Rapid, low-cost, species-specific diagnosis, based upon DNA testing, is becoming important in the treatment of patients with infectious diseases. Here, we demonstrate an innovation that uses origami ...to enable multiplexed, sensitive assays that rival polymerase chain reactions (PCR) laboratory assays and provide high-quality, fast precision diagnostics for malaria. The paper-based microfluidic technology proposed here combines vertical flow sample-processing steps, including paper folding for whole-blood sample preparation, with an isothermal amplification and a lateral flow detection, incorporating a simple visualization system. Studies were performed in village schools in Uganda with individual diagnoses being completed in <50 min (faster than the standard laboratory-based PCR). The tests, which enabled the diagnosis of malaria species in patients from a finger prick of whole blood, were both highly sensitive and specific, detecting malaria in 98% of infected individuals in a double-blind first-in-human study. Our method was more sensitive than other field-based, benchmark techniques, including optical microscopy and industry standard rapid immunodiagnostic tests, both performed by experienced local healthcare teams (which detected malaria in 86% and 83% of cases, respectively). All assays were independently validated using a real-time double-blinded reference PCR assay. We not only demonstrate that advanced, low-cost DNA-based sensors can be implemented in underserved communities at the point of need but also highlight the challenges associated with developing and implementing new diagnostic technologies in the field, without access to laboratories or infrastructure.
Color filters based upon nanostructured metals have garnered significant interest in recent years, having been positioned as alternatives to the organic dye-based filters which provide color ...selectivity in image sensors, as nonfading “printing” technologies for producing images with nanometer pixel resolution, and as ultra-high-resolution, small foot-print optical storage and encoding solutions. Here, we demonstrate a plasmonic filter set with polarization-switchable color properties, based upon arrays of asymmetric cross-shaped nanoapertures in an aluminum thin-film. Acting as individual color-emitting nanopixels, the plasmonic cavity-apertures have dual-color selectivity, transmitting one of two visible colors, controlled by the polarization of the white light incident on the rear of the pixel and tuned by varying the critical dimensions of the geometry and periodicity of the array. This structural approach to switchable optical filtering enables a single nanoaperture to encode two information states within the same physical nanoaperture, an attribute we use here to create micro image displays containing duality in their optical information states.
Cell response to matrix rigidity has been explained by the mechanical properties of the actin-talin-integrin-fibronectin clutch. Here the molecular clutch model is extended to account for cell ...interactions with purely viscous surfaces (i.e., without an elastic component). Supported lipid bilayers present an idealized and controllable system through which to study this concept. Using lipids of different diffusion coefficients, the mobility (i.e., surface viscosity) of the presented ligands (in this case RGD) was altered by an order of magnitude. Cell size and cytoskeletal organization were proportional to viscosity. Furthermore, there was a higher number of focal adhesions and a higher phosphorylation of FAK on less-mobile (more-viscous) surfaces. Actin retrograde flow, an indicator of the force exerted on surfaces, was also seen to be faster on more mobile surfaces. This has consequential effects on downstream molecules; the mechanosensitive YAP protein localized to the nucleus more on less-mobile (more-viscous) surfaces and differentiation of myoblast cells was enhanced on higher viscosity. This behavior was explained within the framework of the molecular clutch model, with lower viscosity leading to a low force loading rate, preventing the exposure of mechanosensitive proteins, and with a higher viscosity causing a higher force loading rate exposing these sites, activating downstream pathways. Consequently, the understanding of how viscosity (regardless of matrix stiffness) influences cell response adds a further tool to engineer materials that control cell behavior.
Despite over 2 million papers published on cancer so far, malignancy still remains a puzzlingly complex disease with overall low survival rates. Expanding our knowledge of the molecular mechanisms of ...malignancy and of resistance to therapy is crucial in guiding the successful design of anti-cancer drugs and new point-of-care diagnostics. The up-and-coming microfluidic Lab-on-a-Chip (LOC) technology and micro-total analysis systems (μTAS) are arguably the most promising platforms to address the inherent complexity of cellular systems with massive experimental parallelization and 4D analysis on a single cell level. This review discusses the emerging applications of microfluidic technologies and their advantages for cancer biology and experimental oncology. We also summarize the recent advances in miniaturized systems to study cancer cell microenvironment, cancer cytomics, and real-time (4D) pharmacological screening. Microfabricated systems, such as cell microarrays, together with on-chip label-less cytometry, and micro-sorting technologies, are all highlighted with the view of describing their potential applications in pharmacological screening, drug discovery, and clinical oncology. It is envisaged that microfluidic solutions may well represent the platform of choice for next generation
in vitro cancer models.
The detection of changes in nucleic acid sequences at specific sites remains a critical challenge in epigenetics, diagnostics and therapeutics. To date, such assays often require extensive time, ...expertise and infrastructure for their implementation, limiting their application in clinical settings. Here we demonstrate a generalizable method, named Specific Terminal Mediated Polymerase Chain Reaction (STEM-PCR) for the detection of DNA modifications at specific sites, in a similar way as DNA sequencing techniques, but using simple and widely accessible PCR-based workflows. We apply the technique to both for site-specific methylation and co-methylation analysis, importantly using a bisulfite-free process - so providing an ease of sample processing coupled with a sensitivity 20-fold better than current gold-standard techniques. To demonstrate the clinical applicability through the detection of single base mutations with high sensitivity and no-cross reaction with the wild-type background, we show the bisulfite-free detection of SEPTIN9 and SFRP2 gene methylation in patients (as key biomarkers in the prognosis and diagnosis of tumours).
•Au@Ag core-shell nanoparticles for highly sensitivite SERS assay.•Fabrication of Au@Ag-pLL on glass nanofibrous paper as SERS substrate.•A paper-based SERS nanosensor for the sensitive detection of ...methamphetamine in sewage.•Evaluation of community-wide illicit drugs consumption ifor wastewater-based epidemiology.
Wastewater-based epidemiology (WBE) is a powerful technique for monitoring illicit drugs of abuse in the community. Here, we report upon a surface-enhanced Raman spectroscopy (SERS) sensor for the sensitive and selective detection of methamphetamine based upon the assembly of noble metal core-shell nanoparticles on a bespoke glassy nanofibrous electrospun paper matrix. The hierarchical structure of the fibrous paper, modified with the synthesized Au@Ag core-shells (Au@Ag) gave strong SERS signalling, enabling us to evaluate the community-wide prevalence of methamphetamine in wastewater treatment plants within Beijing. We show that, when normalized for the daily flow of the wastewater treatment plants and for population density, higher mass loads of drugs are generally found in sewage influent from urban areas, implying greater local methamphetamine usage than that in less populated areas. The user-friendly and disposable paper sensors demonstrate the applicability of rapid on-site illicit drug detection, illustrating the application to wastewater-based epidemiology, which has the potential to inform government agencies regarding societal interventions.
We report a rapid “sample-to-answer” platform that can be used for the quantitative monitoring of genetic biomarkers within communities through the analysis of wastewater. The assay is based on the ...loop-mediated isothermal amplification (LAMP) of nucleic acid biomarkers and shows for the first time the ability to rapidly quantify human-specific mitochondrial DNA (mtDNA) from raw untreated wastewater samples. mtDNA provides a model population biomarker associated with carcinogenesis including breast, renal and gastric cancers. To enable a sample-to-answer, field-based technology, we integrated a filter to remove solid impurities and perform DNA extraction and enrichment into a low cost lateral flow-based test. We demonstrated mtDNA detection over seven consecutive days, achieving a limit of detection of 40 copies of human genomic DNA per reaction volume. The assay can be performed at the site of sample collection, with minimal user intervention, yielding results within 45 min and providing a method to monitor public health from wastewater.
Spatially offset Raman spectroscopy is integrated with a fiber‐coupled spatial heterodyne spectrometer to collect Raman spectra from deep within opaque or scattering materials. The method, named ...spatial heterodyne offset Raman spectroscopy generates a wavenumber‐dependent spatial phase shift of the optical signal as a “spectral” image on a charge‐coupled device detector. The image can be readily processed from the spatial domain using a single, simple, and “on‐the‐fly” Fourier transform to generate Raman spectra, in the frequency domain. By collecting all of the spatially offset Raman scattered photons that pass through the microscope's collection objective lens, the methodology gives an improvement in the Raman sensitivity by an order of magnitude. The instrumentation is both mechanically robust and “movement‐free,” which when coupled with the associated advantages of highly efficient signal collection and ease of data processing, enables rapid interfacial analysis of complex constructs based on established biomaterials models.
Spatial heterodyne offset Raman spectroscopy is a technique which can be implemented as a simple, compact instrument in order to collect undistorted Raman spectra from deep within biomaterials with minimal signal processing. The method, which improves the sensitivity by an order of magnitude, is validated through high throughput measurements using model phantom systems.
Linear cationic antimicrobial peptides are a diverse class of molecules that interact with a wide range of cell membranes. Many of these peptides disrupt cell integrity by forming membrane-spanning ...pores that ultimately lead to their death. Despite these peptides high potency and ability to evade acquired bacterial drug resistance, there is a lack of knowledge on their selectivity and activity mechanisms. Such an understanding would provide an informative framework for rational design and could lead to potential antimicrobial therapeutic targets. In this paper, we use a high-throughput microfluidic platform as a quantitative screen to assess peptide activity and selectivity by precisely controlling exposure to vesicles with lipid compositions that mimic both bacterial and mammalian cell membranes. We explore the complexity of the lipid–peptide interactions governing membrane-disruptive behaviors and establish a link between peptide pore formation and both lipid–peptide charge and topological interactions. We propose a topological model for linear antimicrobial peptide activity based on the increase in membrane strain caused by the continuous adsorption of peptides to the target vesicle coupled with the effects of both lipid–peptide charge and topographical interactions. We also show the validity of the proposed model by investigating the activity of two prototypical linear cationic peptides: magainin 2 amide (which is selective for bacterial cells) and melittin (which targets both mammalian and bacterial cells indiscriminately). Finally, we propose the existence of a negative feedback mechanism that governs the pore formation process and controls the membrane’s apparent permeability.
The early diagnosis of active hepatitis C virus (HCV) infection remains a significant barrier to the treatment of the disease and to preventing the associated significant morbidity and mortality ...seen, worldwide. Current testing is delayed due to the high cost, long turnaround times and high expertise needed in centralised diagnostic laboratories. Here we demonstrate a user-friendly, low-cost pan-genotypic assay, based upon reverse transcriptase loop mediated isothermal amplification (RT-LAMP). We developed a prototype device for point-of-care use, comprising a LAMP amplification chamber and lateral flow nucleic acid detection strips, giving a visually-read, user-friendly result in <40 min. The developed assay fulfils the current guidelines recommended by World Health Organisation and is manufactured at minimal cost using simple, portable equipment. Further development of the diagnostic test will facilitate linkage between disease diagnosis and treatment, greatly improving patient care pathways and reducing loss to follow-up, so assisting in the global elimination strategy.
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