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 100's of picomolar
range (10's of micrograms per liter) for botulinum neurotoxin simulant without any optimization of the microfluidic
device or optical detection approach.
Field-portable sensor system are currently needed for the detection and characterization of biological pathogens in the environment. Nucleic acid analysis is frequently the method of choice for discriminating between pathogenic and non-pathogenic bacteria in environmental samples, however standard protocols are difficult to automate and current microfluidic devices are not configured to analyze environmental samples. In this paper, we describe an automated DNA sample processing system and demonstrate its use for the extraction of bacterial DNA form water and sediment samples. Two challenges in environmental sample analysis are the need to process relatively large sample volumes in order to obtain detectable quantities of DNA present at low concentrations, and the need to purify DNA form a complex sample matrix for downstream detection. These problems are addressed by using sequential injection fluid handling techniques for precise manipulation of the required volumes, and renewable separation columns for automatically trapping and releasing microparticles that are used for sample purification. The renewable microcolumns are used for both bacterial cell concentration and DNA purification. The purified bacterial DNA is then amplified using an on-line PCR module in order to produce detectable quantities of the target DNA.
Automated microfluidic analysis has historically been carried out by flow injection analysis techniques. Sequential injection analysis represents a more versatile method for automated fluid handling. We have explored the use of sequential injection analysis for performing microcolumn separations. These separations can be used as part of a microanalytical procedure, or for sample preparation. In addition, with detection of retained species on the microcolumn, sequential injection separation represents a technique for sensing. Recently, it has been demonstrated that sequential injection separation can be carried out with renewable separation columns--the beads with interactive surfaces can be delivered to the microcolumn, used for processing the sample, and discarded after each measurement. Delivery of new beads for each measurement provides a method for renewable surface separation and renewable surface sensing. Applications in environmental analysis and bioanalytical chemistry will be presented.
The purpose of this work is to develop a rapid, automated system for nucleic acid purification and concentration from environmental and food processing samples. Our current approach involves off-line filtration and cell lysis (ballistic disintegration) functions in appropriate buffers followed by automated nucleic acid capture and purification on renewable affinity matrix microcolumns. Physical cell lysis and renewable affinity microcolumns eliminate the need for toxic organic solvents, enzyme digestions or other time- consuming sample manipulations. Within the renewable affinity microcolumn, we have examined nucleic acid capture and purification efficiency with various microbead matrices (glass, polymer, paramagnetic), surface derivitization (sequence-specific capture oligonucleotides or peptide nucleic acids), and DNA target size and concentration under variable solution conditions and temperatures. Results will be presented comparing automated system performance relative to benchtop procedures for both clean (pure DNA from a laboratory culture) and environmental (soil extract) samples, including results which demonstrate 8 minute purification and elution of low-copy nucleic acid targets from a crude soil extract in a form suitable for PCR or microarray-based detectors. Future research will involve the development of improved affinity reagents and complete system integration, including upstream cell concentration and cell lysis functions and downstream, gene-based detectors. Results of this research will ultimately lead to improved processes and instrumentation for on-line, automated monitors for pathogenic micro-organisms in food, water, air, and soil samples.
Conference Committee Involvement (1)
Optics and Photonics in Global Homeland Security V