Microarrays may be relatively inexpensive to produce, and capture several thousand items of information, but reading the fluorescence signals generated requires the use of dedicated (heavy and expensive) scanning instrumentation. This requirement has largely limited the utility of microarrays to research and drug discovery/development laboratories where such instrumentation is close at hand, said Levi Gheber, Ph.D., senior lecturer in Ben-Gurion University’s department of biotechnology engineering.
“Existing chip technologies generate spots of about 100 microns in diameter, spaced about 300–400 microns apart, and scanners are necessary because, using a standard fluorescence microscope, you can only fit in about nine spots within the field of view of a 20x objective. Yet, overcoming the need for large scanning instrumentation would make chip technology more portable and could even open the way to the development of a new generation of bioarrays for applications including point-of-care diagnostics and environmental monitoring.”
In theory, simply squeezing a greater number of much smaller spots on a slide would allow arrays to be read using a standard optical fluorescence microscope. “If an array had spots of just 1 micron in diameter rather than 100 microns, thousands would potentially be visible in one viewfield of a 20x objective lens,” Dr. Gheber continued. “The ability to generate arrays with much smaller, higher density spots would also require the use of far less biological material, and potentially allow shorter incubation times because of the reduced distances travelled by molecules within the spot.”
The situation is complicated, however, because smaller spots throw up new problems in terms of reduced signal intensity, decreased signal to noise ratio (SNR), and chip surface chemistry, before even considering the technicalities associated with accurately depositing potentially nano-sized spots of biological molecules. It’s a subject being addressed at length by the Ben-Gurion University researchers.
“Fluorescence intensity and signal to noise ratio are the most obvious hurdles,” Dr. Gheber continued. “Fluorescence signal is proportional to the area of the spot, rather than its diameter. If you try to reduce spot diameter 100-fold, from 100 microns to just 1 micron, for example, the spot area and, hence, signal intensity is actually reduced 10,000 times, while background noise stays constant. The problem is compounded further because, in fact, the reduction in signal intensity is even more dramatic due to the limited number of binding molecules per spot.
“Miniaturization must also take into account additional parameters concerning immobilization and chip-surface properties such as autofluorescence and coating homogeneity,” Dr. Gheber said. “There has, as far as we are aware, been little study of the many factors that would impact on how such tiny spots would perform.”
To investigate these parameters further, the Ben-Gurion University team devised mathematical models capable of evaluating multiple factors impacting SNR and predicting the properties of different chip surfaces. Their results suggest that, under suitable conditions, spots with diameter as low as 400 nm are detectable with an SNR above 10, at 1.3 mg/mL target molecule concentration. For spots of 1 micron diameter, detection is feasible at concentrations of just 1 ng/mL. Dr. Gheber presented results from this research at Select Biosciences’ “Advances in Microarray Technology” meeting in Stockholm.