Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay.

Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics. The work presented here aims to provide phenotypic antimicrobial susceptibility testing (AST) information by pairing short growth periods (~3-4 h) with downstream PCR assays to ultimately predict minimum inhibitory concentration (MIC) values of antibiotic treatment. To further simplify the dual workflows of the AST and PCR assays, these reactions are carried out in a single-vessel format (PCR tube) using novel lyophilized reagent beads (LRBs), which store dried PCR reagents along with primers and enzymes, and antibiotic drugs separately. The two reactions are separated in space and time using a melting paraffin wax seal, thus eliminating the need to transfer reagents across different consumables and minimizing user interactions. Finally, these two-step single-vessel reactions are multiplexed by using a microfluidic manifold that allows simultaneous testing of an unknown bacterial sample against different antibiotics at varying concentrations. The LRBs used in the microfluidic system showed no interference with the bacterial growth and PCR assays and provided an innovative platform for rapid point-of-care diagnostics (POC-Dx).

Integrated microfluidic system for rapid forensic DNA analysis: sample collection to DNA profile.

We demonstrate a conduit for the delivery of a step change in the DNA analysis process: A fully integrated instrument for the analysis of multiplex short tandem repeat DNA profiles from reference buccal samples is described and is suitable for the processing of such samples within a forensic environment such as a police custody suite or booking office. The instrument is loaded with a DNA processing cartridge which incorporates on-board pumps and valves which direct the delivery of sample and reagents to the various reaction chambers to allow DNA purification, amplification of the DNA by PCR, and collection of the amplified product for delivery to an integral CE chip. The fluorescently labeled product is separated using micro capillary electrophoresis with a resolution of 1.2 base pairs (bp) allowing laser induced fluorescence-based detection of the amplified short tandem repeat fragments and subsequent analysis of data to produce a DNA profile which is compatible with the data format of the UK DNA database. The entire process from taking the sample from a suspect, to database compatible DNA profile production can currently be achieved in less than 4 h. By integrating such an instrument and microfluidic cartridge with the forensic process, we believe it will be possible in the near future to process a DNA sample taken from an individual in police custody and compare the profile with the DNA profiles held on a DNA Database in as little as 3 h.

An automated instrument for human STR identification: design, characterization, and experimental validation.

The microfluidic integration of an entire DNA analysis workflow on a fully integrated miniaturized instrument is reported using lab-on-a-chip automation to perform DNA fingerprinting compatible with CODIS standard relevant to the forensic community. The instrument aims to improve the cost, duration, and ease of use to perform a "sample-to-profile" analysis with no need for human intervention. The present publication describes the operation of the three major components of the system: the electronic control components, the microfluidic cartridge and CE microchip, and the optical excitation/detection module. Experimental details are given to characterize the level of performance, stability, reliability, accuracy, and sensitivity of the prototype system. A typical temperature profile from a PCR amplification process and an electropherogram of a commercial size standard (GeneScan 500™, Applied Biosystems) separation are shown to assess the relevance of the instrument to forensic applications. Finally, we present a profile from an automated integrated run where lysed cells from a buccal swab were introduced in the system and no further human intervention was required to complete the analysis.

High-Capacity Redox Polymer Electrodes: Applications in Molecular and Cellular Processing.

We present methods to fabricate high-capacity redox electrodes using thick membrane or fiber casting of conjugated polymer solutions. Unlike common solution casting or printing methods used in current organic electronics, the presented techniques enable production of PEDOT:PSS electrodes with high charge capacity and the capability to operate under applied voltages greater than 100 V without electrochemical overoxidation. The electrodes are shown integrated into several electrokinetic components commonly used in automated bioprocess or bioassay workflows, including electrophoretic DNA separation and extraction, cellular electroporation/lysis, and electroosmotic pumping. Unlike current metal electrodes used in these applications, the high-capacity polymer electrodes are shown to function without electrolysis of solvent (i.e., without production of excess H+, OH-, and H2O2 by-products). In addition, each component fabricated using the electrodes is shown to have superior capabilities compared with those fabricated with common metal electrodes. These innovations in electrokinetics include a low-voltage/high-pressure electroosmotic pump, and a "flow battery" (in which electrochemical discharge is used to generate electroosmotic flow in the absence of an applied potential). The novel electrodes (and electrokinetic demonstrations) enable new applications of organic electronics within the biology, health care, and pharmaceutical fields.

A wearable patch for continuous monitoring of sweat electrolytes during exertion.

Implementation of wearable sweat sensors for continuous measurement of fluid based biomarkers (including electrolytes, metabolites and proteins) is an attractive alternative to common, yet intrusive and invasive, practices such as urine or blood analysis. Recent years have witnessed several key demonstrations of sweat based electrochemical sensing in wearable formats, however, there are still significant challenges and opportunities in this space for clinical acceptance, and thus mass implementation of these devices. For instance, there are inherent challenges in establishing direct correlations between sweat-based and gold-standard plasma-based biomarker concentrations for clinical decision-making. In addition, the wearable sweat monitoring devices themselves may exacerbate these challenges, as they can significantly alter sweat physiology (example, sweat rate and composition). Reported here is the demonstration of a fully integrated, wireless, wearable and flexible sweat sensing device for non-obtrusive and continuous monitoring of electrolytes during moderate to intense exertion as a metric for hydration status. The focus of this work is twofold: 1- design of a conformable fluidics systems to suit conditions of operation for sweat collection (to minimize sensor lag) with rapid removal of sweat from the sensing site (to minimize effects on sweat physiology). 2- integration of Na+ and K+ ion-selective electrodes (ISEs) with flexible microfluidics and low noise small footprint electronics components to enable wireless, wearable sweat monitoring. While this device is specific to electrolyte analysis during intense perspiration, the lessons in microfluidics and overall system design are likely applicable across a broad range of analytes.

Optimization of a Microfluidic Mixing Process for Gene Expression-Based Bio-dosimetry.

In recent decades advances in radiation imaging and radiation therapy have led to a dramatic increase in the number of people exposed to radiation. Consequently, there is a clear need for personalized biodosimetry diagnostics in order to monitor the dose of radiation received and adapt it to each patient depending on their sensitivity to radiation exposure (Hall E.J. and Brenner D. J., 2008). Similarly, after a large-scale radiological event such as a dirty bomb attack, there will be a major need to assess, within a few days the radiation doses received by tens of thousands of individuals. Current high throughput devices can handle only a few hundred individuals per day. Hence there is a great need for a very fast self-contained non-invasive biodosimetric device based on a rapid blood test.This paper presents a case study where regression methods and designed experiments are used to arrive at the optimal settings for various factors that impact the kinetics in a biodosimetric device. We use ridge regression to initially identify a set of potentially important variables in the mixing process which is one of the critical sub systems of the device. This was followed by a series of designed experiments to arrive at the optimal setting of the significant microfluidic cartridge and piezoelectric disk (PZT) (D. Sadler, F. Zenhausern, U.S. Patent 6,986,601; Lee, S. Y., Ko, B., Yang, W., 2005) related factors. This statistical approach has been utilized to study the microfluidic mixing to mix water and dye mixtures of 70 µl volume. The outcome of the statistical design, experimentation and analysis was then exploited for optimizing the design, fabrication and assembly of the microfluidic devices. As a result of the experiments that were performed, the system was fine tuned and the mixing time was reduced from 5.5 minutes to 2 minutes.

Biodosimetry on small blood volume using gene expression assay.

This paper reports the development of a biodosimetry device suitable for rapidly measuring expression levels of a low-density gene set that can define radiation exposure, dose and injury in a public health emergency. The platform comprises a set of 14 genes selected on the basis of their abundance and differential expression level in response to radiation from an expression profiling series measuring 41,000 transcripts. Gene expression is analyzed through direct signal amplification using a quantitative Nuclease Protection Assay (qNPA). This assay can be configured as either a high-throughput microplate assay or as a handheld detection device for individual point-of-care assays. Recently, we were able to successfully develop the qNPA platform to measure gene expression levels directly from human whole blood samples. The assay can be performed with volumes as small as 30 microL of whole blood, which is compatible with collection from a fingerstick. We analyzed in vitro irradiated blood samples with qNPA. The results revealed statistically significant discrimination between irradiated and non-irradiated samples. These results indicate that the qNPA platform combined with a gene profile based on a small number of genes is a valid test to measure biological radiation exposure. The scalability characteristics of the assay make it appropriate for population triage. This biodosimetry platform could also be used for personalized monitoring of radiotherapy treatments received by patients.

Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection.

A fully integrated biochip device that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and DNA microarray sensors was developed to perform DNA analysis of complex biological sample solutions. Sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis), polymerase chain reaction, DNA hybridization, and electrochemical detection were performed in this fully automated and miniature device. Cavitation microstreaming was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads and accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from approximately milliliters of whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Hybridization enhancement using cavitation microstreaming.

Conventional DNA microarray hybridization relies on diffusion of target to surface-bound probes, and thus is a rate-limited process. In this paper, a micromixing technique based on cavitation microstreaming principle that was developed to accelerate hybridization process is explained. Fluidic experiments showed that air bubbles resting on a solid surface and set into vibration by a sound field generated steady circulatory flows, resulting in global convection flows and, thus, rapid mixing. The time to fully mix dyed solutions in a 50-microL chamber using cavitation microstreaming was significantly reduced from hours (a pure diffusion-based mixing) to 6 s. Cavitation microstreaming was implemented to enhance DNA hybridization in both fluorescence-detection-based and electrochemical-detection-based DNA microarray chips. The former showed that cavitation microstreaming results in up to 5-fold hybridization signal enhancement with significantly improved signal uniformity, as compared to the results obtained in conventional diffusion-based biochips for a given time (2 h). Hybridization kinetics study in the electrochemical detection-based chips showed that acoustic microstreaming results in up to 5-fold kinetics acceleration. Acoustic microstreaming has many advantages over most existing techniques used for hybridization enhancement, including a simple apparatus, ease of implementation, low power consumption (approximately 2 mW), and low cost.

DNA amplification and hybridization assays in integrated plastic monolithic devices.

PCR amplification, DNA hybridization, and a hybridization wash have been integrated in a disposable monolithic DNA device, containing all of the necessary fluidic channels and reservoirs. These integrated devices were fabricated in polycarbonate plastic material by CO2 laser machining and were assembled using a combination of thermal bonding and adhesive tape bonding. Pluronics polymer phase change valves were implemented in the devices to fulfill the valving requirements. Pluronics polymer material is PCR compatible, and 30% Pluronics polymer valves provide enough holding pressure to ensure a successful PCR amplification. By reducing the temperature locally, to approximately 5 degrees C, Pluronics valves were liquefied and easily opened. A hybridization channel was made functional by oligonucleotide deposition, using Motorola proprietary surface attachment chemistry. Reagent transport on the device was provided by syringe pumps, which were docked onto the device. Peltier thermal electrical devices powered the heating and cooling functionality of the device. Asymmetrical PCR amplification and subsequent hybridization detection of both Escherichia coli K-12 MG1655 and Enterococcus faecalis DNAE genes have been successfully demonstrated in these disposable monolithic devices.

Genotyping on a complementary metal oxide semiconductor silicon polymerase chain reaction chip with integrated DNA microarray.

A novel method for the fast identification of genetic material utilizing a micro-DNA amplification and analysis device (micro-DAAD) consisting of multiple PCR microreactors with integrated DNA microarrays was developed. The device was fabricated in Si-technology and used for the genotyping of Chinese medicinal plants on the basis of differences in the noncoding region of the 5S-rRNA gene. Successful amplification of the genetic material and the consecutive analysis of the fluorescent-labeled amplicons in the micro-DAAD by the integrated oligonucleotide probes were demonstrated. Parallel analysis was performed by loading the four PCR reactors of the micro-DAAD with different samples of 3-microL volume. Temperature sensors and heating elements of the micro-DAAD enable precise temperature control and fast cycling, allowing the rapid completion of a combined amplification and analysis (hybridization) experiment.

Plastic biochannel hybridization devices: a new concept for microfluidic DNA arrays.

Conventional DNA hybridization assay kinetics depends solely on the diffusion of target to surface-bound probes, causing long hybridization times. In this study, we examined the possibilities of accelerating the hybridization process by using microfluidic channels ("biochannels") made of polycarbonate, optionally with an integrated pump. We produced two different devices to study these effects: first, hybridization kinetics was investigated by using an eSensor electrochemical DNA detection platform allowing kinetic measurements in homogenous solution. We fabricated an integrated cartridge for the chip comprising the channel network and a micropump for the oscillation of the hybridization mixture to further overcome diffusion limitations. As a model assay, we used an assay for the detection of single-nucleotide polymorphisms in the HFE-H gene. Second, based on the biochannel approach, we constructed a plastic microfluidic chip with a network of channels for optical detection of fluorescent-labeled targets. An assay for the simultaneous detection of four pathogenic bacteria surrogate strains from multiple samples was developed for this device. We observed high initial hybridization velocities and a fast attainment of equilibrium for the biochannel with integrated pump. Experimental results were compared with predictions generated by computer simulations.