Studies On Quantum Dots And Quantum Dot-Tagged Fluorescent Microspheres For Biomedical Application

Semiconductor quantum dots (QDs), as called nanocrystals are often composed of elements from the II–VI or III–V groups (such as CdSe,CdTe,ZnSe,InP,InAs). QDs in the 1.5–10 nm size range have drawn widespread attention in the past decade. The development of functionalized QDs promoted the development of novel diagnostic tools and methods. The advantages of the organometallic method are the ability to create nearly perfect crystal structures and the possibility of high quantum yields. There has been much research on biocompatible QDs for bioanalytical labeling applications. Research has shown that QDs can be linked with biomolecules such as peptides, antibodies, and nucleic acids for use as fluorescent probes. QD-biomolecules in different conjugation protocols have been greatly exploited in a variety of fluoroimmunoassay, cell imaging, and DNA sequencing techniques.In this work, QD-protein conjugates were prepared with IgG as the binding bridge, which was attached to two different luminescent colloidal semiconductor quantum dots (QDs) via electrostatic self-assembly. The formation of the bio-conjugates resulted in fluorescence resonance energy transfer (FRET) between the two different QDs (green and red emitting). The luminescence of green-emitting QDs was quenched whereas the emission of the red-emitting QDs was enhanced. FRET efficiency (E) and the distance between the donor-acceptor were calculated and the influences of the ionic strength, and pH on the FRET process of QDs (G)–mouse IgG-QDs(R) bioconjugate system were studied. The use of polystyrene fluorescent microspheres conjugated to antigens and CdTe quantum dots (QDs) conjugated to antibodies as sensitive fluorescent labels in fluoroimmunoassay was studied. CdTe QDs were attached to antibodies via electrostatic/hydrophilic self-assembly. Both CdTe QDs labeled antibodies (anti IgGQD) and microspheres labeled antigens (IgGFM) were used to form a typical immunoassay on the surface of fluorescent microspheres. Because fluorescent QDs have broad excitation spectra and narrow emission bandwidths, the immunoreaction system with different emission of microspheres and QDs can be detected effectively at a single excitation wavelength. This approach allowed detection of goat-anti-rabbit IgG in the range of 0.5 to 10μg/mL. Furthermore, the immunocomplexes of anti IgGQD-IgGFM were confirmed by coincidence detection of the fluorescence signals of CdTe QDs and microspheres on a microfluidic on-chip device.Quantum dot-tagged microspheres are emerging as a new class of fluorescent labels and are expected to open new opportunities in nanotechnology and biology. For ultrasensitive high-throughput detection, the development of QD-tagged microspheres as a remarkable biomolecular probes can provide a more significant means to surmount the limitations and increase the advantages of QDs in biomedical applications. The basic concept is to develop smart microspheres that have not only conjugated biomolecules on the surface but also built-in codes for rapid target identification inside the microspheres. Polystyrene fluorescent microspheres prepared by deposition of CdTe Quantum Dots (QDs) are used in immunoassay. CdTe QDs/polyelectrolyte multilayers on the surface of polystyrene microspheres have been formed layer-by-layer self-assembly via electrostatic interactions. Rabbit IgG have been bound on the outermost layer of the fluorescent microspheres. The immunoreaction between fluorescent microspheres/rabbit IgG and corresponding antibody was confirmed by the change of the fluorescent spectra. This approach allowed detection of the rabbit IgG based on the change of fluorescent intensity. A novel microfluidic chip device with a laser-induced fluorescence system was established and used for the detection of fluorescent microspheres.In this work, multicolor quantum QD-encoded microspheres have been prepared via the layer-by-layer assembly approach. Polystyrene microspheres of 3μm diameter were used as templates for the deposition of different sized CdTe QDs/polyelectrolyte multilayers via electrostatic interactions. Two kinds of biofuntional multicolor microspheres with two different antibodies, anti-human IgG and anti-rabbit IgG were prepared. Human IgG and rabbit IgG can be detected as target antigens in the multiplexed fluoroimmunoassays. Furthermore, a novel microfluidic on-chip device was developed to detect two kinds of antigen-conjugated multicolor QD-encoded microspheres; the microspheres can be distinguished from each other based on their fluorescence signals, and we detected BVDV as actual sample.A study on the adsorption of rabbit immunoglobulin G onto CdTe quantum dots (QDs)/ polystyrene microspheres was presented. The adsorption appears to be sensitive to pH conditions and ionic strength. Maximum adsorption for protein was obtained near the isoelectric point. Adsorption isotherm analysis demonstrated that the electrostatic interaction plays an important role in the adsorption of protein. The thickness of adsorbed layer calculated from the maximal adsorption amounts (qm) is 6.5 nm, which indicates that the rabbit IgG molecules exist between the side-on and end-on mode in the monolayer. The bio-functional rabbit IgG/fluorescent microspheres were further used for the detection of antibody in fluoroimmunoassays. This approach allowed detection of goat-anti-rabbit IgG in the range of 1-100 ng/mL.