Environmental Sample Processor (ESP)
Arrays on the 2G ESP
The Second Generation (2G) ESP can run two types of assays within the core: sandwich hybridization arrays (SHAs) and enzyme-linked immunosorbent assays (ELISAs).
Sandwich hybridization arrays
In eukaryotes, RNA is transcribed to make ribosomal RNA (rRNA), the main constituent of ribosomes. Because the number of ribosomes per cell can be so much greater than gene copies in DNA, rRNA can be a good target for the sometimes-low concentration of target organisms in the ocean.
SHA detects rRNA by the following steps:
- A capture probe is printed on some type of solid-support surface. The pattern is printed repeatedly on sheets of nitro-cellulose; when finished, we cut out 25-millimeter disks to sit in array pucks.
- The ESP passes water through a filter, concentrating the (particulate) organisms contained in the sample, and creates a nucleic acid extract. That extract is pumped into the array puck, washing over the probe array. If the target rRNA is present (wavy white lines), it sticks to the capture probe while the rest of the extract sample is washed away. The sample extract is usually full of junk material, but the probes are specific, and very fine discrimination is possible.
- The next solution washed across the array substrate contains another probe (orange lines)—the digital signal probe—which attaches to a different region of the target molecule. This completes the “sandwich”of probe/target/probe.
- The signal probe generates a signal in the form of light, which is then recorded by a camera.
- The image is transmitted via radio modem to shore where it is processed to determine what sequences were detected and how intense the reaction was. The intensity of the light recorded is directly proportional to the amount of the target that is present. (Brighter spots indicate more target.)
Later, on shore, the assay can be verified by a matching 96-well bench run. In the benchtop assay, the presence of a target rRNA is indicated by a color change in the wells.
The advantage of this system is that sequences are captured directly from the homogenate (extract), which is not manipulated in any way. No purification is required, and the reagents are stable.