Richard M. Ozanich

Rapid Multiplexed Flow Cytometric Assay for Botulinum Neurotoxin Detection Using an Automated Fluidic Microbead-Trapping Flow Cell for Enhanced Sensitivity

A bead-based sandwich immunoassay for botulinum neurotoxin serotype A (BoNT/A) has been developed and demonstrated using a recombinant 50 kDa fragment (BoNT/A-HC-fragment) of the BoNT/A heavy chain (BoNT/A-HC) as a structurally valid simulant. Three different anti-BoNT/A antibodies were attached to three different fluorescent dye encoded flow cytometry beads for multiplexing. The assay was conducted in two formats: a manual microcentrifuge tube format and an automated fluidic system format. Flow cytometry detection was used for both formats. The fluidic system used a novel microbead-trapping flow cell to capture antibody-coupled beads with subsequent sequential perfusion of sample, wash, dye-labeled reporter antibody, and final wash solutions. After the reaction period, the beads were collected for analysis by flow cytometry. Sandwich assays performed on the fluidic system gave median fluorescence intensity signals on the flow cytometer that were 2-4 times higher than assays performed manually in the same amount of time. Limits of detection were estimated at 1 pM (approximately 50 pg/mL for BoNT/A-HC-fragment) for the 15 min fluidic assay in buffer.

A Flow-Through Ultrasonic Lysis Module for the Disruption of Bacterial Spores

An automated, flow-through ultrasonic lysis module that is capable of disrupting bacterial spores to increase the DNA available for biodetection is described. The system uses a flow-through chamber that allows for direct injection of the sample without the need for a chemical or enzymatic pretreatment step to disrupt the spore coat before lysis. Lysis of Bacillus subtilis spores, a benign simulant of Bacillus anthracis, is achieved by flowing the sample through a tube whose axis is parallel to the faces of two transducers that deliver 10 W cm−2 to the surface of the tube at 1.4-MHz frequency. Increases in amplifiable DNA were assessed by real-time PCR analysis that showed at least a 25-fold increase in amplifiable DNA after ultrasonic treatment with glass beads, compared with controls with no ultrasonic power applied. The ultrasonic system and integrated fluidics are designed as a module that could be incorporated into multistep, automated sample treatment and detection systems for pathogens.

Defining cell culture conditions to improve human norovirus infectivity assays

Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional (3-D) tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that lead to more reproducible hNoV infectivity in vitro requires that the cell line be (1) of human gastrointestinal origin, (2) expresses apical microvilli, and (3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log(10) increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microscopy. In our hands, using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using qRT-PCR that measures all RNA vs. plaque assays that measure infectious virus.

Use of a Novel Fluidics Microbead Trap/Flow-Cell Enhances Speed and Sensitivity of Bead-Based Bioassays

Automated devices and methods for biological sample preparation often use surface functionalized microbeads (superparamagnetic or nonmagnetic) to allow capture, purification, and preconcentration of trace amounts of proteins, cells, or nucleic acids (DNA/RNA) from complex samples. We have developed unique methods and hardware for trapping either magnetic or nonmagnetic functionalized beads that allow samples and reagents to be efficiently perfused over a microcolumn of beads. This approach yields enhanced mass transport and up to fivefold improvements in assay sensitivity or speed, dramatically improving assay capability relative to assays conducted in more traditional “batch modes” (i.e., in tubes or microplate wells). Summary results are given that highlight the analytical performance improvements obtained for automated microbead processing systems using novel microbead trap/flow-cells for various applications including (1) simultaneous capture of multiple cytokines using an antibody-coupled polystyrene bead assay with subsequent flow cytometry detection; (2) capture of nucleic acids using oligonucleotide-coupled polystyrene beads with flow cytometry detection; and (3) capture of Escherichia coli 0157:H7 from 50-mL sample volumes using antibody-coupled superparamagnetic microbeads with subsequent culturing to assess capture efficiency.

A next-generation countermeasure architecture to prevent explosives attacks at large public events

A concept has been developed for a next-generation integrated countermeasure architecture to detect improvised explosive devices hidden on people or left behind in unstructured crowds. The work is part of the Standoff Technology Integration and Demonstration Program of the U.S. Department of Homeland Securitys (DHSs) Science and Technology Directorate. The architecture uses a layered-defense approach that automates screening operations, prioritizes threats, and mobilizes resources accordingly. A system tracks people in motion, integrating and automating sensor control and scan acquisition to optimize threat-identification accuracy and allocation of screening resources. A threat-based decision module prioritizes screening targets based on user-defined rules. Operators manage system-wide risk and mobilize field teams for interdiction. DHS is working with industry on technology development and testing to achieve the required level of system integration and economics in crowd conditions.

Preconcentrating Minicolumn Sensors for Trace Environmental Monitoring

Preconcentrating minicolumn sensors represent an effective approach to achieve a substantial preconcentration factor within a sensing device, enabling very low detection limits as is required for environmental sensing. A selectively sorbent phase collects and concentrates analyte molecules as sample liquid is pumped through the column, ultimately equilibrating the entire sorbent phase with the analyte concentration in the sample. At equilibration, the amount on the column will be proportional to the analyte concentration when working at trace concentrations. We have demonstrated radiometric detection for the detection of radionuclides with this sensor, incorporating scintillating material in the column with the sorbent phase. The pertechnetate form of 99-technetium can be sensed using a mixed column bed containing anion exchange resin and scintillating plastic beads, collecting scintillation light with a pair of photomultiplier (PMT) tubes external to the transparent column body. For hexavalent chromium, we can also collect anions with anion exchange resin. In this case, optical fibers are coupled to the column body to measure the optical absorbance as a function of the amount of hexavalent chromium captured by the column. Both sensors can detect their respective analytes to levels below water standards.

Bead-based assays for biodetection: from flow-cytometry to microfluidics

The potential for the use of biological agents by terrorists is a real threat. Two approaches for antibody-based detection of biological species are described in this paper: 1) The use of microbead arrays for multiplexed flow cytometry detection of cytokines and botulinum neurotoxin simulant, and 2) a microfluidic platform for capture and separation of different size superparamagnetic nanoparticles followed by on-chip fluorescence detection of the sandwich complex. These approaches both involve the use of automated fluidic systems for trapping antibody-functionalized microbeads, which allows sample, assay reagents, and wash solutions to be perfused over a micro-column of beads, resulting in faster and more sensitive immunoassays. The automated fluidic approach resulted in up to five-fold improvements in immunoassay sensitivity/speed as compared to identical immunoassays performed in a typical manual batch mode. A second approach for implementing multiplexed bead-based immunoassays without using flow cytometry detection is currently under development. The goal of the microfluidic-based approach is to achieve rapid (<20 minutes), multiplexed (≥ 3 bioagents) detection using a simple and low-cost, integrated microfluidic/optical detection platform. Using fiber-optic guided laser-induced fluorescence, assay detection limits were shown to be in the 100s of picomolar range (10s of micrograms per liter) for botulinum neurotoxin simulant without any optimization of the microfluidic device or optical detection approach.