On the design of low-cost fluorescent protein biosensors.

There is a large body of knowledge on proteins and their ligands that is available to the sensor researcher for the successful design of fluorescent biosensors. Chemically synthesized receptors rarely match the sensitivity and selectivity of proteins.Additionally, proteins are easily produced and manipulated through recombinant protein techniques. Although limitations exist in the prediction of signal response of proteins labeled with fluorescent probes, thoughtful experimentation can lead to useful, highly responsive fluorescent protein assays. Conversion of these assays into sensor devices may require additional manipulation of the fluorescence properties of the labeled proteins. We have shown that this can be achieved by a second fluorophore serving as a reference for ratiometric measurements. The choice of reference is contingent on the low-cost, miniaturized design of the device. Accordingly, the reference fluorophore is excitable with the same LED as the signal transducing probe and has a fluorescence decay lifetime that is orders of magnitude longer.Alternating illumination with intensity modulated light at two frequencies allows for ratiometric sensing without the need for bulky filter wheels while collecting the signals over a wide range of emission wavelengths. The result is a simple optoelectronics design that is cost-effective and small enough to be portable.In summary, the process of designing protein-based fluorescent biosensors for practical applications requires the systematic collaboration of a cross-disciplinary group of molecular biologists, chemists and engineers.