Low-High-Low Rotationally Pulse-Actuated Serial Dissolvable Film Valves Applied to Solid Phase Extraction and LAMP Isothermal Amplification for Plant Pathogen Detection on a Lab-on-a-Disc.

The ability of the centrifugal Lab-on-a-Disc (LoaD) platform to closely mimic the "on bench" liquid handling steps (laboratory unit operations (LUOs)) such as metering, mixing, and aliquoting supports on-disc automation of bioassay without the need for extensive biological optimization. Thus, well-established bioassays, normally conducted manually using pipettes or using liquid handling robots, can be relatively easily automated in self-contained microfluidic chips suitable for use in point-of-care or point-of-use settings. The LoaD's ease of automation is largely dependent on valves that can control liquid movement on the rotating disc. The optimum valving strategy for a true low-cost and portable device is rotationally actuated valves, which are actuated by changes in the disc spin-speed. However, due to tolerances in disc manufacturing and variations in reagent properties, most of these valving technologies have inherent variation in their actuation spin-speed. Most valves are actuated through stepped increases in disc spin-speed until the motor reaches its maximum speed (rarely more than 6000 rpm). These manufacturing tolerances combined with this "analogue" mechanism of valve actuation limits the number of LUOs that can be placed on-disc. In this work, we present a novel valving mechanism called low-high-low serial dissolvable film (DF) valves. In these valves, a DF membrane is placed in a dead-end pneumatic chamber. Below an actuation spin-speed, the trapped air prevents liquid wetting and dissolving the membrane. Above this spin-speed, the liquid will enter and wet the DF and open the valve. However, as DFs take ∼40 s to dissolve, the membrane can be wetted, and the disc spin-speed reduced before the film opens. Thus, by placing valves in a series, we can govern on which "digital pulse" in spin-speeding a reagent is released; a reservoir with one serial valve will open on the first pulse, a reservoir with two serial valves on the second, and so on. This "digital" flow control mechanism allows the automation of complex assays with high reliability. In this work, we first describe the operation of the valves, outline the theoretical basis for their operation, and support this analysis with an experiment. Next, we demonstrate how these valves can be used to automate the solid-phase extraction of DNA on on-disc LAMP amplification for applications in plant pathogen detection. The disc was successfully used to extract and detect, from a sample lysed off-disc, DNA indicating the presence of thermally inactivated Clavibacter michiganensis ssp. michiganensis (Cmm), a bacterial pathogen on tomato leaf samples.

Plant pathogen detection on a lab-on-a-disc using solid-phase extraction and isothermal nucleic acid amplification enabled by digital pulse-actuated dissolvable film valves.

By virtue of its ruggedness, portability, rapid processing times, and ease-of-use, academic and commercial interest in centrifugal microfluidic systems has soared over the last decade. A key advantage of the LoaD platform is the ability to automate laboratory unit operations (LUOs) (mixing, metering, washing etc.) to support direct translation of 'on-bench' assays to 'on-chip'. Additionally, the LoaD requires just a low-cost spindle motor rather than specialized and expensive microfluidic pumps. Furthermore, when flow control (valves) is implemented through purely rotational changes in this same spindle motor (rather than using additional support instrumentation), the LoaD offers the potential to be a truly portable, low-cost and accessible platform. Current rotationally controlled valves are typically opened by sequentially increasing the disc spin-rate to a specific opening frequency. However, due lack of manufacturing fidelity these specific opening frequencies are better described as spin frequency 'bands'. With low-cost motors typically having a maximum spin-rate of 6000 rpm (100 Hz), using this 'analogue' approach places a limitation on the number of valves, which can be serially actuated thus limiting the number of LUOs that can be automated. In this work, a novel flow control scheme is presented where the sequence of valve actuation is determined by architecture of the disc while its timing is governed by freely programmable 'digital' pulses in its spin profile. This paradigm shift to 'digital' flow control enables automation of multi-step assays with high reliability, with full temporal control, and with the number of LUOs theoretically only limited by available space on the disc. We first describe the operational principle of these valves followed by a demonstration of the capability of these valves to automate complex assays by screening tomato leaf samples against plant pathogens. Reagents and lysed sample are loaded on-disc and then, in a fully autonomous fashion using only spindle-motor control, the complete assay is automated. Amplification and fluorescent acquisition take place on a custom spin-stand enabling the generation of real-time LAMP amplification curves using custom software. To prevent environmental contamination, the entire discs are sealed from atmosphere following loading with internal venting channels permitting easy movement of liquids about the disc. The disc was successfully used to detect the presence of thermally inactivated Clavibacter michiganensis. Michiganensis (CMM) bacterial pathogen on tomato leaf samples.

Label- and amplification-free electrochemical detection of bacterial ribosomal RNA.

Current approaches to molecular diagnostics rely heavily on PCR amplification and optical detection methods which have restrictions when applied to point of care (POC) applications. Herein we describe the development of a label-free and amplification-free method of pathogen detection applied to Escherichia coli which overcomes the bottleneck of complex sample preparation and has the potential to be implemented as a rapid, cost effective test suitable for point of care use. Ribosomal RNA is naturally amplified in bacterial cells, which makes it a promising target for sensitive detection without the necessity for prior in vitro amplification. Using fluorescent microarray methods with rRNA targets from a range of pathogens, an optimal probe was selected from a pool of probe candidates identified in silico. The specificity of probes was investigated on DNA microarray using fluorescently labeled 16S rRNA target. The probe yielding highest specificity performance was evaluated in terms of sensitivity and a LOD of 20 pM was achieved on fluorescent glass microarray. This probe was transferred to an EIS end point format and specificity which correlated to microarray data was demonstrated. Excellent sensitivity was facilitated by the use of uncharged PNA probes and large 16S rRNA target and investigations resulted in an LOD of 50 pM. An alternative kinetic EIS assay format was demonstrated with which rRNA could be detected in a species specific manner within 10-40min at room temperature without wash steps.

Genotypic assessment of drug-resistant tuberculosis in Baghdad and other Iraqi provinces using low-cost and low-density DNA microarrays.

We report on a molecular investigation carried out to ascertain the prevalence of drug-resistant tuberculosis (TB) and the specific gene mutations responsible for resistance to rifampicin (RIF) and/or isoniazid (INH) in Iraq. In total, 110 clinical isolates from category II TB cases from Baghdad (58%) and several Iraqi provinces (42%) were analysed using colorimetric, low-cost and low-density (LCD) microarrays (MYCO-Direct and MYCO-Resist LCD array kits) to identify the point mutations responsible for resistance in Mycobacterium tuberculosis isolates. We found 76 patients (69.1%) had resistant strains, of which 40 (36%) were multidrug-resistant (MDR)-TB. Where mono-resistance was identified, it was found to be predominantly to RIF (83%). The most common mutations were rpoB S531L (50%), inhA C15T (25%) and katG S315T (15%). The most common MDR-TB genotypes were rpoB S531L with inhA C15T (60%) and rpoB S531L with katG S315T (20%). Where phenotypic analysis of clinical isolates was also performed, genotypic data were found to show excellent correlation with phenotypic results. Correlation was found between the MYCO-Resist LCD array and GenoType MTBDRplus for detection of resistance to RIF. Our study shows MDR-TB in 36% of category II TB cases in Baghdad and surrounding Iraqi provinces, which reflects the World Health Organization findings based on phenotypic studies. Diagnosis of TB and MDR-TB using culture-based tests is a significant impediment to global TB control. The LCD arrays investigated herein are easy to use, sensitive and specific molecular tools for TB resistance profiling in resource-limited laboratory settings.

Rapid Electrochemical Detection of New Delhi Metallo-beta-lactamase Genes To Enable Point-of-Care Testing of Carbapenem-Resistant Enterobacteriaceae.

The alarming rate at which antibiotic resistance is occurring in human pathogens causes a pressing need for improved diagnostic technologies aimed at rapid detection and point-of-care testing to support quick decision making regarding antibiotic therapy and patient management. Here, we report the successful development of an electrochemical biosensor to detect bla(NDM), the gene encoding the emerging New Delhi metallo-beta-lactamase, using label-free electrochemical impedance spectroscopy (EIS). The presence of this gene is of critical concern because organisms harboring bla(NDM) tend to be multiresistant, leaving very few treatment options. For the EIS assay, we used a bla(NDM)-specific PNA probe that was designed by applying a new approach that combines in silico probe design and fluorescence-based DNA microarray validation with electrochemical testing on gold screen-printed electrodes. The assay was successfully demonstrated for synthetic targets (LOD = 10 nM), PCR products (LOD = 100 pM), and direct, amplification-free detection from a bla(NDM)-harboring plasmid. The biosensor's specificity, preanalytical requirements, and performance under ambient conditions were demonstrated and successfully proved its suitability for further point-of-care test development.

Impedimetric detection of single-stranded PCR products derived from methicillin resistant Staphylococcus aureus (MRSA) isolates.

Using electrochemical impedance spectroscopy (EIS) the sensitive and specific detection of the antibiotic resistance gene mecA has been demonstrated. The gene sequence was obtained from clinical Staphylococcus aureus isolates. Initially a mecA specific probe was selected from hybridisation tests with a 3' and 5' version of a previously published probe sequence. When immobilised on a gold electrode in PNA form it was possible to detect hybridisation of mecA PCR product electrochemically at concentrations as low as 10nM. By incorporating an undecane-thiol and 1.8 nm glycol spacer into the PNA probe it was possible to extend the limit of detection for mecA to 10 pM. Most published studies on EIS and nucleic acid detection report the use of short artificial DNA sequences or novel signal amplification schemes which improve sensitivity whereas this study reports the successful detection of long DNA fragments produced by PCR following extraction from clinical isolates. Finally, using screen printed electrodes the paper demonstrates hybridisation monitoring of mecA in an "on-line" assay format under ambient conditions which paves the way for rapid mecA detection in point of care scenarios.

Development of immunosensors for direct detection of three wound infection biomarkers at point of care using electrochemical impedance spectroscopy.

A method for label-free, electrochemical impedance immunosensing for the detection and quantification of three infection biomarkers in both buffer and directly in the defined model matrix of mock wound fluid is demonstrated. Triggering Receptor-1 Expressed on Myeloid cells (TREM-1) and Matrix MetalloPeptidase 9 (MMP-9) are detected via direct assay and N-3-oxo-dodecanoyl-l-HomoSerineLactone (HSL), relevant in bacterial quorum sensing, is detected using a competition assay. Detection is performed with gold screen-printed electrodes modified with a specific thiolated antibody. Detection is achieved in less than 1h straight from mock wound fluid without any extensive sample preparation steps. The limits of detection of 3.3 pM for TREM-1, 1.1 nM for MMP-9 and 1.4 nM for HSL are either near or below the threshold required to indicate infection. A relatively large dynamic range for sensor response is also found, consistent with interaction between neighbouring antibody-antigen complexes in the close-packed surface layer. Together, these three novel electrochemical immunosensors demonstrate viable multi-parameter sensing with the required sensitivity for rapid wound infection detection directly from a clinically relevant specimen.

Dielectrophoretic manipulation of ribosomal RNA.

The manipulation of ribosomal RNA (rRNA) extracted from E. coli cells by dielectrophoresis (DEP) has been demonstrated over the range of 3 kHz-50 MHz using interdigitated microelectrodes. Quantitative measurement using total internal reflection fluorescence microscopy of the time dependent collection indicated a positive DEP response characterized by a plateau between 3 kHz and 1 MHz followed by a decrease in response at higher frequencies. Negative DEP was observed above 9 MHz. The positive DEP response below 1 MHz is described by the Clausius-Mossotti model and corresponds to an induced dipole moment of 3300 D with a polarizability of 7.8×10(-32) F m(2). The negative DEP response above 9 MHz indicates that the rRNA molecules exhibit a net moment of -250 D, to give an effective permittivity value of 78.5 ε(0), close to that of the aqueous suspending medium, and a relatively small surface conductance value of ∼0.1 nS. This suggests that our rRNA samples have a fairly open structure accessible to the surrounding water molecules, with counterions strongly bound to the charged phosphate groups in the rRNA backbone. These results are the first demonstration of DEP for fast capture and release of rRNA units, opening new opportunities for rRNA-based biosensing devices.