Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.

In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2) pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.

Erratum to: An integrated disposable device for DNA extraction and helicase dependent amplification.

In the original manuscript, we reported the demonstration of an integrated microfluidic chip that performed helicase dependent amplification (HDA) on samples containing live bacteria. Bacterial lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter were incorporated into a disposable polymer cartridge format. We reported that the device was able to detect as few as 10 colony-forming units (CFU) of E. coli in growth medium. While the main conclusions of the original paper remain sound, the data presented in support of those conclusions contained errors that we detail, discuss and correct here. In short, we misidentified a non-specific product as a specific product of our HDA reaction. We incorrectly called reactions containing the non-specific product (length 70 bp) positive. Further investigation demonstrated that our primer set was faulty and not capable of amplifying the specific product. Here we redesigned primers, sequenced all of the products and reran all of the experiments reported previously to generate a new, verified dataset.

Rapid point-of-care concentration of bacteria in a disposable microfluidic device using meniscus dragging effect.

We report a low cost, disposable polymer microfluidic sample preparation device to perform rapid concentration of bacteria from liquid samples using enhanced evaporation targeted at downstream detection using surface enhanced Raman spectroscopy (SERS). The device is composed of a poly(dimethylsiloxane) (PDMS) liquid sample flow layer, a reusable metal airflow layer, and a porous PTFE (Teflon™) membrane sandwiched in between the liquid and air layers. The concentration capacity of the device was successfully demonstrated with fluorescently tagged Escherichia coli (E. coli). The recovery concentration was above 85% for all initial concentrations lower than 1 × 10(4) CFU mL(-1). In the lowest initial concentration cases, 100 µL initial volumes of bacteria solution at 100 CFU mL(-1) were concentrated into 500 nL droplets with greater than 90% efficiency in 15 min. Subsequent tests with SERS on clinically relevant Methicillin-Sensitive Staphylococcus aureus (MSSA) after concentration in this device proved more than 100-fold enhancement in SERS signal intensity compared to the signal obtained from the unconcentrated sample. The concentration device is straightforward to design and use, and as such could be used in conjunction with a number of detection technologies.

An integrated disposable device for DNA extraction and helicase dependent amplification.

Here we report the demonstration of an integrated microfluidic chip that performs helicase dependent amplification (HDA) on samples containing live bacteria. Combined chip-based sample preparation and isothermal amplification are attractive for world health applications, since the need for instrumentation to control flow rate and temperature changes are reduced or eliminated. Bacteria lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter are incorporated into a disposable polymer cartridge format. Smart passive fluidic control using a flap valve and a hydrophobic vent (with a nanoporous PTFE membrane) with a simple on-chip mixer eliminates multiple user operations. The device is able to detect as few as ten colony forming units (CFU) of E. coli in growth medium.

The precise self-assembly of individual carbon nanotubes using magnetic capturing and fluidic alignment.

A new method for the self-assembly of a carbon nanotube (CNT) using magnetic capturing and fluidic alignment has been developed and characterized in this work. In this new method, the residual iron (Fe) catalyst positioned at one end of the CNT was utilized as a self-assembly driver to attract and position the CNT, while the assembled CNT was aligned by the shear force induced from the fluid flow through the assembly channel. The self-assembly procedures were successfully developed and the electrical properties of the assembled multi-walled carbon nanotube (MWNT) and single-walled carbon nanotube (SWNT) were fully characterized. The new assembly method developed in this work shows its feasibility for the precise self-assembly of parallel CNTs for electronic devices and nanobiosensors.

On chip cell separator using magnetic bead-based enrichment and depletion of various surface markers.

This paper presents an on-chip magnetic cell sorting system for the sorting of cells based on a variety of surface markers. A polymer lab on a chip integrated with an electroplated array of Ni/Fe permalloy has been designed, fabricated, and characterized for the separation of cell substitutes at a variety of flow rates and incubation times. The system sequentially labels cell substitutes with magnetic beads and sorts them, repeating this process to sort for a variety of surface markers. Flow rates and incubation times were varied to characterize the system and produce the best combination of high specific capture and low nonspecific capture. The separation system developed on polymer is selective and efficient while being low cost, portable, and fabricated in a modular structure that can be integrated with other cell handling processes.

Development of functional lab-on-a-chip on polymer for point-of-care testing of metabolic parameters.

This paper presents the development of an easy-to-handle and disposable clinical diagnostic lab-on-a-chip using fully integrated plastic microfluidic components, which has the sampling/identifying capability to make fast and reliable measurements of metabolic parameters from human whole blood. A smart and functional lab-on-a-chip cartridge, which incorporates a full on-chip auto-calibration function for in the field applications, has been developed, and then fully characterized using a portable analyzer (3 (1/4)''x 5''x 1'') with multi-analyte detection capability. In addition, several new approaches in realizing smart and functional lab-on-a-chips on polymer have been adopted, which include the pinch valve for automatic fluidic sealing, a by-pass channel as the sampling indicator, and a robust connector design for long analyzer lifetimes. Metabolic parameters such as glucose, lactate, and partial oxygen from human whole blood have been successfully measured using the functional polymer lab-on-a-chips and the portable analyzer developed in this work.

A polymer lab-on-a-chip for magnetic immunoassay with on-chip sampling and detection capabilities.

This paper presents a new polymer lab-on-a-chip for magnetic bead-based immunoassay with fully on-chip sampling and detection capabilities, which provides a smart platform of magnetic immunoassay-based lab-on-a-chip for point-of-care testing (POCT) toward biochemical hazardous agent detection, food inspection or clinical diagnostics. In this new approach, the polymer lab-on-a-chip for magnetic bead-based immunoassay consists of a magnetic bead-based separator, an interdigitated array (IDA) micro electrode, and a microfluidic system, which are fully incorporated into a lab-on-a-chip on cyclic olefin copolymer (COC). Since the polymer lab-on-a-chip was realized using low cost, high throughput polymer microfabrication techniques such as micro injection molding and hot embossing method, a disposable polymer lab-on-a-chip for the magnetic bead-based immunoassay can be successfully realized in a disposable platform. With this newly developed polymer lab-on-a-chip, an enzyme-labelled electrochemical immunoassay (ECIA) was performed using magnetic beads as the mobile solid support, and the final enzyme product produced from the ECIA was measured using chronoamperometry. A sampling and detection of as low as 16.4 ng mL(-1) of mouse IgG has been successfully performed in 35 min for the entire procedure.