This is second part on Barcoding technologies for multiplexed assays. Luminex paved the way for multiplexed assays using encoded particles and certainly more will follow the success. Optically encoded particles like Luminex beads are limited to 100 beads probably because they run into wavelength problems. Luminex beads are made by filling small polystyrene balls with mix of two flurophore with similar excitation wavelength but different emission wavelength. In addition, one extra fluorescent molecule is needed to detect biomolecular interaction. Very soon instrumentation needed to keep track of all fluorescent molecules become expensive and bulky. Several other variations on optical encoding technologies have been proposed/tested
- Encoding particles with quantum dots of varying intensity levels as well as emission wavelengths
- Encoding particles with fluorescent dyes with different fluorescent decay time and then use time resolved fluorescence spectroscopy
- Various combinations of fluorescently doped silica particles
In addition to the wavelength problems, other challenges of for optically encoded particles are to manufacture them reproducibly at reasonable cost and the instrumentation.
Radio frequency encoding is another possibility and has the possibility of making vary large number (>1012) of distinct particles. PharmaSeq is developing this technology.
Dot coded particles uses acrylate polymers and photopolymerization to encode particles. Another novelty in this technology is that it impregnated each particle with probes. This scheme can possibly generate 1020 unique codes (http://web.mit.edu/doylegroup). Another approach imprints colloids with unique surface pattern that reminds me of my trip to grocery store where each apple is stamped with unique code that can be read by scanner (http://www.engin.umich.edu/research/lahann/lahann.htm). Barcoded metal rods with different pattern of gold and silver particles have different reflectance to identify each particles and fluorescence is used for bio-molecular interaction readout (http://research.chem.psu.edu/cdkgroup/index.htm).
Though promising the technical challenges for all encoding technologies are
- reproducibly making these particles at reasonable cost
- easy readout/instrumentation again at reasonable cost
- conjugation chemistry to attach the biomolecules to particles
Sensitivity and selectivity compared to conventional ELISA based technologies
These technologies must also pass the “customer need” test. The “coolness” of the technology is not in doubt as is evident for publications in high profile journals but in the process of writing this I realized that after the initial hype most of these technologies slowly fade from lime light. A possible reason is that they may be trying to solve a problem that may not exist. Do we really need to measure 1020 different analytes. Is more the better?