Semiquantitative Nucleic Acid Test with Simultaneous Isotachophoretic Extraction and Amplification.

Nucleic acid amplification tests (NAATs) provide high diagnostic accuracy for infectious diseases and quantitative results for monitoring viral infections. The majority of NAATs require complex equipment, cold chain dependent reagents, and skilled technicians to perform the tests. This largely confines NAATs to centralized laboratories and can significantly delay appropriate patient care. Low-cost, point-of-care (POC) NAATs are especially needed in low-resource settings to provide patients with diagnosis and treatment planning in a single visit to improve patient care. In this work, we present a rapid POC NAAT with integrated sample preparation and amplification using electrokinetics and paper substrates. We use simultaneous isotachophoresis (ITP) and recombinase polymerase amplification (RPA) to rapidly extract, amplify, and detect target nucleic acids from serum and whole blood in a paper-based format. We demonstrate simultaneous ITP and RPA can consistently detect 5 copies per reaction in buffer and 10 000 copies per milliliter of human serum with no intermediate user steps. We also show preliminary extraction and amplification of DNA from whole blood samples. Our test is rapid (results in less than 20 min) and made from low-cost materials, indicating its potential for detecting infectious diseases and monitoring viral infections at the POC in low resource settings.

Translating diagnostic assays from the laboratory to the clinic: analytical and clinical metrics for device development and evaluation.

As lab-on-a-chip health diagnostic technologies mature, there is a push to translate them from the laboratory to the clinic. For these diagnostics to achieve maximum impact on patient care, scientists and engineers developing the tests should understand the analytical and clinical statistical metrics that determine the efficacy of the test. Appreciating and using these metrics will benefit test developers by providing consistent measures to evaluate analytical and clinical test performance, as well as guide the design of tests that will most benefit clinicians and patients. This paper is broken into four sections that discuss metrics related to general stages of development including: (1) laboratory assay development (analytical sensitivity, limit of detection, analytical selectivity, and trueness/precision), (2) pre-clinical development (diagnostic sensitivity, diagnostic specificity, clinical cutoffs, and receiver-operator curves), (3) clinical use (prevalence, predictive values, and likelihood ratios), and (4) case studies from existing clinical data for tests relevant to the lab-on-a-chip community (HIV, group A strep, and chlamydia). Each section contains definitions of recommended statistical measures, as well as examples demonstrating the importance of these metrics at various stages of the development process. Increasing the use of these metrics in lab-on-a-chip research will improve the rigor of diagnostic performance reporting and provide a better understanding of how to design tests that will ultimately meet clinical needs.

NAIL: Nucleic Acid detection using Isotachophoresis and Loop-mediated isothermal amplification.

Nucleic acid amplification tests are the gold standard for many infectious disease diagnoses due to high sensitivity and specificity, rapid operation, and low limits of detection. Despite the advantages of nucleic acid amplification tests, they currently offer limited point-of-care (POC) utility due to the need for complex instruments and laborious sample preparation. We report the development of the Nucleic Acid Isotachophoresis LAMP (NAIL) diagnostic device. NAIL uses isotachophoresis (ITP) and loop-mediated isothermal amplification (LAMP) to extract and amplify nucleic acids from complex matrices in less than one hour inside of an integrated chip. ITP is an electrokinetic separation technique that uses an electric field and two buffers to extract and purify nucleic acids in a single step. LAMP amplifies nucleic acids at constant temperature and produces large amounts of DNA that can be easily detected. A mobile phone images the amplification results to eliminate the need for laser fluorescent detection. The device requires minimal user intervention because capillary valves and heated air chambers act as passive valves and pumps for automated fluid actuation. In this paper, we describe NAIL device design and operation, and demonstrate the extraction and detection of pathogenic E. coli O157:H7 cells from whole milk samples. We use the Clinical and Laboratory Standards Institute (CLSI) limit of detection (LoD) definitions that take into account the variance from both positive and negative samples to determine the diagnostic LoD. According to the CLSI definition, the NAIL device has a limit of detection (LoD) of 1000 CFU mL(-1) for E. coli cells artificially inoculated into whole milk, which is two orders of magnitude improvement to standard tube-LAMP reactions with diluted milk samples and comparable to lab-based methods. The NAIL device potentially offers significant reductions in the complexity and cost of traditional nucleic acid diagnostics for POC applications.

Colorimetric detection of catalytic reactivity of nanoparticles in complex matrices.

There is a need for new methodologies to quickly assess the presence and reactivity of nanoparticles (NPs) in commercial, environmental, and biological samples since current detection techniques require expensive and complex analytical instrumentation. Here, we investigate a simple and portable colorimetric detection assay that assesses the surface reactivity of NPs, which can be used to detect the presence of NPs, in complex matrices (e.g., environmental waters, serum, urine, and in dissolved organic matter) at as low as part per billion (ppb) or ng/mL concentration levels. Surface redox reactivity is a key emerging property related to potential toxicity of NPs with living cells, and is used in our assays as a key surrogate for the presence of NPs and a first tier analytical strategy toward assessing NP exposures. We detect a wide range of metal (e.g., Ag and Au) and oxide (e.g., CeO2, SiO2, VO2) NPs with a diameter range of 5 to 400 nm and multiple capping agents (tannic acid (TA), polyvinylpyrrolidone (PVP), branched polyethylenimine (BPEI), polyethylene glycol (PEG)). This method is sufficiently sensitive (ppb levels) to measure concentrations typically used in toxicological studies, and uses inexpensive, commercially available reagents.

Simple, low-cost styrene-ethylene/butylene-styrene microdevices for electrokinetic applications.

Styrene-ethylene/butylene-styrene (SEBS) copolymers combine thermoplastic and elastomeric properties to provide microdevices with the advantageous properties of hard thermoplastics and ease of fabrication similar to PDMS. This work describes the electrical surface properties of SEBS block copolymers using current monitoring experiments to determine zeta potential. We show that SEBS exhibits a stable and relatively high zeta potential magnitude compared to similar polymers. The zeta potential of SEBS is stable when stored in air over time, and no significant differences are observed between different batches and devices, demonstrating reproducibility of results. We show zeta potential trends for varying pH and counterion concentration and demonstrate that SEBS has a repeatable surface potential comparable to glass. Oxygen plasma treatment greatly increases the zeta potential magnitude immediately following treatment before undergoing a moderate hydrophobic recovery to a stable zeta potential. SEBS copolymers also offer simple rapid prototyping fabrication and mass production potential. The presented electrokinetic properties combined with simple, low-cost fabrication of microdevices make SEBS a quality material for electrokinetic research and application development.

Simple replica micromolding of biocompatible styrenic elastomers.

In this work, we introduce a simple solvent-assisted micromolding technique for the fabrication of high-fidelity styrene-ethylene/butylene-styrene (SEBS) microfluidic devices with high polystyrene (PS) content (42 wt% PS, SEBS42). SEBS triblock copolymers are styrenic thermoplastic elastomers that exhibit both glassy thermoplastic and elastomeric properties resulting from their respective hard PS and rubbery ethylene/butylene segments. The PS fraction gives SEBS microdevices many of the appealing properties of pure PS devices, while the elastomeric properties simplify fabrication of the devices, similar to PDMS. SEBS42 devices have wettable, stable surfaces (both contact angle and zeta potential) that support cell attachment and proliferation consistent with tissue culture dish substrates, do not adsorb hydrophobic molecules, and have high bond strength to wide range of substrates (glass, PS, SEBS). Furthermore, SEBS42 devices are mechanically robust, thermally stable, as well as exhibit low auto-fluorescence and high transmissivity. We characterize SEBS42 surface properties by contact angle measurements, cell culture studies, zeta potential measurements, and the adsorption of hydrophobic molecules. The PS surface composition of SEBS microdevices cast on different substrates is determined by time-of-flight secondary ion mass spectrometry (ToF-SIMS). The attractive SEBS42 material properties, coupled with the simple fabrication method, make SEBS42 a quality substrate for microfluidic applications where the properties of PS are desired but the ease of PDMS micromolding is favoured.