Capillary-based multiplexed isothermal nucleic acid-based test for sexually transmitted diseases in patients

We demonstrate a multiplexed loop mediated isothermal amplification (LAMP) assay for infectious disease diagnostics, where the analytical process flow of target pathogens genomic DNA is performed manually by moving magnetic beads through a series of plugs in a capillary. Heat is provided by a water bath and the results read by the naked eye, enabling applications in low resource settings

Monitoring genetic population biomarkers for wastewater-based epidemiology

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

Cycling of rational hybridization chain reaction to enable enzyme-free DNA-based clinical diagnosis

In order to combat the growing threat of global infectious diseases, there is the need for rapid diagnostic technologies that are sensitive and that can provide species specific information (as might be needed to direct therapy as resistant strains of microbes emerge). Here, we present a convenient, enzyme-free amplification mechanism for a rational hybridization chain reaction (HCR), which is implemented in a simple format for isothermal amplification and sensing, applied to the DNA-based diagnosis of Hepatitis B virus (HBV) in 54 patients. During the cycled amplification process, DNA monomers self-assemble in an organized and controllable way, only when a specific target HBV sequence is present. This mechanism is confirmed using super-resolution Stochastic Optical Reconstruction Microscopy (STORM). The enabled format is designed in a manner analogous to an enzyme-linked immunosorbent assay (ELISA), generating coloured products with distinct tonality and with a limit of detection of ca. 5 copies/reaction. This routine assay also showed excellent sensitivity (>97%) in clinical samples demonstrating the potential of this convenient, low cost, enzyme-free method for use in low resource settings

Branched hybridization chain reaction – using highly dimensional DNA nanostructures for label-free, reagent-less multiplexed molecular diagnostics

The specific and multiplexed detection of DNA underpins many analytical methods, including the detection of microorganisms that are important in the medical, veterinary, and environmental sciences. To achieve such measurements generally requires enzyme-mediated amplification of the low concentrations of the target nucleic acid sequences present, together with the precise control of temperature, as well as the use of enzyme-compatible reagents. This inevitably leads to compromises between analytical performance and the complexity of the assay. The hybridization chain reaction (HCR) provides an attractive alternative, as a route to enzyme-free DNA amplification. To date, the linear nucleic acid products, produced during amplification, have not enabled the development of efficient multiplexing strategies, nor the use of label-free analysis. Here, we show that by designing new DNA nanoconstructs, we are able, for the first time, to increase the molecular dimensionality of HCR products, creating highly branched amplification products, which can be readily detected on label-free sensors. To show that this new, branching HCR system offers a route for enzyme-free, label-free DNA detection, we demonstrate the multiplexed detection of a target sequence (as the initiator) in whole blood. In the future, this technology will enable rapid point-of-care multiplexed clinical analysis or in-the-field environmental monitoring

Paper-based microfluidics for DNA diagnostics of malaria in low resource underserved rural communities

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

Sequence terminus dependent PCR for site-specific mutation and modification

This dataset provides all the data associated with the development of a biotechnology assay on detecting nucleic acid modifications using a PCR workflow

Rapid veterinary diagnosis of bovine reproductive infectious diseases from semen using paper-origami DNA microfluidics

The health and well-being of cattle is a significant concern for global agricultural output. In dairy production within low and middle income countries (LMICs), there is a significant biosensing challenge in detecting sexually transmitted infection (STI) pathogens during animal husbandry, due in part to difficulties associated with the limited infrastructure for veterinary medicine. Here we demonstrate low-cost, multiplexed and sample-to-answer paper-origami tests for the detection of three bovine infectious reproductive diseases in semen samples, collected at a test site in rural India. Pathogen DNA from one viral pathogen, Bovine Herpes virus-1 (BoHV-1) and two bacteria (Brucella and Leptospira) was extracted, amplified (using loop-mediated isothermal amplification, LAMP) and detected fluorescently, enabling <1 pg (~ from 115 to 274 copies per reaction) of target genomic DNA to be measured. Data was collected as a fluorescence signal either visually, using a low-cost hand-held torch, or digitally with a mobile-phone camera. Limits of detection and sensitivities of the paper-origami device for the three pathogens were also evaluated using pathogen-inoculated semen samples and were as few as 50 Leptospira organisms, 50 CFU Brucella and 1 TCID50. BoHV-1. Semen samples from elite bulls at a germplasm centre were also tested in double-blind tests, as a demonstrator for a low cost, user-friendly point-of-care sensing platform, for in-the-field resource-limited regions. The sensors showed excellent levels of sensitivity and specificity, and for the first of time a demonstrated ability of the application of paper-origami devices for the diagnosis multiple infectious diseases from semen samples

Genetic and phenotypic profiling of single living circulating tumour cells from patients with microfluidics

Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non–small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction