhesion forces and the frictional forces measured by the probe.

A Polarised Population of Dynamic Microtubules Mediates Homeostatic Length Control in Animal Cells

An analysis of cells grown on micro-patterned lines, and of cells during zebrafish development, identifies a population of microtubules that align along the long axis of cells to mediate homeostatic length control.

Differential stress induced by thiol adsorption on facetted nanocrystals

Polycrystalline gold films coated with thiol-based self-assembled monolayers (SAM) form the basis of a wide range of nanomechanical sensor platforms. The detection of adsorbates with such devices relies on the transmission of mechanical forces, which is mediated by chemically derived stress at the organic-inorganic interface. Here, we show that the structure of a single 300-nm-diameter facetted gold nanocrystal, measured with coherent X-ray diffraction, changes profoundly after the adsorption of one of the simplest SAM-forming organic molecules. On self-assembly of propane thiol, the crystals flat facets contract radially inwards relative to its spherical regions. Finite-element modelling indicates that this geometry change requires large stresses that are comparable to those observed in cantilever measurements. The large magnitude and slow kinetics of the contraction can be explained by an intermixed gold-sulphur layer that has recently been identified crystallographically. Our results illustrate the importance of crystal edges and grain boundaries in interface chemistry and have broad implications for the application of thiol-based SAMs, ranging from nanomechanical sensors to coating technologies.

Frequency modulation atomic force microscopy: a dynamic measurement technique for biological systems

Frequency modulation atomic force microscopy (FM-AFM) has been modified to operate in a liquid environment within an atomic force microscope specifically designed for investigating biological samples. We demonstrate the applicability of FM-AFM to biological samples using the spectroscopy mode to measure the unbinding forces of a single receptor–ligand (biotin–avidin) interaction. We show that quantitative adhesion force measurements can only be obtained provided certain modifications are made to the existing theory, which is used to convert the detected frequency shifts to an interaction force. Quantitative force measurements revealed that the unbinding forces for the biotin–avidin interaction were greater than those reported in previous studies. This finding was due to the use of high average tip velocities, which were calculated to be two orders of magnitude greater than those typically used in unbinding receptor–ligand experiments. This study therefore highlights the potential use of FM-AFM to study a range of biological systems, including living cells and/or single biomolecule interactions.

Detecting Disease Outbreaks in Mass Gatherings Using Internet Data

Background Mass gatherings, such as music festivals and religious events, pose a health care challenge because of the risk of transmission of communicable diseases. This is exacerbated by the fact that participants disperse soon after the gathering, potentially spreading disease within their communities. The dispersion of participants also poses a challenge for traditional surveillance methods. The ubiquitous use of the Internet may enable the detection of disease outbreaks through analysis of data generated by users during events and shortly thereafter. Objective The intent of the study was to develop algorithms that can alert to possible outbreaks of communicable diseases from Internet data, specifically Twitter and search engine queries. Methods We extracted all Twitter postings and queries made to the Bing search engine by users who repeatedly mentioned one of nine major music festivals held in the United Kingdom and one religious event (the Hajj in Mecca) during 2012, for a period of 30 days and after each festival. We analyzed these data using three methods, two of which compared words associated with disease symptoms before and after the time of the festival, and one that compared the frequency of these words with those of other users in the United Kingdom in the days following the festivals. Results The data comprised, on average, 7.5 million tweets made by 12,163 users, and 32,143 queries made by 1756 users from each festival. Our methods indicated the statistically significant appearance of a disease symptom in two of the nine festivals. For example, cough was detected at higher than expected levels following the Wakestock festival. Statistically significant agreement (chi-square test, P<.01) between methods and across data sources was found where a statistically significant symptom was detected. Anecdotal evidence suggests that symptoms detected are indeed indicative of a disease that some users attributed to being at the festival. Conclusions Our work shows the feasibility of creating a public health surveillance system for mass gatherings based on Internet data. The use of multiple data sources and analysis methods was found to be advantageous for rejecting false positives. Further studies are required in order to validate our findings with data from public health authorities.

Who Owns the Data? Open Data for Healthcare.

Research on large shared medical datasets and data-driven research are gaining fast momentum and provide major opportunities for improving health systems as well as individual care. Such open data can shed light on the causes of disease and effects of treatment, including adverse reactions side-effects of treatments, while also facilitating analyses tailored to an individual’s characteristics, known as personalized or “stratified medicine.” Developments, such as crowdsourcing, participatory surveillance, and individuals pledging to become “data donors” and the “quantified self” movement (where citizens share data through mobile device-connected technologies), have great potential to contribute to our knowledge of disease, improving diagnostics, and delivery of healthcare and treatment. There is not only a great potential but also major concerns over privacy, confidentiality, and control of data about individuals once it is shared. Issues, such as user trust, data privacy, transparency over the control of data ownership, and the implications of data analytics for personal privacy with potentially intrusive inferences, are becoming increasingly scrutinized at national and international levels. This can be seen in the recent backlash over the proposed implementation of care.data, which enables individuals’ NHS data to be linked, retained, and shared for other uses, such as research and, more controversially, with businesses for commercial exploitation. By way of contrast, through increasing popularity of social media, GPS-enabled mobile apps and tracking/wearable devices, the IT industry and MedTech giants are pursuing new projects without clear public and policy discussion about ownership and responsibility for user-generated data. In the absence of transparent regulation, this paper addresses the opportunities of Big Data in healthcare together with issues of responsibility and accountability. It also aims to pave the way for public policy to support a balanced agenda that safeguards personal information while enabling the use of data to improve public health.

Chemical Force Microscopy with Active Enzymes

The adhesion forces have been measured between an atomic force microscope tip derivatized with an active enzyme, shikimate kinase, and an ATP mimic immobilized on a gold surface. Experiments with competitive binding of other ligands in solution show that the observed adhesion forces arise predominantly from specific interactions between the immobilized enzyme and surface-bound adenine derivative. These experiments represent a step in the development of a screening methodology based upon chemical force microscopy.