Advances in microfluidics for environmental analysis.

During the past few years, a growing number of groups have recognized the utility of microfluidic devices for environmental analysis. Microfluidic devices offer a number of advantages and in many respects are ideally suited to environmental analyses. Challenges faced in environmental monitoring, including the ability to handle complex and highly variable sample matrices, lead to continued growth and research. Additionally, the need to operate for days to months in the field requires further development of robust, integrated microfluidic systems. This review examines recently published literature on the applications of microfluidic systems for environmental analysis and provides insight in the future direction of the field.

Design and development of a field-deployable single-molecule detector (SMD) for the analysis of molecular markers.

Single-molecule detection (SMD) has demonstrated some attractive benefits for many types of biomolecular analyses including enhanced processing speed by eliminating processing steps, elimination of ensemble averaging and single-molecule sensitivity. However, it's wide spread use has been hampered by the complex instrumentation required for its implementation when using fluorescence as the readout modality. We report herein a simple and compact fluorescence single-molecule instrument that is straightforward to operate and consisted of fiber optics directly coupled to a microfluidic device. The integrated fiber optics served as waveguides to deliver the laser excitation light to the sample and collecting the resulting emission, simplifying the optical requirements associated with traditional SMD instruments by eliminating the need for optical alignment and simplification of the optical train. Additionally, the use of a vertical cavity surface emitting laser and a single photon avalanche diode serving as the excitation source and photon transducer, respectively, as well as a field programmable gate array (FPGA) integrated into the processing electronics assisted in reducing the instrument footprint. This small footprint SMD platform was tested using fluorescent microspheres and single AlexaFluor 660 molecules to determine the optimal operating parameters and system performance. As a demonstration of the utility of this instrument for biomolecular analyses, molecular beacons (MBs) were designed to probe bacterial cells for the gene encoding Gram-positive species. The ability to monitor biomarkers using this simple and portable instrument will have a number of important applications, such as strain-specific detection of pathogenic bacteria or the molecular diagnosis of diseases requiring rapid turn-around-times directly at the point-of-use.

Fabrication of a cyclic olefin copolymer planar waveguide embedded in a multi-channel poly(methyl methacrylate) fluidic chip for evanescence excitation.

The fabrication and characterization of a novel cyclic olefin copolymer (COC) waveguide embedded in a poly(methyl methacrylate), PMMA, fluidic chip configured in a multi-channel format with an integrated monolithic prism for evanescent fluorescence excitation are reported. The fabrication approach allowed the embedded waveguide to be situated orthogonal to a series of fluidic channels within the PMMA wafer to sample fluorescent solutions in these channels using the evanescence properties of the waveguide. Construction of the device was achieved using several fabrication techniques including high precision micromilling, hot embossing and stenciling of a polymer melt to form the waveguide and coupling prism. A waveguide channel was fabricated in the fluidic chip's cover plate, also made from PMMA, and was loaded with a COC solution using a pre-cast poly(dimethylsiloxane), PDMS, stencil containing a prism-shaped recess. The PMMA substrate contained multiple channels (100 microm wide x 30 microm deep with a pitch of 100 microm) that were situated orthogonal to the waveguide to allow penetration of the evanescent field into the sampling solution. The optical properties of the waveguide in terms of its transmission properties and penetration depth of the evanescent field in the adjacent solution were evaluated. Finally, the device was used for laser-induced fluorescence evanescent excitation of a dye solution hydrodynamically flowing through multiple microfluidic channels in the chip and processed using a microscope equipped with a charge-coupled device (CCD) for parallel readout. The device and optical system were able to image 11 channels simultaneously with a limit-of-detection of 7.1 x 10(-20) mol at a signal-to-noise ratio of 2. The waveguide was simple to manufacture and could be scaled to illuminate much higher channel numbers making it appropriate for high-throughput measurements using evanescent excitation.

Charge-coupled device operated in a time-delayed integration mode as an approach to high-throughput flow-based single molecule analysis.

Single molecule detection (SMD) readouts are particularly attractive for assays geared toward high-throughput processing, because they can potentially reduce assay time by eliminating various processing steps. Unfortunately, most flow-based SMD experiments have generated low throughputs due primarily to the fact that they are configured in single assay formats. The use of a charge-coupled device (CCD) with flow-based SMD can image multiple single molecule assays simultaneously to realize high-throughput processing capabilities. We present, for the first time, the ability to simultaneously track and detect single molecules in multiple microfluidic channels by employing a CCD camera operated in time-delayed integration (TDI) mode as a means for increasing the throughput of any single molecule measurement. As an example of the technology, we have configured a CCD to operate in a TDI mode to detect single double-stranded DNA molecules (lambda and pBR322) labeled with an intercalating dye (TOTO-3) in a series of microfluidic channels poised on a poly(methyl methacrylate), PMMA, chip. A laser beam was launched into the side of the chip, which irradiated a series of fluidic channels (eight) with the resulting fluorescence imaged onto a CCD. Using this system, we were able to identify single DNA molecules based on the fluorescence burst intensity arising from differences in the extent of dye labeling associated with the DNA molecule length. The CCD/TDI approach allowed increasing sample throughput by a factor of 8 compared to a single-assay SMD experiment. A sampling throughput of 276 molecules s (-1) per channel and 2208 molecules s (-1) for an eight channel microfluidic system was demonstrated. Operated in its full capacity, this multichannel format was projected to yield a sample throughput of 1.7 x 10 (7) molecules s (-1), which represents a 170-fold improvement over previously reported single molecule sampling rates.