OPTICAL DIAGNOSTICS/FLOW CYTOMETRY: Advances in optical biodetection

By Mike May

Light-based techniques reveal biological entities from DNA to pathogens, enabling such advances as point-of-care flow cytometry, super-sensitive diagnostics, and real-time gene analysis.

Optical biodetection encompasses light-based technologies that can image, sense, or analyze biological specimens or processes. From that gigantic range of possibilities, this article will cover three very different examples that demonstrate the diversity of techniques able to shine light on biological components: A point-of-care diagnostic platform based on microfluidics and flow cytometry from PARC, a Xerox company (Palo Alto, CA); a surface plasmon resonance (SPR) approach from BiOptix (Boulder, CO); and advanced optics for the real-time detection of the polymerase chain reaction (PCR) in an instrument made by Bio-Rad Laboratories (Hercules, CA).

Parsing pathogens

"Pathogen detection must be specific, and you need optics for an immediate response," says Peter Kiesel, principal scientist at PARC. "The main target of our work is a sophisticated point-of-care diagnostic."

To develop such a device, Kiesel and Noble Johnson, manager of PARC's optoelectronic materials and devices area, developed an innovative form of flow cytometry. In traditional flow cytometry, fluorescently labeled particles flow through an optical detector one by one, but Kiesel and Noble created a large detection area that tracks multiple particles. To provide spatial resolution, they added a mask that blocks the fluorescent light in a chosen random pattern between the targets and the detector. The mask creates a characteristic blinking signal from the particles. "We can extract that signal from the noise," says Kiesel.

For the labeling, says Noble, "we utilize existing assays. So we are relying on chemical selectivity to identify what it is blinking." He adds, "The key point is that we can realize a compact, robust optofluidic system that has the same level of performance as the large commercial flow cytometers." This compact system will not provide as many channels of detection as the robust cytometers designed for use in a lab—or as many parameters that can be adjusted—but it makes an ideal device for diagnosing pathogens on site.

The current version of this device is about as big as a shoebox, and consists of only a few hundred dollars worth of components. A smartphone provides enough computing power to do the analysis. With a black-and-white shadow mask, one substance—say, one type of pathogen—can be detected. "A patterned multi-color mask," Kiesel says, "enables recording several fluorescence channels with a single large-area detector, and detection of two substances has been demonstrated."

Beyond human medicine, this device could also be used in agricultural and veterinary sciences, plus industrial food processing, water-purity testing, and much more (see Fig. 1).

The handheld PARC prototype flow cytometer can detect biological components in a wide range of settings
FIGURE 1. The handheld PARC prototype flow cytometer can detect biological components in a wide range of settings. (Image courtesy of Peter Kiesel and Noble Johnson)

Sensing surface changes

Surface plasmon resonance (SPR) is "an advanced optical technology that can measure refractive-index changes on a sensor chip's gold surface due to a change in mass that occurs during a binding event," according to Ken Wilczek, vice president, sales and marketing at BiOptix. That binding can be a biological component attaching to a receptor, such as a protein binding an antibody.

In this way, SPR can determine many features of a sample, says Wilczek, including the concentration of target molecules, kinetic rate, and affinity constants." He continues: "Additional benefits of surface plasmon resonance include: the ability to identify binding events without the use of labels, high-sensitivity, higher-throughput capability, and the ability to monitor association and dissociation events in a real-time manner."

To enhance the capabilities of SPR, BiOptix developed a version of the technique called enhanced SPR (E-SPR). As Wilczek explains: "E-SPR, or phase-based SPR, differs from standard surface plasmon resonance detection methods in that it employs the inherently high optical sensitivity of interferometry with the low noise of leading SPR systems—utilizing an internal reference for increased measurement confidence." With E-SPR, a researcher can also track multiple components in a single sample (see Fig. 2). "This new class of SPR instrumentation from BiOptix has been designed to measure protein-protein and protein-small molecule interactions—down to 100 Daltons—in a high-throughput 96- or 384-well platform utilizing four parallel channels rather than a single flow cell," Wilczek says. This technology can unravel questions related to many scenarios, including small molecule-screening studies, protein-protein kinetics, antibody affinities and epitope mapping, oligosaccharide characterization, nucleic acid (DNA-DNA or DNA-RNA) hybridization, fragment screening, and biomolecule concentration measurements.

With the enhanced surface plasmon resonance employed in this platform, multiple components can be tracked in one sample
FIGURE 2. With the enhanced surface plasmon resonance employed in this platform, multiple components can be tracked in one sample. (Image courtesy of BiOptix)

Probing PCR

With polymerase chain reaction (PCR) nearing its 30th birthday, it's no surprise that this DNA-replicating technique serves as a workhorse in many biology laboratories. "PCR is a fundamental technique used not only by pure molecular biologists, but also by researchers in diverse disciplines who are examining the way the genetic code influences health, disease, and cell biology," according to Rachel Scott, Ph.D., amplification instruments business unit marketing manager, gene expression division at Bio-Rad Laboratories. Moreover, PCR goes beyond research labs. Scott says, "One of the key trends in real-time PCR is its adoption for industrialized applications, like food testing and molecular diagnostics."

Real-time PCR (RT-PCR) technology monitors the increasing fluorescent signal as the amount of replicated DNA increases during the PCR reaction. Probes bound to specific sequence stretches of the DNA or generic DNA-binding dyes provide the increasing strength in an optical signal, which correlates with the amount of DNA.

As an example, Scott describes Bio-Rad's CFX96 Touch Real-Time PCR Detection System: "Scanning just above the sample plate, the optics shuttle individually illuminates and detects fluorescence from each well." This system can also simultaneously track up to five targets per sample well. The technology behind the CFX detection uses LEDs. "The LED's and optical filter set's design maximize fluorescence detection for specific dyes in specific channels. Centering the optics shuttle above each well for every read, the light path is always fixed and optimal."

In all approaches to optically based biodetection, researchers must take special care in developing and adjusting the light-related components on a system. That fine-tuning lets researchers watch and record processes that seem unimaginable, even with an optical device as complex as the human eye.

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