Superfund Research Program
Nanoparticle Immunoassays - Advances & Spin-offs
Background: The Superfund Basic Research Program at the University of California, Davis has developed a range of bioassays for the detection of environmental toxins, their metabolites or tracers. Drs. Ian Kennedy and Bruce Hammock are leading an SBRP project to take the next step in the evolution of these assays into small scale, rapid, sensitive multi-analyte instruments for use in the laboratory and ultimately in field portable instruments.
High sensitivity detection can be achieved in miniaturized immunoassay systems with new, optically efficient luminescent labels for biomolecules. Nanoparticles made of lanthanide oxides (e.g., europium, terbium) are promising fluorophores as a new class of tag. These particles have unique spectral properties including large Stokes shift (i.e., low overlap between emission and absorption spectra), sharp emission spectra, long lifetime and good photo-stability. These properties greatly reduce background signal-to-noise, vastly improve detection limits, increase sample stability, reduce sample volume requirements, facilitate quantitation and allow for new inexpensive assay formats.
Advances: The UC Davis team applied flame spray pyrolysis to engineer a novel type of nanoparticle that has both luminescent and magnetic properties. The particles have magnetic cores of iron oxide doped with cobalt and neodymium, and luminescent shells of europium-doped gadolinium oxide (Eu:Gd2O3). The composite particles’ magnetic characteristics were evaluated with a Vibrating Sample Magnetometer and results showed an overall paramagnetic response (i.e., their motion can be influenced by an externally applied magnetic field). Fluorescence spectroscopy demonstrated spectra typical of the Eu ion in a Gd2O3 host – a narrow emission peak centered near 615 nm. The UC Davis synthesis method offers low-cost, high-rate synthesis, allowing a wide range of biological applications of magnetic/fluorescent core/shell particles.
The researchers demonstrated the potential for application of these nanoparticles to the visualization of protein micropatterns using a standard assay (the interaction between avidin and biotin). First, they coated the nanoparticles with avidin through physical adsorption. Next, they patterned biotinylated Bovine Serum Albumin (BSA-b) on a silicon wafer using a micro-contact printing technique. The wafer was incubated in a solution containing the avidin-coated nanoparticles and the luminescent core of the nanoparticle provided a signal that indicated the number of avidin-biotin complexes. The specific interaction between biotin and avidin was confirmed by fluorescent microscopy and atomic-force microscopy (AFM). The fluorescent microscopic images revealed that the nanoparticles were organized onto the areas defined by the micro-contact printing process – non-specific binding of the avidin-coated nanoparticles to bare substrate was negligible. In contrast to organic dyes, the fluorescent nanoparticle tag did not suffer any photobleaching during the observation, demonstrating the suitability of Eu:Gd2O3 nanoparticles as fluorescent labels with extended excitation periods. More detailed studies were performed using AFM at a single nanoparticle level. The specific and the non-specific binding densities of the particles were qualitatively evaluated.
The UC Davis researchers also explored binding functional groups on the surface of the Eu:Gd2O3 nanoparticles. The nanoparticles were encapsulated in poly-L-lysine (PL) and then covalently bound to antibodies. The bioconjugate nanoparticles successfully served as reporters in a competitive fluorescence microimmunoassay for a biomarker of human exposure to pyrethroid insecticides (phenoxybenzoic acid, PBA). The microarray immunoassay demonstrated a limit of detection of 1.4 µg/L PBA. This work suggests the potential application of lanthanide oxide nanoparticles as fluorescent probes in microarray and biosensor technology, immunodiagnostics and high-throughput screening.
The research team has also applied magnetic/phosphor nanoparticles to a “lab-on-a-chip”. Manipulation of the nanoparticles and separation was achieved with external electromagnets. The external magnets also served to accelerate immuno-reactions by increasing mass transfer rates for rapid detection while consuming very little reagent volume. This work established that using external electromagnets for mixing for an assay inside a micro-channel shortens the incubation time, as compared to assay with static reagents, by a factor of about 10.
Significance: To understand the impact of chemical exposures on human populations, environmental levels as well as the internal doses of the chemicals must be quantified. Much environmental health research is hindered by the large numbers of samples required and the high costs of analyses. Antibody-based detection method, particularly miniaturized “lab-on-a-chip” systems such as those being developed by the UC Davis SBRP, could yield high-throughput, inexpensive, portable devices that can analyze small sample volumes (micron-sized droplets) and reduce time-consuming sample preparation/analysis procedures. Although initially developed for environmental applications, the general utility as an analytical detection method for other fields, including clinical medicine and homeland security, is evident.
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To learn more about this research, please refer to the following sources:
- Nichkova MJ, Dosev D, Perron R, Gee SJ, Hammock BD, Kennedy IM. 2006. Eu3+-doped Gd2O3 nanoparticles as reporters for optical detection and visualization of antibodies patterned by microcontact printing. Anal Bioanal Chem 384(3):631-637. PMID:16416096
- Dosev D, Nichkova MJ, Liu M, Guo B, Liu G, Hammock BD, Kennedy IM. 2005. Application of luminescent Eu:Gd2O3 nanoparticles to the visualization of protein micropatterns. J Biomed Opt 10(6):064006. doi:10.1117/1.2136347 PMID:16409071
- Feng J, Shan G, Maquieira A, Koivunen ME, Guo B, Hammock BD. 2003. Functionalized europium oxide nanoparticles used as a fluorescent label in an immunoassay for atrazine. Anal Chem 75(19):5282-5286.
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