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Lab-On-a-Chip Applications Development (LOCAD) Science

©Kjell Ove Storvik

Bacterial cell walls

All bacterial cells are contained by membranes and molecules that are thicker than those of human or animal cells. Such solid cell walls allow bacteria to survive in environments that would be harmful to human or animal cells. Interestingly, most bacteria can be grouped into two categories – Gram positive or Gram negative – based on whether the result of a technique called the Gram stain is positive or negative. If the result is positive, the bacteria are a deep purple color. If the result is negative, they are a light red or pink. These results are based on the use of two dyes – purple and red – to stain the bacteria. Gram positive (purple) bacteria have a very thick cell wall (see Gram positive picture below) that allows purple dye to flow into the cell, but not out. Gram negative (light red/pink) bacteria have a double-membrane cell wall (see Gram negative picture below), that allows the purple dye to flow in and back out of the cell. They may then be stained by a red dye. The bodies of many animals recognize molecules in bacterial cell walls as substances that could be harmful to them. LOCAD-PTS has modified a reaction like this from the horseshoe crab in order to detect lipopolysaccharide (see below, Gram negative), lipoteichoic acid (see below, Gram positive), and beta-glucan from fungi.

Gram negative bacterial cell wall

Gram positive bacterial cell wall

Horseshoe Crabs (Limulus polyphemus)

Horseshoe crabs are invertebrates that live in salt water marshes and are related to spiders, ticks, and scorpions (see picture below). Two large compound eyes are located on the top of its shell, and seven pairs on appendages on the underside allow it to move and sense the presence of food. In order to protect the horseshoe crab from getting an infection, a series of proteins, called enzymes, in the crabs’ blood or hemolymph are able to detect even a single bacterium and signal the formation of a blood clot to immobilize the bacterial invader.

Horseshoe crabs on a beach

These horseshoe crab enzymes form the basis for the LOCAD-PTS technology, allowing the unit to be small, rapid, and sensitive. The liquid to be tested is placed into the PTS cartridge with the horseshoe crab enzymes. If even a small amount of a bacterial molecule is present, these enzymes detect it and have been modified to create a change in color rather than a blood clot (see enzyme cascade picture below).

Enzyme “cascade” from the horseshoe crab that is used in the
LOCAD-PTS to create a change in color.

Future Technology:
1) Microfluidics

About ten years ago, the field of microfluidics began to grow. More researchers became aware of the potential applications of these tiny chips etched with minute channels. On a microfluidic chip, experiments that would have taken days in a standard laboratory and large volumes of sample now required only hours and a very small volume. Because the channels and wells on a microfluidic chip are so small, reactions proceed more quickly and smaller sample volumes are needed. Experiments may be combined end-to-end on a microfludic chip so that a sample proceeds from one test to the next.

Caliper 42 microfluidic workstation

These little chips indeed have the potential to become little “laboratories-on-a-chip”. Future LOCAD technologies plan to use microfluidic chips to contain possibly hazardous samples, decrease sample volumes, and provide rapid results. Additionally, integrated chips could prepare a sample for future tests that the sample will undergo on the same chip.

Two different colored fluids meet in a microfluidic channel.

2) Microarrays

Microarrays are a molecular biology technology employed by researchers the world over. In a microarray, thousands of DNA or protein molecules are “arrayed” or printed in rows and columns in a particular pattern.

Example of a DNA microarray.
Courtesy of Verena Stake, Carnegie Institute for Science.

Each DNA or protein spot in the array will only react with one other DNA or protein molecule, making microarrays a very rapid method for checking a sample for specific DNA or protein molecules. Future LOCAD technology intends to employ this technique to permit rapid, specific testing of a sample for thousands of different molecules at the same time. Left: A protein microarray. Each row filled with small spots represents one type of antibody that specifically recognizes a particular molecule. The second row of antibodies has positively recognized their specific molecule.