Automated dynamic inlet microfluidics system: 3D printer adaptation for cost-effective, low volume, on-demand multi-analyte droplet generator.

The paper demonstrates an adaptation of a 3D printer (Prusa Mini+) with novel modules to develop a droplet generation system that generates combinatorial droplets from a standard 96 well plate. The calibration methodology developed would allow any fused deposition modeling (FDM) printer to generate monodisperse droplets (coefficient of variance (CV%) < 5%) from well plates or vials of any geometry. The system maintains precision across various volumes while maintaining a C.V. range of 0.81% to 3.61%, with an increased precision for larger volumes. The cost of the system developed is 70% less than commercially available droplet generation packages. Successful droplet library storage is accomplished via 3D printed cartridge connectors. The implemented system has been calibrated for Tygon® and PTFE at different velocities and volumetric configurations.

Characteristics of Bow-Tie Antenna Structures for Semi-Insulating GaAs and InP Photoconductive Terahertz Emitters.

This work presents a study of photoconductive (PC) terahertz (THz) emitters based upon varied bow-tie (BT) antenna structures on the semi-insulating (SI) forms of GaAs and InP. The BT antennas have electrodes in the form of a Sharp BT, a Broad BT, an Asymmetric BT, a Blunted BT, and a Doubled BT. The study explores the main features of PC THz emitters for spectroscopic studies and sensors application in terms of THz field amplitude and spectral bandwidth. The emitters' performance levels are found to depend strongly upon the PC material and antenna structure. The SI-InP emitters display lower THz field amplitude and narrower bandwidth compared to the SI-GaAs emitters with the same structure (and dimensions). The characterized Doubled BT structure yields a higher THz field amplitude, while the characterized Asymmetric BT structure with flat edges yields a higher bandwidth in comparison to the sharp-edged structures. This knowledge on the PC THz emitter characteristics, in terms of material and structure, can play a key role in future implementations and applications of THz sensor technology.

Photoconductive terahertz generation in semi-insulating GaAs and InP under the extremes of bias field and pump fluence.

This Letter analyzes photoconductive (PC) terahertz (THz) emitters based on the semi-insulating (SI) forms of GaAs and InP. The dependencies of the emitters are studied under the extremes of the bias field and pump fluence to reveal the underlying physics of charge carrier photoexcitation, transport, and emission. The bias field dependence shows that SI-GaAs PC THz emitters are preferentially subject to space-charge-limited current, under the influence of trap states, while SI-InP PC THz emitters are preferentially subject to sustained current, due to a prolonged charge carrier lifetime and the ensuing joule heating. The pump fluence dependence shows space-charge and near-field screening for all emitters, with SI-GaAs predisposed to near-field screening (under the influence of transient mobility) and SI-InP predisposed to space-charge screening. Such findings can support a deeper understanding of the underlying physics and optimal performance of SI-GaAs and SI-InP PC THz emitters.

Developments in the integration and application of terahertz spectroscopy with microfluidics.

This work presents an overview of terahertz (THz) spectroscopy with a focus on its implementation within microfluidic platforms. Such platforms are of great interest because they can enable label-free and reagent-free sensing. However, they must be implemented with thought towards the incorporated materials and structures as they can greatly impact the bandwidth, frequency resolution, signal-to-noise ratio, and dynamic range of the measurements. This review explores such relationships with insight given on the design and material considerations for the effective integration of THz spectroscopy in microfluidic platforms. The review also describes recent work on the application of THz spectroscopy to biomaterial analyses on increasing scales, targeting DNA, proteins, cells, and tissues.

Characterization and Integration of Terahertz Technology within Microfluidic Platforms.

In this work, the prospects of integrating terahertz (THz) time-domain spectroscopy (TDS) within polymer-based microfluidic platforms are investigated. The work considers platforms based upon the polar polymers polyethylene terephthalate (PET), polycarbonate (PC), polymethyl-methacrylate (PMMA), polydimethylsiloxane (PDMS), and the nonpolar polymers fluorinated ethylene propylene (FEP), polystyrene (PS), high-density polyethylene (HDPE), and ultra-high-molecular-weight polyethylene (UHMWPE). The THz absorption coefficients for these polymers are measured. Two microfluidic platforms are then designed, fabricated, and tested, with one being based upon PET, as a representative high-loss polar polymer, and one being based upon UHMWPE, as a representative low-loss nonpolar polymer. It is shown that the UHMWPE microfluidic platform yields reliable measurements of THz absorption coefficients up to a frequency of 1.75 THz, in contrast to the PET microfluidic platform, which functions only up to 1.38 THz. The distinction seen here is attributed to the differing levels of THz absorption and the manifestation of differing f for the systems. Such findings can play an important role in the future integration of THz technology and polymer-based microfluidic systems.

High-Throughput Incubation and Quantification of Agglutination Assays in a Microfluidic System.

In this paper, we present a two-phase microfluidic system capable of incubating and quantifying microbead-based agglutination assays. The microfluidic system is based on a simple fabrication solution, which requires only laboratory tubing filled with carrier oil, driven by negative pressure using a syringe pump. We provide a user-friendly interface, in which a pipette is used to insert single droplets of a 1.25-µL volume into a system that is continuously running and therefore works entirely on demand without the need for stopping, resetting or washing the system. These assays are incubated by highly efficient passive mixing with a sample-to-answer time of 2.5 min, a 5⁻10-fold improvement over traditional agglutination assays. We study system parameters such as channel length, incubation time and flow speed to select optimal assay conditions, using the streptavidin-biotin interaction as a model analyte quantified using optical image processing. We then investigate the effect of changing the concentration of both analyte and microbead concentrations, with a minimum detection limit of 100 ng/mL. The system can be both low- and high-throughput, depending on the rate at which assays are inserted. In our experiments, we were able to easily produce throughputs of 360 assays per hour by simple manual pipetting, which could be increased even further by automation and parallelization. Agglutination assays are a versatile tool, capable of detecting an ever-growing catalog of infectious diseases, proteins and metabolites. A system such as this one is a step towards being able to produce high-throughput microfluidic diagnostic solutions with widespread adoption. The development of analytical techniques in the microfluidic format, such as the one presented in this work, is an important step in being able to continuously monitor the performance and microfluidic outputs of organ-on-chip devices.

On-Chip Magnetic Bead Manipulation and Detection Using a Magnetoresistive Sensor-Based Micro-Chip: Design Considerations and Experimental Characterization.

The remarkable advantages micro-chip platforms offer over cumbersome, time-consuming equipment currently in use for bio-analysis are well documented. In this research, a micro-chip that includes a unique magnetic actuator (MA) for the manipulation of superparamagnetic beads (SPBs), and a magnetoresistive sensor for the detection of SPBs is presented. A design methodology, which takes into account the magnetic volume of SPBs, diffusion and heat transfer phenomena, is presented with the aid of numerical analysis to optimize the parameters of the MA. The MA was employed as a magnetic flux generator and experimental analysis with commercially available COMPEL™ and Dynabeads(®) demonstrated the ability of the MA to precisely transport a small number of SPBs over long distances and concentrate SPBs to a sensing site for detection. Moreover, the velocities of COMPEL™ and Dynabead(®) SPBs were correlated to their magnetic volumes and were in good agreement with numerical model predictions. We found that 2.8 µm Dynabeads(®) travel faster, and can be attracted to a magnetic source from a longer distance, than 6.2 µm COMPEL™ beads at magnetic flux magnitudes of less than 10 mT. The micro-chip system could easily be integrated with electronic circuitry and microfluidic functions, paving the way for an on-chip biomolecule quantification device.

Comparison of capacitive and radio frequency resonator sensors for monitoring parallelized droplet microfluidic production.

Scaled-up production of microfluidic droplets, through the parallelization of hundreds of droplet generators, has received a lot of attention to bring novel multiphase microfluidics research to industrial applications. However, apart from droplet generation, other significant challenges relevant to this goal have never been discussed. Examples include monitoring systems, high-throughput processing of droplets and quality control procedures among others. In this paper, we present and compare capacitive and radio frequency (RF) resonator sensors as two candidates that can measure the dielectric properties of emulsions in microfluidic channels. By placing several of these sensors in a parallelization device, the stability of the droplet generation at different locations can be compared, and potential malfunctions can be detected. This strategy enables for the first time the monitoring of scaled-up microfluidic droplet production. Both sensors were prototyped and characterized using emulsions with droplets of 100-150 µm in diameter, which were generated in parallelization devices at water-in-oil volume fractions (φ) between 11.1% and 33.3%.Using these sensors, we were able to measure accurately increments as small as 2.4% in the water volume fraction of the emulsions. Although both methods rely on the dielectric properties of the emulsions, the main advantage of the RF resonator sensors is the fact that they can be designed to resonate at multiple frequencies of the broadband transmission line. Consequently with careful design, two or more sensors can be parallelized and read out by a single signal. Finally, a comparison between these sensors based on their sensitivity, readout cost and simplicity, and design flexibility is also discussed.

A remotely operated drug delivery system with an electrolytic pump and a thermo-responsive valve.

Implantable drug delivery devices are becoming attractive due to their abilities of targeted and controlled dose release. Currently, two important issues are functional lifetime and non-controlled drug diffusion. In this work, we present a drug delivery device combining an electrolytic pump and a thermo-responsive valve, which are both remotely controlled by an electromagnetic field (40.5 mT and 450 kHz). Our proposed device exhibits a novel operation mechanism for long-term therapeutic treatments using a solid drug in reservoir approach. Our device also prevents undesired drug liquid diffusions. When the electromagnetic field is on, the electrolysis-induced bubble drives the drug liquid towards the Poly (N-Isopropylacrylamide) (PNIPAM) valve that consists of PNIPAM and iron micro-particles. The heat generated by the iron micro-particles causes the PNIPAM to shrink, resulting in an open valve. When the electromagnetic field is turned off, the PNIPAM starts to swell. In the meantime, the bubbles are catalytically recombined into water, reducing the pressure inside the pumping chamber, which leads to the refilling of the fresh liquid from outside the device. A catalytic reformer is included, allowing more liquid refilling during the limited valve's closing time. The amount of body liquid that refills the drug reservoir can further dissolve the solid drug, forming a reproducible drug solution for the next dose. By repeatedly turning on and off the electromagnetic field, the drug dose can be cyclically released, and the exit port of the device is effectively controlled.

A cyclically actuated electrolytic drug delivery device.

This work, focusing on an implantable drug delivery system, presents the first prototype electrolytic pump that combines a catalytic reformer and a cyclically actuated mode. These features improve the release performance and extend the lifetime of the device. Using our platinum (Pt)-coated carbon fiber mesh that acts as a catalytic reforming element, the cyclical mode is improved because the faster recombination rate allows for a shorter cycling time for drug delivery. Another feature of our device is that it uses a solid-drug-in-reservoir (SDR) approach, which allows small amounts of a solid drug to be dissolved in human fluid, forming a reproducible drug solution for long-term therapies. We have conducted proof-of-principle drug delivery studies using such an electrolytic pump and solvent blue 38 as the drug substitute. These tests demonstrate power-controlled and pulsatile release profiles of the chemical substance, as well as the feasibility of this device. A drug delivery rate of 11.44 ± 0.56 µg min(-1) was achieved by using an input power of 4 mW for multiple pulses, which indicates the stability of our system.

The Pseudomonas aeruginosa generalized transducing phage phiPA3 is a new member of the phiKZ-like group of 'jumbo' phages, and infects model laboratory strains and clinical isolates from cystic fibrosis patients.

Pseudomonas aeruginosa is an important pathogen in cystic fibrosis patients, and a model organism for the study of nosocomially acquired infections, biofilms and intrinsic multidrug resistance. In this study we characterize ϕPA3, a new generalized transducing bacteriophage for P. aeruginosa. ϕPA3 transduced chromosomal mutations between PAO1 strains, and infected multiple P. aeruginosa clinical isolates as well as the P. aeruginosa model laboratory strains PAK and PA14. Electron microscopy imaging was used to classify ϕPA3 in the order Caudovirales and the family Myoviridae. The genome of ϕPA3 was sequenced and found to contain 309,208 bp, the second-largest bacteriophage currently deposited in GenBank. The genome contains 378 ORFs and five tRNAs. Many ORF products in the ϕPA3 genome are similar to proteins encoded by P. aeruginosa phage ϕKZ and Pseudomonas chlororaphis phage 201ϕ2-1, and so ϕPA3 was classified genetically as a member of the ϕKZ-like group of phages. This is the first report of a member of this group of phages acting as a generalized transducer. Given its wide host range, high transduction efficiency and large genome size, the 'jumbo' phage ϕPA3 could be a powerful tool in functional genomic analysis of diverse P. aeruginosa strains of fundamental and clinical importance.

RoboSCell: an automated single cell arraying and analysis instrument.

Single cell research has the potential to revolutionize experimental methods in biomedical sciences and contribute to clinical practices. Recent studies suggest analysis of single cells reveals novel features of intracellular processes, cell-to-cell interactions and cell structure. The methods of single cell analysis require mechanical resolution and accuracy that is not possible using conventional techniques. Robotic instruments and novel microdevices can achieve higher throughput and repeatability; however, the development of such instrumentation is a formidable task. A void exists in the state-of-the-art for automated analysis of single cells. With the increase in interest in single cell analyses in stem cell and cancer research the ability to facilitate higher throughput and repeatable procedures is necessary. In this paper, a high-throughput, single cell microarray-based robotic instrument, called the RoboSCell, is described. The proposed instrument employs a partially transparent single cell microarray (SCM) integrated with a robotic biomanipulator for in vitro analyses of live single cells trapped at the array sites. Cells, labeled with immunomagnetic particles, are captured at the array sites by channeling magnetic fields through encapsulated permalloy channels in the SCM. The RoboSCell is capable of systematically scanning the captured cells temporarily immobilized at the array sites and using optical methods to repeatedly measure extracellular and intracellular characteristics over time. The instrument's capabilities are demonstrated by arraying human T lymphocytes and measuring the uptake dynamics of calcein acetoxymethylester--all in a fully automated fashion.

A novel permalloy based magnetic single cell micro array.

Devices capable of automatically aligning cells onto geometrical arrays are of great interest to biomedical researchers. Such devices can facilitate the study of numerous cells while the cells remain physically separated from one another. In this way, cell arrays reduce cell-to-cell interactions while the cells are all subjected to common stimuli, which allows individual cell behaviour to be revealed. The use of arrays allows for the parallel analysis of single cells, facilitates data logging, and opens the door to the use of automated machine-based single cell analysis techniques. A novel permalloy based magnetic single cell micro array (MSCMA) is presented in this paper. The MSCMA creates an array of magnetic traps by generating magnetic flux density peaks at predefined locations. When using cells labelled with immunomagnetic labels, the cells will interact with the magnetic fields, and can be captured at the magnetic trap sites. Prototypes of the MSCMA have been successfully fabricated and tested using both fixed and live Jurkat cells (10 microm average diameter) that were labelled. The prototypes performed as predicted during experimental trials. The experimental results show that the MSCMA can randomly array up to 136 single cells per square mm. The results also show that the number of single cells captured is a function of the trap site density of the MSCMA design and the cell density in the fluid sample.

The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair.

Various mechanisms exist that enable bacteria to resist bacteriophage infection. Resistance strategies include the abortive infection (Abi) systems, which promote cell death and limit phage replication within a bacterial population. A highly effective 2-gene Abi system from the phytopathogen Erwinia carotovora subspecies atroseptica, designated ToxIN, is described. The ToxIN Abi system also functions as a toxin-antitoxin (TA) pair, with ToxN inhibiting bacterial growth and the tandemly repeated ToxI RNA antitoxin counteracting the toxicity. TA modules are currently divided into 2 classes, protein and RNA antisense. We provide evidence that ToxIN defines an entirely new TA class that functions via a novel protein-RNA mechanism, with analogous systems present in diverse bacteria. Despite the debated role of TA systems, we demonstrate that ToxIN provides viral resistance in a range of bacterial genera against multiple phages. This is the first demonstration of a novel mechanistic class of TA systems and of an Abi system functioning in different bacterial genera, both with implications for the dynamics of phage-bacterial interactions.

A generalized transducing phage for the murine pathogen Citrobacter rodentium.

A virulent phage (phiCR1) capable of generalized transduction in Citrobacter rodentium was isolated from the environment and characterized. C. rodentium is a natural pathogen of mice, causing transmissible murine colonic hyperplasia. Sequencing of its genome has recently been completed and will soon be fully annotated and published. C. rodentium is an important model organism for infections caused by the human pathogens enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC). phiCR1 uses a lipopolysaccharide receptor, has a genome size of approximately 300 kb, and is able to transduce a variety of markers. phiCR1 is the first reported transducing phage for C. rodentium and will be a useful tool for functional genomic analysis of this important natural murine pathogen.

Quorum sensing has an unexpected role in virulence in the model pathogen Citrobacter rodentium.

The bacterial mouse pathogen Citrobacter rodentium causes attaching and effacing (AE) lesions in the same manner as pathogenic Escherichia coli, and is an important model for this mode of pathogenesis. Quorum sensing (QS) involves chemical signalling by bacteria to regulate gene expression in response to cell density. E. coli has never been reported to have N-acylhomoserine lactone (AHL) QS, but it does utilize luxS-dependent signalling. We found production of AHL QS signalling molecules by an AE pathogen, C. rodentium. AHL QS is directed by the croIR locus and a croI mutant is affected in its surface attachment, although not in Type III secretion. AHL QS has an important role in virulence in the mouse as, unexpectedly, the QS mutant is hypervirulent; by contrast, we detected no impact of luxS inactivation. Further study of QS in Citrobacter should provide new insights into AE pathogenesis. As the croIR locus might have been horizontally acquired, AHL QS might exist in some strains of pathogenic E. coli.

A generalized transducing phage (phiIF3) for the genomically sequenced Serratia marcescens strain Db11: a tool for functional genomics of an opportunistic human pathogen.

A bacteriophage (phiIF3) capable of mediating generalized transduction in Serratia marcescens strain Db11 has been isolated and characterized. The genome of this Serratia strain has recently been sequenced and is likely to become the reference strain for S. marcescens researchers. phiIF3 is most likely a virulent phage, which can transduce markers at frequencies of 10(-6) transductants per p.f.u. It has a lipopolysaccharide receptor and was determined to have a latent period of 50 min and a burst size of approximately 100 phages. The phage DNA was resistant to digestion with restriction enzymes. Electron microscopy showed phiIF3 to be a member of the family Myoviridae. This is the first report of a generalized transducing phage able to infect Db11 and this phage will be a valuable tool for functional genomic analysis of the pathogen host.

The Serratia gene cluster encoding biosynthesis of the red antibiotic, prodigiosin, shows species- and strain-dependent genome context variation.

The prodigiosin biosynthesis gene cluster (pig cluster) from two strains of Serratia (S. marcescens ATCC 274 and Serratia sp. ATCC 39006) has been cloned, sequenced and expressed in heterologous hosts. Sequence analysis of the respective pig clusters revealed 14 ORFs in S. marcescens ATCC 274 and 15 ORFs in Serratia sp. ATCC 39006. In each Serratia species, predicted gene products showed similarity to polyketide synthases (PKSs), non-ribosomal peptide synthases (NRPSs) and the Red proteins of Streptomyces coelicolor A3(2). Comparisons between the two Serratia pig clusters and the red cluster from Str. coelicolor A3(2) revealed some important differences. A modified scheme for the biosynthesis of prodigiosin, based on the pathway recently suggested for the synthesis of undecylprodigiosin, is proposed. The distribution of the pig cluster within several Serratia sp. isolates is demonstrated and the presence of cryptic clusters in some strains shown. The pig cluster of Serratia marcescens ATCC 274 is flanked by cueR and copA homologues and this configuration is demonstrated in several S. marcescens strains, whilst these genes are contiguous in strains lacking the pig cluster.