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

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

Developing new synthetic tools for nucleic acid based diagnostics

With increasing globalization, new infectious diseases are being discovered and spread quickly around the world. The traditional ways for the detection and identification of infectious pathogens are time-consuming and usually require specific facilities. This may delay effective treatment and lead to the spread of infectious disease, especially in the early stages of an epidemic. Therefore, accurate and efficient methods for identification of causative pathogens are very important. Here, we take advantages of new synthetic tools and nucleic acid technologies to develop novel infectious disease diagnostic methods that are user-friendly and have high sensitivity and specificity. These include: 1). The development of a rapid ultrasonic DNA isothermal amplification method with multiplexed melting analysis; 2). The development of an origami device based nucleic acid multiplexed detection method; 3). The development of a novel branched Hybridization Chain Reaction (HCR) assay. 1. Rapid ultrasonic isothermal amplification of DNA with multiplexed melting analysis-applications in the clinical diagnosis of sexually transmitted diseases (1) In this project, surface acoustic wave (SAW) signals are generated by interdigitated transducer (IDT) on LiNbO3 and propagated into disposable silicon superstrates on which a droplet of Loop-mediated isothermal amplification (LAMP) reaction mixture has been placed. As SAW interacts with the LAMP mixture, its energy is transferred into the LAMP mixture and causes the temperature of the mixture to increase. By controlling the SAW generation voltage, the temperature of the LAMP mixture could be maintained within a certain range as necessary for the LAMP reaction. During the process of LAMP amplification, a lot of double-strand DNA (dsDNA) is produced; this can incorporate specific fluorescent dyes and result in an exponential increase in fluorescence signal intensity. Also, by gradually increasing the SAW generation voltage, a SAW actuation-based DNA melting method could be used for multiplex detection. Ten-fold serially diluted targets from 105 copies/reaction to 10 copies/reaction were used to quantify the analytical sensitivity of the SAW-LAMP system and measurable signals were found down to 10 copies/reaction. Compared to a Peltier-LAMP system, SAW actuation enables the amplification to be performed more rapidly, about 18.23 % +/- 2.5 faster. Six clinical samples were used to demonstrate the clinical validation of SAW-LAMP by comparing with results from qPCR. The use of a SAW actuation-based DNA melting method distinguished the difference between melting temperatures of C. trachomatis amplicon (79.65 +/- 0.14 °C), and N. gonorrhoea (82.55 +/- 0.53 °C). 2. Development of paper origami device based nucleic acid multiplex detection for infectious diseases diagnostics In this project, a paper-folding origami device to manipulate malaria-infected blood samples was described. Through the simple process of paper folding, the nucleic acid of parasites in the blood sample could be extracted, purified and eluted. The extracted nucleic acid was then amplified with a multiplexed colorimetric LAMP assay in a plastic plate. Finally, the amplification products of multiplexed colorimetric LAMP assay were detected within an array with a low cost hand-held torch by naked eye. The multiplexed colorimetric LAMP assays for Plasmodium pan, Plasmodium falciparum, Plasmodium vivax with an internal control (IC) were investigated. The analytical sensitivity of colorimetric LAMP assays was tested by WHO International Standard DNA, with the limit of detection down to 105 IU/ml. Serially diluted quantified hCMV genomic DNA was used to demonstrate the DNA recovery of our origami device, which was between 60-70%. Serial dilution of a known infected blood samples (from 100 parasites/μl to 1 parasites/μl) were used to study the analytical sensitivity and obtain an LOD of the origami device to 5 parasites/μl. 80 fully characterised fresh malaria infected blood samples were used to assess the clinical validation effect of our origami device through a double blind, randomized controlled, clinical trial. All samples were also tested with commercially available LAMP kit and benchmark real-time PCR assay. The coincidence of our method and benchmark PCR were 88.75% (71/80) for Plasmodium pan, 90% (72/80) for P. falciparum and 93.75% (75/80) for P. vivax. Similarly, the coincidence between our method and the LAMP kit for Plasmodium pan and P. falciparum were 90% (72/80) and 92.50% (74/80) respectively. Using benchmark PCR as a gold standard for the detection of Plasmodium pan, P. ovale, P. falciparum and P. vivax, the sensitivity for our tests was of 85.5%, 92.9%, 61.1% and 81.0%, respectively. While the specificity are 100%, 94.2%, 98.5% and 98.30% respectively. We also established our origami device can diagnose species type from stored samples (either frozen, fixed, or dried). 3. Development of a novel branched Hybridization chain reaction (HCR) By increasing the dimensionality of an HCR system, a novel branched HCR product with complex branched structures instead of linear constructs has been developed. To validate the principle of a transition from a 1D chain to a higher dimension, we adapted a 3-arm branching construct to enable it to form a chain reaction by incorporating hybridization tails onto its sequences. The novel branched HCR reaction can form three-arm junction units with the introduction of a specific initiator. The three-armed units formed not only freed initiators to start of another cycle of HCR, but also bonded to each other to form complex and branched products. We also show that the highly branched polymers produced allow label-free acoustic mass sensing. The product of branched HCR was detected by Love Wave (LW) biosensor with the limit of detection at 2 nM, meaning 0.1% - 0.2% working frequency shift. Based on the branched HCR, we also design a new multiplexed HCR mechanism, where a single reaction is able to detect the presence of different initiators. It is based on designing primers that carry additional hairpin structures, which cross-react specifically upon initiation, yielding branching, thus opening up new applications for this enzyme-free, label-free DNA detection system