Label-less fluorescence-based method to detect hybridization with applications to DNA micro-array.
By coupling scattered light from DNA to excite fluorescence in a polymer, we describe a quantitative, label-free assay for DNA hybridization detection. Since light scattering is intrinsically proportional to number of molecules, the change in (scattering coupled) fluorescence is highly linear with respect to percent binding of single stranded DNA (ssDNA) target with the immobilized ssDNA probes. The coupling is achieved by immobilizing ssDNA on a fluorescent polymer film at optimum thickness in nanoscale. The fluorescence from the underlining polymer increases due to proportionate increase in scattering from double stranded DNA (dsDNA) (i.e., probe-target binding) compared to ssDNA (i.e., probe). Because the scattering is proportional to fourth power of refractive index, the detection of binding is an order of magnitude more sensitive compared to other label-free optical methods, such as, reflectivity, interference, ellipsometry and surface-plasmon resonance. Remarkably, polystyrene film of optimum thickness 30 nm is the best fluorescent agent since its excitation wavelength matches (within 5 nm) with wavelength for the maximum refractive index difference between ssDNA and dsDNA. A quantitative model (with no fitting parameters) explains the observations. Potential dynamic range is 1 in 10(4) at signal-to-noise ratio of 3:1.
Niu
Sanjun
S
Department of Chemical Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
Singh
Gaurav
G
Saraf
Ravi F
RF
D004247:DNA / Q000737:chemistry*; D004563:Electrochemistry / Q000295:instrumentation; D005453:Fluorescence; D005456:Fluorescent Dyes; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis
D005456:Fluorescent Dyes; D004247:DNA