Joel P. Golden

The good, the bad, and the tiny: a review of microflow cytometry

Recent developments in microflow cytometry have concentrated on advancing technology in four main areas: (1) focusing the particles to be analyzed in the microfluidic channel, (2) miniaturization of the fluid-handling components, (3) miniaturization of the optics, and (4) integration and applications development. Strategies for focusing particles in a narrow path as they pass through the detection region include the use of focusing fluids, nozzles, and dielectrophoresis. Strategies for optics range from the use of microscope objectives to polymer waveguides or optical fibers embedded on-chip. While most investigators use off-chip fluidic control, there are a few examples of integrated valves and pumps. To date, demonstrations of applications are primarily used to establish that the microflow systems provide data of the same quality as laboratory systems, but new capabilities—such as automated sample staining—are beginning to emerge. Each of these four areas is discussed in detail in terms of the progress of development, the continuing limitations, and potential future directions for microflow cytometers.

Array biosensor for detection of biohazards

A fluorescence-based biosensor has been developed for simultaneous analysis of multiple samples for multiple biohazardous agents. A patterned array of antibodies immobilized on the surface of a planar waveguide is used to capture antigen present in samples; bound analyte is then quantified by means of fluorescent tracer antibodies. Upon excitation of the fluorophore by a small diode laser, a CCD camera detects the pattern of fluorescent antibody:antigen complexes on the waveguide surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. This array biosensor has been used to detect toxins, toxoids, and killed or non-pathogenic (vaccine) strains of pathogenic bacteria. Limits of detection in the mid-ng/ml range (toxins and toxoids) and in the 10(3)-10(6) cfu/ml range (bacterial analytes) were achieved with a facile 14-min off-line assay. In addition, a fluidics and imaging system has been developed which allows automated detection of staphylococcal enterotoxin B (SEB) in the low ng/ml range.

Multi-wavelength microflow cytometer using groove-generated sheath flow

A microflow cytometer was developed that ensheathed the sample (core) fluid on all sides and interrogated each particle in the sample stream at four different wavelengths. Sheathing was achieved by first sandwiching the core fluid with the sheath fluid laterally via fluid focusing. Chevron-shaped groove features fabricated in the top and bottom of the channel directed sheath fluid from the sides to the top and bottom of the channel, completely surrounding the sample stream. Optical fibers inserted into guide channels provided excitation light from diode lasers at 532 and 635 nm and collected the emission wavelengths. Two emission collection fibers were connected to PMTs through a multimode fiber splitter and optical filters for detection at 635 nm (scatter), 665 nm and 700 nm (microsphere identification) and 565 nm (phycoerythrin tracer). The cytometer was capable of discriminating microspheres with different amounts of the fluorophores used for coding and detecting the presence of a phycoerythrin antibody complex on the surface of the microspheres. Assays for Escherichia coli were compared with a commercial Luminex flow cytometer.

Simultaneous detection of six biohazardous agents using a planar waveguide array biosensor

Recently, we demonstrated that an array biosensor could be used with cocktails of fluorescent antibodies to perform three assays simultaneously on a single substrate, and that multiple samples could be analyzed in parallel. We extend this technology to demonstrate the simultaneous analysis of six samples for six different hazardous analytes, including both bacteria and protein toxins. The level of antibody cross-reactivity is explored, revealing a possible common epitope in two of the toxins. A panel of environmental interferents was added to the samples; these interferents neither prevented the detection of the analytes nor caused false-positive responses.

Multiplexed detection of bacteria and toxins using a microflow cytometer.

A microfabricated flow cytometer was used to demonstrate multiplexed detection of bacteria and toxins using fluorescent coded microspheres. Antibody-coated microspheres bound biothreat targets in a sandwich immunoassay format. The microfluidic cytometer focused the microspheres in three dimensions within the laser interrogation region using passive groove structures to surround the sample stream with sheath fluid. Optical analysis at four different wavelengths identified the coded microspheres and quantified target bound by the presence of phycoerythrin tracer. The multiplexed assays in the microflow cytometer had performance approaching that of a commercial benchtop flow cytometer. The respective limits of detection for bacteria (Escherichia coli, Listeria, and Salmonella) were found to be 10(3), 10(5), and 10(4) cfu/mL for the microflow cytometer and 10(3), 10(6), and 10(5) cfu/mL for the commercial system. Limits of detection for the toxins (cholera toxin, staphylococcal enterotoxin B, and ricin) were 1.6, 0.064, and 1.6 ng/mL for the microflow cytometer and 1.6, 0.064, and 8.0 ng/mL for the commercial system.

Two simple and rugged designs for creating microfluidic sheath flow

A simple design capable of 2-dimensional hydrodynamic focusing is proposed and successfully demonstrated. In the past, most microfluidic sheath flow systems have often only confined the sample solution on the sides, leaving the top and bottom of the sample stream in contact with the floor and ceiling of the channel. While relatively simple to build, these designs increase the risk of adsorption of sample components to the top and bottom of the channel. A few designs have been successful in completely sheathing the sample stream, but these typically require multiple sheath inputs and several alignment steps. In the designs presented here, full sheathing is accomplished using as few as one sheath input, which eliminates the need to carefully balance the flow of two or more sheath inlets. The design is easily manufactured using current microfabrication techniques. Furthermore, the sample and sheath fluid can be subsequently separated for recapture of the sample fluid or re-use of the sheath fluid. Designs were demonstrated in poly(dimethylsiloxane) (PDMS) using soft lithography and poly(methyl methacrylate) (PMMA) using micromilling and laser ablation.

Detection of multiple toxic agents using a planar array immunosensor

A planar array immunosensor, equipped with a charge-coupled device (CCD) as a detector, was used to simultaneously detect 3 toxic analytes. Wells approximately 2 mm in diameter were formed on glass slides using a photoactivated optical adhesive. Antibodies against staphylococcal enterotoxin B (SEB), ricin, and Yersinia pestis were covalently attached to the bottoms of the circular wells to form the sensing surface. Rectangular wells containing chicken immunoglobulin were used as alignment markers and to generate control signals. After removing the optical adhesive, the slides were mounted over a scientific grade CCD operating at ambient temperature in inverted (multipin phasing) mode. A two-dimensional graded index of refraction lens array was used to focus the sensing surface onto the CCD. Solutions of toxins were then placed on the slide. After rinsing, Cy5-labeled antibodies were introduced. The identity and amount of toxin bound at each location on the slide were determined by quantitative image analysis. Concentrations as low as 25 ng/mL of ricin, 15 ng/mL of pestis F1 antigen, and 5 ng/mL of SEB could be routinely measured.

Array biosensor: optical and fluidics systems.

Optical and fluidics systems have been developed as central components for an automated array biosensor. Disposable planar waveguides are patterned with immobilized capture antibodies using a physically isolated patterning (PIP) method. The PIP method enables simultaneous deposition of several antibodies and completely circumvents cross-immobilization problems encountered with other array deposition processes. A multi-channel fluidics cell allows numerous assays to be performed on the patterned waveguide. The sensing arrays are optically interrogated using a diode laser with a tailored output to optimize coupling to and maximize excitation uniformity within the waveguide. A patterned cladding is employed to optically isolate the waveguide from perturbations induced by the permanently attached flow cells. Compact optics image the evanescently excited fluorescence onto a large area, cooled CCD array. The image data is processed and automated signal analysis corrects for local background and noise variations.

A Portable Array Biosensor for Detecting Multiple Analytes in Complex Samples

The Multi-Analyte Array Biosensor (MAAB) has been developed at the Naval Research Laboratory (NRL) with the goal of simultaneously detecting and identifying multiple target agents in complex samples with minimal user manipulation. This paper will focus on recent improvements in the biochemical and engineering aspects of this instrument. These improvements have enabled the expansion of the repertoire of analytes detected to include Salmonella typhimurium and Listeria monocytogenes, and also expanded the different sample matrices tested. Furthermore, all components of the biochemical assays could be prepared well in advance of sample testing, resulting in a “plug-and-play” methodology. Simultaneous detection of three toxins (ricin, staphylococcal enterotoxin B, and cholera toxin) was demonstrated using a novel fluidics cube module that limits the number of manipulations to only the initial sample loading. This work demonstrates the utility of the MAAB for rapid analysis of complex samples with multianalyte capability, with a minimum of operator manipulations required for either sample preparation or final analysis.

A fiber optic biosensor: combination tapered fibers designed for improved signal acquisition

Abstract One of the most critical features of an evanescent wave fiber optic biosensor is the design of the fiber probes sensing region. The initial fibers tested exhibited poor sensitivity, primarily due to loss of fluorescent signal which was collected by the sensing region but which failed to propagate back in the clad fiber due to V-number mismatch. Earlier work has shown that tapering of the sensing region improves coupling of the fluorescence signal. While sensitivity was increased, there was a lack of reproducibility of signal magnitude from fiber to fiber. To produce a more consistent fiber probe which maintained sensitivity, signal return along the length of the probe was investigated. An effective design, termed a combination taper, was found which provided an even signal return along the fibers length. The fiber tapers down to the V-number matching radius over 1 cm, maintaining total internal reflection, then continues to taper gently along the following 9 cm to near the distal end. The combination taper fiber provides not only the desired sensitivity, but also improved reproducibility.