Rapid Microbiological Methods for a New Generation

These are exciting times, as 19th-century microbiological methods make way for rapid detection, quantification and characterization technologies.

By Michael J. Miller, Ph.D., Senior Research Fellow, Eli Lilly and Co.

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Microarrays are usually fabricated using a variety of technologies, including printing with fine-pointed pins onto glass slides, photolithography, ink-jet printing or electrochemistry. Other methods may also be used, such as in situ synthesis, whereby the probes are synthesized directly on the chip instead of spotting them on the array. An example would be CombiMatrix’s (Mukilteo, Wash.) CustomArray.

Applications for microarrays include nucleic acid sequence identification and measuring expression levels of genes. For example, Affymetrix’s (Santa Clara, Calif.) GeneChip contains the entire human genome (~50,000 known genes and gene variants) on a single chip, while CombiMatrix’s CustomArray contains probes that can detect influenza A and Avian H5N1 (bird flu) strains. Both of these platforms are commercially available.

Biosensors

A biosensor can detect an analyte that is comprised of a biological component combined with a physicochemical detector component. Nineteenth-century miners used a canary in a cage as a biosensor to detect lethal concentrations of gas. Today, the most widespread example of a commercially available biosensor is the blood glucose monitor, but future uses will include remote sensing of airborne bacteria and detection of pathogens.

The biological component of a biosensor may contain tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids or whole cells. The detector element works in a physicochemical manner and may include optical, electrochemical, thermometric, piezoelectric or magnetic technologies. An example of a currently available biosensor that performs immunological assays is the ICS Chip by Ambri (Chatswood, NSW, Australia). In this platform, the immunoassay components are spotted in 1-micron tall wells using less than 1 nanoliter of material.

Combination systems

Combining multiple MEMS platforms may result in a hybrid system that is superior to the individual platforms themselves. An example is ST Microelectronics’ (Geneva, Switzerland) In-Check silicon chip, which combines microfluidics and microarrays. This technology platform provides a fully integrated PCR reactor and a microarray used to hybridize and detect the PCR amplicons. The chip is mounted on a one-by-three-inch plastic slide that provides the necessary mechanical, thermal, electrical and fluidic connections.

A sample size of 2-8 µl is used, PCR reactions are three times faster than conventional thermocyclers, and a portable, customized fluorescent-based optical reader analyzes the microarray in a few seconds. The currently available platform hosts a pathogen panel to identify 10 sepsis-causing bacterial species as well as methicillin-resistant strains of Staphylococcus aureus from positive blood culture samples.

Nanotechnology

Nanotechnology operates at the atomic, molecular or macromolecular range of approximately 1 to 100 nanometers to create and use structures, devices, and systems that have novel properties. The key to this technology is high-voltage electron-beam (or E-beam) lithography, in which a beam of electrons is scanned across a surface covered with a thin film, called a resist. The electrons produce a chemical change in the resist, which allows the surface to be patterned.

 

Rapid Microbiological Methods: BioForce Nanosciences' nanoarrays
Nanoarrays rapidly screen minute biological samples, including proteins, DNA, RNA and whole viruses, as well as non-biological samples such as chemical solutions and particle suspensions. Courtesy of BioForce Nanosciences, Inc.

Nanotechnology has allowed us to fabricate nanoarrays, with molecules placed at defined locations on a surface with nanometer spatial resolution. Nanoarray spots can include biological samples such as proteins, DNA, RNA and whole viruses, as well as non-biological samples such as chemical solutions, colloids and particle suspensions.

Nanoarrays are the next evolutionary step in the miniaturization of bioaffinity tests for proteins, nucleic acids and receptor-ligand pairs. These arrays utilize approximately 1/10,000th of the surface area occupied by a conventional microarray, and over 1,500 nanoarray spots would occupy the area required for a single microarray spot.

For example, BioForce Nanosciences’ (Ames, Iowa) NanoArray prints biological and non-biological materials onto silicon chips (at right) and other surfaces with ultra-micro spot sizes ranging from 1-20 µm, and in the nanometer range to 250 nm. This technology utilizes surface patterning tools, or SPTs, microcantilever-based micro-fluidic handling devices. The droplet volumes these microcantilevers deliver to the nanoarray are in the femtoliter and attoliter range (10-12 milliliter and 10-15 milliliter, respectively).

Nanoarrays offer a number of advantages, including label-free detection (via atomic force microscopy). They work in both solutions and biological liquids, retaining biological activity for subsequent analyses.

Optical spectroscopy and instantaneous detection

Optical spectroscopy measures the interactions between light and the material being studied. Light scattering is a phenomenon in which the propagation of light is disturbed by its interaction with particles.

In a Mie scattering particle detector, airborne particles intersect a light beam emanating from a laser diode. If the air is free of particles, a “beam blocker” at the center of the first convex lens stops this laser beam. If the air contains particles, they will scatter the laser beam and cause part of the light to deviate at an angle from the incident beam. This scattered light will be collected by the second lens and focused onto a photo detector, which converts the light intensity to an electrical signal. This is the basis for a novel, instantaneous and continuous microbial detector, currently in development at BioVigilant Systems (Tucson, Ariz.).

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