Droplet-Based Single-Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno-Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min.

Empiric broad-spectrum antimicrobial treatments of urinary tract infections (UTIs) have contributed to widespread antimicrobial resistance. Clinical adoption of evidence-based treatments necessitates rapid diagnostic methods for pathogen identification (ID) and antimicrobial susceptibility testing (AST) with minimal sample preparation. In response, a microfluidic droplet-based platform is developed for achieving both ID and AST from urine samples within 30 min. In this platform, fluorogenic hybridization probes are utilized to detect 16S rRNA from single bacterial cells encapsulated in picoliter droplets, enabling molecular identification of uropathogenic bacteria directly from urine in as little as 16 min. Moreover, in-droplet single-bacterial measurements of 16S rRNA provide a surrogate for AST, shortening the exposure time to 10 min for gentamicin and ciprofloxacin. A fully integrated device and screening workflow were developed to test urine specimens for one of seven unique diagnostic outcomes including the presence/absence of Gram-negative bacteria, molecular ID of the bacteriaas Escherichia coli, an Enterobacterales, or other organism, and assessment of bacterial susceptibility to ciprofloxacin. In a 50-specimen clinical comparison study, the platform demonstrates excellent performance compared to clinical standard methods (areas-under-curves, AUCs >0.95), within a small fraction of the turnaround time, highlighting its clinical utility.

Evidence of Long-range nerve pathways connecting and coordinating activity in secondary lymph organs.

Background: Peripheral nerve reflexes enable organ systems to maintain long-term physiological homeostasis while responding to rapidly changing environmental conditions. Electrical nerve stimulation is commonly used to activate these reflexes and modulate organ function, giving rise to an emerging class of therapeutics called bioelectronic medicines. Dogma maintains that immune cell migration to and from organs is mediated by inflammatory signals (i.e. cytokines or pathogen associated signaling molecules). However, nerve reflexes that regulate immune function have only recently been elucidated, and stimulation of these reflexes for therapeutic effect has not been fully investigated. Methods: We utilized both electrical and ultrasound-based nerve stimulation to activate nerve pathways projecting to specific lymph nodes. Tissue and cell analysis of the stimulated lymph node, distal lymph nodes and immune organs is then utilized to measure the stimulation-induced changes in neurotransmitter/neuropeptide concentrations and immune cellularity in each of these sites. Results and conclusions: In this report, we demonstrate that activation of nerves and stimulated release of neurotransmitters within a local lymph node results in transient retention of immune cells (e.g. lymphocytes and neutrophils) at that location. Furthermore, such stimulation results in transient changes in neurotransmitter concentrations at distal organs of the immune system, spleen and liver, and mobilization of immune cells into the circulation. This report will enable future studies in which stimulation of these long-range nerve connections between lymphatic and immune organs can be applied for clinical purpose, including therapeutic modulation of cellularity during vaccination, active allergic response, or active auto-immune disease.

Microfluidic means of achieving attomolar detection limits with molecular beacon probes.

We used inline, micro-evaporators to concentrate and transport DNA targets to a nanoliter single molecule fluorescence detection chamber for subsequent molecular beacon probe hybridization and analysis. This use of solvent removal as a unique means of target transport in a microanalytical platform led to a greater than 5000-fold concentration enhancement and detection limits that pushed below the femtomolar barrier commonly reported using confocal fluorescence detection. This simple microliter-to-nanoliter interconnect for single molecule counting analysis resolved several common limitations, including the need for excessive fluorescent probe concentrations at low target levels and inefficiencies in direct handling of highly dilute biological samples. In this report, the hundreds of bacteria-specific DNA molecules contained in approximately 25 microliters of a 50 aM sample were shuttled to a four nanoliter detection chamber through micro-evaporation. Here, the previously undetectable targets were enhanced to the pM regime and underwent probe hybridization and highly-efficient fluorescent event analysis via microfluidic recirculation through the confocal detection volume. This use of microfluidics in a single molecule detection (SMD) platform delivered unmatched sensitivity and introduced compliment technologies that may serve to bring SMD to more widespread use in replacing conventional methodologies for detecting rare target biomolecules in both research and clinical labs.

Integration and application of vitrified collagen in multilayered microfluidic devices for corneal microtissue culture.

This paper describes the fabrication and application of microfluidic devices containing collagen vitrigel (CV) used as both a functional and sacrificial cell growth substrate for the development of corneal microtissue patches. Within the device, vacuum fixation of the CV in a dehydrated state enables quick integration with standard multilayer soft lithographic techniques, while on-chip rehydration results in a gel-like collagen substrate for microfluidic cell culture. Fluidic connectivity to both the apical and basal side of the CV permits bilayered culture of epithelium and supporting stromal cell layers. In addition, microfluidic introduction of a collagenase etching media enables sacrificial degradation of the supporting CV membrane for development of barrier tissue constructs containing minimal synthetic substrate. The utility of this platform was evaluated by miniaturizing the standard transepithelial permeability (TEP) assay in order to measure the integrity of an array of corneal tissue micropatches.

DNA methylation analysis on a droplet-in-oil PCR array.

We performed on-chip DNA methylation analysis using methylation-specific PCR (MSP) within an arrayed micro droplet-in-oil platform that is designed for more practical application of microfluidic droplet technologies in clinical applications. Unique features of this ready-to-use device include arrayed primers that are pre-deposited into open micro-reaction chambers and use of the oil phase as a companion fluid for both sample actuation and compartmentalization. These technical advantages allow for infusion of minute amounts of sample for arrayed MSP analysis, without the added complexities inherent in microfluidic droplet-based studies. Ease of use of this micro device is exemplified by analysis of two tumor suppressor promoters, p15 and TMS1 using an on-chip methylation assay. These results were consistent with standard MSP protocols, yet the simplicity of the droplet-in-oil microfluidic PCR platform provides an easy and efficient tool for DNA methylation analysis in a large-scale arrayed manner.

Tunable blinking kinetics of cy5 for precise DNA quantification and single-nucleotide difference detection.

Fluorescence correlation spectroscopy (FCS) can resolve the intrinsic fast-blinking kinetics (FBKs) of fluorescent molecules that occur on the order of microseconds. These FBKs can be heavily influenced by the microenvironments in which the fluorescent molecules are contained. In this work, FCS is used to monitor the dynamics of fluorescence emission from Cy5 labeled on DNA probes. We found that the FBKs of Cy5 can be tuned by having more or less unpaired guanines (upG) and thymines (upT) around the Cy5 dye. The observed FBKs of Cy5 are found to predominantly originate from the isomerization and back-isomerization processes of Cy5, and Cy5-nucleobase interactions are shown to slow down these processes. These findings lead to a more precise quantification of DNA hybridization using FCS analysis, in which the FBKs play a major role rather than the diffusion kinetics. We further show that the alterations of the FBKs of Cy5 on probe hybridization can be used to differentiate DNA targets with single-nucleotide differences. This discrimination relies on the design of a probe-target-probe DNA three-way-junction, whose basepairing configuration can be altered as a consequence of a single-nucleotide substitution on the target. Reconfiguration of the three-way-junction alters the Cy5-upG or Cy5-upT interactions, therefore resulting in a measurable change in Cy5 FBKs. Detection of single-nucleotide variations within a sequence selected from the Kras gene is carried out to validate the concept of this new method.

A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin.

The transcription factor (TF) Sp1 is a well-known RNA polymerase II transcription activator that binds to GC-rich recognition sites in a number of essential cellular and viral promoters. In addition, direct interference of Sp1 binding to DNA cognate sites using DNA-interacting compounds may provide promising therapies for suppression of cancer progression and viral replication. In this study, we present a rapid, sensitive and cost-effective evaluation of a GC intercalative drug, doxorubicin (DOX), in dissociating the Sp1-DNA complex using fluorescence correlation spectroscopy (FCS) in a microfluidic system. FCS allows assay miniaturization without compromising sensitivity, making it an ideal analytical method for integration of binding assays into high-throughput, microfluidic platforms. A polydimethylsiloxane (PDMS)-based microfluidic chip with a mixing network is used to achieve specific drug concentrations for drug titration experiments. Using FCS measurements, the IC50 of DOX on the dissociation of Sp1-DNA complex is estimated to be 0.55 microM, which is comparable to that measured by the electrophoretic mobility shift assay (EMSA). However, completion of one drug titration experiment on the proposed microfluidic-FCS platform is accomplished using only picograms of protein and DNA samples and less than 1 h total assay time, demonstrating vast improvements over traditional ensemble techniques.

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.

Applications of MEMS technologies in tissue engineering.

The success of therapeutic strategies within the fields of regenerative medicine, including tissue engineering, biomaterials engineering, and cell and tissue transplantation science, relies on researchers' understanding of the complex cellular microenvironments that occur within functional tissue. Microfabricated biomedical platforms provide tools for researchers to study cellular response to various stimuli with micro- and nanoscale spatial control. Initial studies utilizing relatively passive means of microenvironmental control have provided the fundamental knowledge required to begin to design microculture platforms that closely mimic these biological systems. In this review, we discuss second-generation cell and tissue culture platforms that utilize active components, borrowed from work in the development of microelectromechanical systems (MEMS). These microsystems offer the unprecedented opportunity to fabricate culture platforms designed to match tissue-specific growth parameters. In addition, the adoption of MEMS components opens up the door for future integration with the burgeoning field of microanalytical systems, providing analytical platforms that retain the sensitivity and resolution required within low-volume, microfluidic culture technologies.

Characterization of pulmonary cell growth parameters in a continuous perfusion microfluidic environment.

In vitro models of the alveolo-pulmonary barrier consist of microvascular endothelial cells and alveolar epithelial cells cultured on opposing sides of synthetic porous membranes. However, these simple models do not reflect the physiological microenvironment of pulmonary cells, wherein cells are exposed to a complex milieu of mechanical and soluble stimuli. In this report, we studied alveolar epithelial (A549) and microvascular endothelial (HMEC-1) cells within varying microfluidic environments as a first step towards building a microfluidic analog of the gas-exchange interface. We fabricated polydimethylsiloxane (PDMS) microdevices for parallel studies of cell growth under multiple flow rates. Cells adhered and proliferated in the microculture chambers for shear stresses up to approximately 2 x 10(-3) dynes/cm(2), corresponding to media turnover rates of approximately 53 seconds. Proliferation of these cells into confluent monolayers and expression of cell-specific markers (SP-A and CD-31) demonstrated successful pulmonary cell culture in microscale devices, a first for alveolar epithelial cells. These results represent the initial steps towards the development of microfluidic analogs of the alveolo-pulmonary barrier and tissue engineering of the lung.