Differentiating Live Versus Dead Gram-Positive and Gram-Negative Bacteria With and Without Oxidative Stress Using Buoyant Mass Measurements.

Expeditious and accurate determination of pathogenic bacteria cell viability is of great importance to public health for numerous areas including medical diagnostics, food safety, and environmental monitoring. In this work a cell buoyant mass classifier approach is presented to assess bacteria cell viability in real time. Buoyant mass measurements for live and dead Gram-positive and Gram-negative bacteria populations were acquired with a commercial suspended microchannel resonator, Archimedes, to generate receiver operating characteristic (ROC) curves. To quantitatively assess the difference in buoyant mass for live and dead bacteria populations, ROC curves were generated to demonstrate cell viability determination. The results are presented as a binary classifier with a decision boundary, above which cells are considered live and below which cells are considered dead. A decision threshold value is evaluated with consideration that a certain true positive rate (correct classification of a live cell) is maintained with an acceptable false positive rate. The potential for this approach to monitor cell viability in real time is significant, especially when considering multiple classifier dimensions such as buoyant mass and density. This classifier approach represents a next generation technique for rapid and label-free diagnostics based on cell feature measurements.

High-Throughput Flow-Through Direct Immunoassays for Targeted Bacteria Detection.

Regulatory authorities require analytical methods for bacteria detection to analyze large sample volumes (typically 100 mL). Currently only the Membrane Filtration and the Most Probable Number assays analyze such large volumes, while other assays for bacteria detection (ELISA, lateral flow assays, etc.) typically analyze volumes 1000 times smaller. This study describes flow-through direct immunoassays (FTDI), a new methodology for the targeted detection of bacteria in liquid samples of theoretically any volume. Flow-through direct immunoassays are performed in fluid-permeable microwells (e.g., wells of a filter well plate) that have a membrane on their bottom where the bacteria are trapped before their detection using a direct immunoassay. Two versions of FTDI assays for the detection of E. coli in 10 mL of sample were developed. A rapid FTDI assay that can be completed in less than 2.5 h can detect E. coli bacteria in levels down to 17 CFU/mL, and an ultrasensitive FTDI assay that employs an additional bacteria culturing step to boost the sensitivity can detect E. coli bacteria in levels lower than 1 CFU/mL in less than 5.5 h. All the steps of the assays, including the immunoassay steps, the culturing step, and the analytical signal measurement step are performed inside the well plate to decrease the chance of contamination and ensure a safe, easy process for the user. The assays were assessed and validated in tap water, river water, and apple juice samples, and the results suggests that the assays are robust, precise, and accurate. When the assays are performed in 96-well filter plates, a filter well plate vacuum manifold and a multichannel peristaltic pump are also used, so multiple samples can be analyzed in parallel to allow high-throughput analysis of samples.

3D printed self-supporting elastomeric structures for multifunctional microfluidics.

Microfluidic devices fabricated via soft lithography have demonstrated compelling applications such as lab-on-a-chip diagnostics, DNA microarrays, and cell-based assays. These technologies could be further developed by directly integrating microfluidics with electronic sensors and curvilinear substrates as well as improved automation for higher throughput. Current additive manufacturing methods, such as stereolithography and multi-jet printing, tend to contaminate substrates with uncured resins or supporting materials during printing. Here, we present a printing methodology based on precisely extruding viscoelastic inks into self-supporting microchannels and chambers without requiring sacrificial materials. We demonstrate that, in the submillimeter regime, the yield strength of the as-extruded silicone ink is sufficient to prevent creep within a certain angular range. Printing toolpaths are specifically designed to realize leakage-free connections between channels and chambers, T-shaped intersections, and overlapping channels. The self-supporting microfluidic structures enable the automatable fabrication of multifunctional devices, including multimaterial mixers, microfluidic-integrated sensors, automation components, and 3D microfluidics.

Isolation of intact bacteria from blood by selective cell lysis in a microfluidic porous silica monolith.

Rapid and efficient isolation of bacteria from complex biological matrices is necessary for effective pathogen identification in emerging single-cell diagnostics. Here, we demonstrate the isolation of intact and viable bacteria from whole blood through the selective lysis of blood cells during flow through a porous silica monolith. Efficient mechanical hemolysis is achieved while providing passage of intact and viable bacteria through the monoliths, allowing size-based isolation of bacteria to be performed following selective lysis. A process for synthesizing large quantities of discrete capillary-bound monolith elements and millimeter-scale monolith bricks is described, together with the seamless integration of individual monoliths into microfluidic chips. The impact of monolith morphology, geometry, and flow conditions on cell lysis is explored, and flow regimes are identified wherein robust selective blood cell lysis and intact bacteria passage are achieved for multiple gram-negative and gram-positive bacteria. The technique is shown to enable rapid sample preparation and bacteria analysis by single-cell Raman spectrometry. The selective lysis technique presents a unique sample preparation step supporting rapid and culture-free analysis of bacteria for the point of care.

Graphene Nanoplatelet-Polymer Chemiresistive Sensor Arrays for the Detection and Discrimination of Chemical Warfare Agent Simulants.

A cross-reactive array of semiselective chemiresistive sensors made of polymer-graphene nanoplatelet (GNP) composite coated electrodes was examined for detection and discrimination of chemical warfare agents (CWA). The arrays employ a set of chemically diverse polymers to generate a unique response signature for multiple CWA simulants and background interferents. The developed sensors' signal remains consistent after repeated exposures to multiple analytes for up to 5 days with a similar signal magnitude across different replicate sensors with the same polymer-GNP coating. An array of 12 sensors each coated with a different polymer-GNP mixture was exposed 100 times to a cycle of single analyte vapors consisting of 5 chemically similar CWA simulants and 8 common background interferents. The collected data was vector normalized to reduce concentration dependency, z-scored to account for baseline drift and signal-to-noise ratio, and Kalman filtered to reduce noise. The processed data was dimensionally reduced with principal component analysis and analyzed with four different machine learning algorithms to evaluate discrimination capabilities. For 5 similarly structured CWA simulants alone 100% classification accuracy was achieved. For all analytes tested 99% classification accuracy was achieved demonstrating the CWA discrimination capabilities of the developed system. The novel sensor fabrication methods and data processing techniques are attractive for development of sensor platforms for discrimination of CWA and other classes of chemical vapors.

Impedimetric Immunosensing in a Porous Volumetric Microfluidic Detector.

A sensitive and rapid impedemetric immunosensor is demonstrated utilizing porous volumetric microfluidic detection elements and silver enhanced gold nanoparticle probes. The porous detection elements significantly increase capture probe density and decrease diffusion length scales compared to conventional planar sensors to improve target capture efficiency and enhance impedance signal. In this work, a packed bed of silica beads functionalized with antibody probes serves as a porous sensor element within a thermoplastic microchannel, with an interdigitated gold electrode microarray used to measure impedance changes caused by the concentration dependent formation of silver aggregates. The measured impedance change is independent of electrode spacing, enabling a device with low resolution electrodes to achieve a sandwich immunoassay detection limit between 1-10 ng/mL with a 4-log dynamic range, with a total assay time of 75 min.

Surface functionalization of electrospun nanofibers for detecting E. coli O157:H7 and BVDV cells in a direct-charge transfer biosensor.

Electrospinning is a versatile and cost effective method to fabricate biocompatible nanofibrous materials. The novel nanostructure significantly increases the surface area and mass transfer rate, which improves the biochemical binding effect and sensor signal to noise ratio. This paper presents the electrospinning method of nitrocellulose nanofibrous membrane and its antibody functionalization for application of bacterial and viral pathogen detection. The capillary action of the nanofibrous membrane is further enhanced using oxygen plasma treatment. An electrospun biosensor is designed based on capillary separation and conductometric immunoassay. The silver electrode is fabricated using spray deposition method which is non-invasive for the electrospun nanofibers. The surface functionalization and sensor assembly process retain the unique fiber morphology. The antibody attachment and pathogen binding effect is verified using the confocal laser scanning microscope (CLSM) and scanning electronic microscope (SEM). The electrospun biosensor exhibits linear response to both microbial samples, Escherichia coli O157:H7 and bovine viral diarrhea virus (BVDV) sample. The detection time of the biosensor is 8 min, and the detection limit is 61 CFU/mL and 10(3)CCID/mL for bacterial and viral samples, respectively. With the advantage of efficient antibody functionalization, excellent capillary capability, and relatively low cost, the electrospinning process and surface functionalization method can be implemented to produce nanofibrous capture membrane for different immuno-detection applications.