Bioassays are becoming ever more powerful. Among the latest developments are a micro-sonicator capable of lysing difficult cells such as anthrax spores, and an ultra high throughput screening technique which allows one operator to perform a million assays every day.
Californian company MicroFluidic Systems Incorporated (MFSI) has secured an exclusive license to US patent 6100084 entitled aMicro-sonicator for spore lysis' from Lawrence Livermore National Laboratory.
The licensed patent describes a device that couples ultrasonic energy to a fluid-filled chamber for the lysis, or bursting open, of cells or spores to release the internal cellular components for analysis. This is particularly critical for the use of the cell's nucleic acid, such as DNA, for specific genomic-based identification of the organism. Spores, which are formed by certain bacteria including anthrax, and were the subject of the mail-based bioterrorist threat last autumn, are particularly hard to lyse.
The patented device has been shown to lyse such spore-forming pathogenic bacteria in 30 seconds or less providing for the rapid, highly specific and sensitive, DNA-based identification of the organism. Other spore lysis methods can require over 45 minutes and special chemical treatments to obtain similar results.
The use of the DNA for analysis is recognised by many as the best, most specific method for pathogen identification, and the licensed device provides for key biodefence and other pathogen identification methods to be performed very efficiently.
"We are excited about the opportunity to develop pathogen sample processing systems for the US government and industry based on our exclusive rights to this powerful technology,“ said Phil Belgrader, co-inventor and vice president of MFSI. "We have already proven the high efficiency of this technology and are working with industrial partners to develop it into a product,“ he added.
MFSI was founded in 2001 and is focused on the development of automated microfluidic systems for biological assays. Among the company's other developments are what is believed to be the world's first miniaturised, portable, battery-operated, real-time, polymerase-chain-reaction (PCR)-based pathogen detection system.
Ultra high throughput screening
BioTrove has completed one million cell assays with Living Chip technology as part of a study to demonstrate its use in the ultra high throughput screening of genetic diversity.
The Living Chip-25K is a high-throughput screening tool that allows about 25000 nanolitre-volume reactions to be initiated and monitored simultaneously. One operator can perform as many as one million nanolitre-volume assays per day using a minimum of automation.
In the validation experiment, a mutagenised Bacillus library was screened by processing over 40 pairs of nanoplates for discovery of mutant strains with high protease expression.
Each nanoplate is a high-density array of 24576 isolated reaction containers in the same footprint as a standard microplate and each reaction container holds 35 nanolitres of fluid. Cells from the library were loaded onto a nanoplate at a density such that there is on average one cell clone in each container. A second nanoplate is loaded with a fluorescently-labelled substrate and stacked onto the cell colony plate to initiate protease hydrolysis in each reaction container of the array in a simultaneous and massively parallel manner.
The stacked plates are imaged and the high activity mutants are robotically cherry-picked for further analysis. Up to one million diversity assays can be performed by one scientist per day resulting in a tremendous increase in efficiency and productivity.
"We are very pleased with the outcome of this study as a validation of our technology for diversity discovery,“ said Tom Morrison, director of biomolecular discovery. "We plan to apply the technology to the discovery and optimisation of high-value biomolecules such as antibodies, peptides and enzymes,“ he added.
Earlier this year the Massachusetts Institute of Technology was granted US patent number 6 387 331 on the chip, which is now exclusively licensed worldwide to BioTrove.
The Living Chip consists of a precisely constructed, high-density array of micro-channels, or bottomless wells, in a plate. It can be thought of as a dense array of test-tubes that are easily filled and washed. The system combines the advantages of high-density arrays on glass slides, with the added functionality that liquids and gases may easily pass into and out of the channels. Other benefits include:
* Easy replication and storage of chemical and genetic libraries.
* Accurate, reliable dispensing and recovery. No gas bubbles.
* Sample densityof a microarray, higher probe/spot density, functionality of microplate.
* Instantaneous loading of 10000 to 1000000 precise aliquots of liquid.
* Massively parallel initiation of reactions by stacking chips.
* Homogeneous or inhomogeneous assays.
* Low background, sensitive optical imaging including fluorescence, absorbance and luminescence.
* Potential for integrated synthesis and screening operations.
