A nanoarray based-single molecule detection system was developed for detecting proteins with extremely high
sensitivity. The nanoarray was able to effectively trap nanoparticles conjugated with biological sample into
nanowells by integrating with an electrophoretic particle entrapment system (EPES). The nanoarray/EPES is
superior to other biosensor using immunoassays in terms of saving the amounts of biological solution and enhancing
kinetics of antibody binding due to reduced steric hindrance from the neighboring biological molecules. The
nanoarray patterned onto a layer of PMMA and LOL on conductive and transparent indium tin oxide (ITO)-glass
slide by using e-beam lithography. The suspension of 500 nm-fluorescent (green emission)-carboxylated polystyrene
(PS) particles coated with protein-A followed by BDE 47 polyclonal antibody was added to the chip that was
connected to the positive voltage. The droplet was covered by another ITO-coated-glass slide and connected to a
ground terminal. After trapping the particles into the nanowells, the solution of different concentrations of anti-rabbit-
IgG labeled with Alexa 532 was added for an immunoassay. A single molecule detection system could
quantify the anti-rabbit IgG down to atto-mole level by counting photons emitted from the fluorescent dye bound to
a single nanoparticle in a nanowell.
Core-shell nanoparticles with an upconverting phosphorescent, lanthanide core and plasmonic gold shell are employed
for Förster Resonance Energy Transfer (FRET). FRET is demonstrated from a highly efficient upconverting fluoride
nanoparticle doped with Ytterbium (Yb) and Erbium (Er) ions to Streptavidin conjugated with Tetramethyl rhodamine
fluorophore.
A nanoarray, integrated with an electrophoretic system, was developed to trap nanoparticles into their corresponding
nanowells. This nanoarray overcomes the complications of losing the function and activity of the protein binding to the
surface in conventional microarrays by using minimum amounts of sample. The nanoarray is also superior to other
biosensors that use immunoassays in terms of lowering the limit of detection to the femto- or atto-molar level. In
addition, our electrophoretic particle entrapment system (EPES) is able to effectively trap the nanoparticles using a low
trapping force for a short duration. Therefore, good conditions for biological samples conjugated with particles can be
maintained. The channels were patterned onto a bi-layer consisting of a PMMA and LOL coating on conductive indium
tin oxide (ITO)-coated glass slide by using e-beam lithography. The suspensions of 170 nm-nanoparticles then were
added to the chip that was connected to a positive voltage. On top of the droplet, another ITO-coated-glass slide was
covered and connected to a ground terminal. Negatively charged fluorescent nanoparticles (blue emission) were
selectively trapped onto the ITO surface at the bottom of the wells by following electric field lines. Numerical modeling
was performed by using commercially available software, COMSOL Multiphysics to provide better understanding about
the phenomenon of electrophoresis in a nanoarray. Simulation results are also useful for optimally designing a nanoarray
for practical applications.
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