Application of magnetic/phosphorescent nanoparticles in immunoassays

Transcription

1 Application of magnetic/phosphorescent nanoparticles in immunoassays Ian M. Kennedy and Dosev Dosev, Mikaela Nichkova, Bruce Hammock, Shirley Gee, R. Dumas, Kai Liu Department of Mechanical and Aeronautical Engineering, Department of Entomology, Department of Physics University of California Davis Introduction Magnetic particles are widely used in biochemistry for a variety of biochemical and biomedical applications such as magnetic separation, MRI imaging, drug delivery etc Lanthanide oxides as phosphors Narrow emission spectra due to electronic transitions Single nanoparticle of lanthanide oxide can contain thousands of luminescent ions Large Stokes shift Long lifetime Excellent photostability Possibility of low price synthesis (good for environmental application, large amounts of samples) 1

2 ropium oxide spectra ropium 80x Excitation spectrum observed at 610 nm Emission spectrum excited at 466 nm Intensity (arbitrary units) Wavelength (nm) Intensity (arbitrary units) 4x Excitation spectrum observed at 548 nm Emission spectrum excited at 524 nm Rhodamine Wavelength (nm) Spray pyrolysis Nitrates of the precursors are dissolved in alcohol. The solution is sprayed in a hydrogen flame. Oxide particles are formed in the flame and are collected thermophoretically on a cold finger. easily controlled chemical composition large variety of available precursors reproducible results no need for post-synthesis annealing high production rate Temperature controls phase Fast quench yields non equilibrium phases 2

3 Spectral properties of :Gd 2 Exc: UV<280 nm, 395 nm, 466 nm Em: nm ( 5 D 0 7 F 2 transition of the 3+ ion) Lifetime: 1-2 msec Optimal doping concentration achieved 20 at.% Greater doping concentration than achieved with other synthesis methods Spectral properties of Tb:Gd 2 and Sm:Gd 2 Tb:Gd 2 emission spectrum Exc. 260 nm 5 D 4 7 F 5 (545 nm) 4 G 5/2 6 H 7/2 (597 nm) 4 G 5/2 6 H 9/2 (645 nm) 3

4 Spectral properties mixed doping Doping of Y 2 with more than one dopant. - same excitation wavelength simultaneous emission Example - UV - different excitation wavelength different emissions from one type of particles Example 466 nm Sm:Gd2O3 nanoparticles produces by spray pyrolysis fluorescent properties Emission under UV excitation Upconverting emission under various excitation wavelengths D. Dosev, I. M. Kennedy, M. Godlewski, K. Tomsia, I. Gryczynski, E.M. Goldys; Fluorescence upconversion in Sm doped Gd 2, Applied Physics Letters

5 :Y 2 lifetime Intensity, arb. units Intensity, Arb. units Wavelength, nm Time, µs Wavelength, nm Time, µs Pure 2 :Y 2 Tb:Y 2 lifetime Intensity, arb. units Wavelength, nm Time, µs 5

6 Time gated detection Micropattern based immunoassay using :Gd2O3 nanoparticles as fluorescent labels Stage 1 Avidin biotin system: optimization of protocols, control of non-specific binding Stage 2 Specific recognition using IgGs, IgG particle coating, control of binding sites, qualitative immunoassay, SEM evaluation Stage 3 Introducing internal fluorescent standard, new polymer coating of the :Gd 2 nanoparticles, quantitative immunoassay for detection of PBA. 6

7 Stage 1 Avidin biotin system: optimization of protocols, control of non-specific binding Epifluorescent microscope image Dosi Dosev, Mikaela Nichkova, Maozi Liu, Bing Guo, Gangyu Liu, Younan Xia, Bruce D. Hammock, Ian M. Kennedy; Application of fluorescent :Gd 2 nanoparticles to the visualization of protein micropatterns, Journal of Biomedical Optics 2006 Stage 2 Specific recognition using IgGs, IgG particle coating, control of binding sites, qualitative immunoassay, SEM evaluation -:Gd 2 particles were coated with anti-rabbit IgG via physical adsorption TEM characterization bare 100 nm particle Resulting binding sites pmol IgG/ mg nanoparticles µg IgG / mg nanoparticles Coating IgG concentration Saturation of particle surface coated 100 nm particle Mikaela Nichkova, Dosi Dosev, Richard Perron, Shirley J. Gee, Bruce D. Hammock, Ian M. Kennedy 3+ - doped Gd 2 nanoparticles as reporters for optical detection and visualization of antibodies patterned by microcontact printing, Analytical Biochemistry

8 Stage 2 Specific recognition using IgGs, IgG particle coating, control of binding sites, qualitative immunoassay, SEM evaluation Confocal microscope image - printed rabbit IgG - blocking BSA - anti-rabbit IgG labeled with :Gd 2 Mikaela Nichkova, Dosi Dosev, Richard Perron, Shirley J. Gee, Bruce D. Hammock, Ian M. Kennedy 3+ - doped Gd 2 nanoparticles as reporters for optical detection and visualization of antibodies patterned by microcontact printing, Analytical Biochemistry 2006 Stage 2 Specific recognition using IgGs, IgG particle coating, control of binding sites, qualitative immunoassay, SEM evaluation SEM characterization - printed rabbit IgG - blocking BSA - anti-rabbit IgG labeled with :Gd 2 Mikaela Nichkova, Dosi Dosev, Richard Perron, Shirley J. Gee, Bruce D. Hammock, Ian M. Kennedy 3+ - doped Gd 2 nanoparticles as reporters for optical detection and visualization of antibodies patterned by microcontact printing, Analytical Biochemistry 8

9 Stage 3 Introducing internal fluorescent standard, new polymer coating of the :Gd 2 nanoparticles, quantitative immunoassay for detection of PBA. bare particle PL (A) NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 NH3Br O H3N O NH n O FITC (B) IgG (C) (d) NH3Br PL PL coated particle fluorescein antigen - fluorescein A- PL coating; B- Evaluation of surface amino groups C- Covalent binding of antibodies D- Evaluation of active binding sites Mikaela Nichkova, Dosi Dosev, Shirley J. Gee, Bruce D. Hammock, Ian M. Kennedy; Microarray immunoassay for phenoxybenzoic acid using polymer encapsulated :Gd 2 nanoparticles as fluorescent labels, Analytical Chemistry Stage 3 Introducing internal fluorescent standard, new polymer coating of the :Gd 2 nanoparticles, quantitative immunoassay for detection of PBA. (A) (B) A- microcontact printing of the coating antigen (BSA-PBA) B- immobilization of the internal standard (BSA-fluorescein) (C) + + C- preincubation of the labeled antibody with the analyte (PBA) D- incubation of the micropatterns with the labeled antibody (D) -PBA -BSA-PBA - BSA-fluorescein -:Gd 2 nanoparticles Mikaela Nichkova, Dosi Dosev, Shirley J. Gee, Bruce D. Hammock, Ian M. Kennedy; Microarray immunoassay for phenoxybenzoic acid using polymer encapsulated :Gd 2 nanoparticles as fluorescent labels, Analytical Chemistry 9

10 Stage 3 Introducing internal fluorescent standard, new polymer coating of the :Gd 2 nanoparticles, quantitative immunoassay for detection of PBA. 0 µg L -1 5 µg L µg L µg L -1 The detection limit of the PBA is in the low ppb range. Mikaela Nichkova, Dosi Dosev, Shirley J. Gee, Bruce D. Hammock, Ian M. Kennedy; Microarray immunoassay for phenoxybenzoic acid using polymer encapsulated :Gd 2 nanoparticles as fluorescent labels, Analytical Chemistry Current status of heterogeneous immunoassays Primary antibodies are immobilized on plain surface such as 96- well plate, glass slide, microarray, etc. 10

11 Current status of heterogeneous immunoassays Primary antibodies are immobilized on plain surface such as 96- well plate, glass slide, microarray, etc. Current status of heterogeneous immunoassays Primary antibodies are immobilized on plain surface such as 96- well plate, glass slide, microarray, etc. Measurements rely on absolute fluorescence intensity reading. 11

12 Novel format for immunoassay using magnetic/luminescent particles Primary antibodies are immobilized on the particle s surface. Measurements rely on relative (ratiometric) fluorescence intensity reading. Novel format for immunoassay using magnetic/luminescent particles Emission spectra of the Co:Nd:Fe 2 / :Gd 2 core/shell particles and the IgG-Alexa Fluor 350 bound to their surface (excitation at 350 nm). 12

13 Internal standard normalization Novel format for immunoassay using magnetic/luminescent particles 20 Alexa Fluor 350/ rabbit IgG concentration, µg/ml Calibration curve for the competitive magnetic immunoassay for rabbit IgG. The signal of the labeled antigen (rabbit IgG-Alexa Fluor 350) bound on the surface of the magnetic nanoparticles is normalized vs the luminescence of the particles. 13

14 1. Magnetic & Fluorescent particles synthesized by spray pyrolysis 1. Synthesis of paramagetic Nd:Fe2O3 nanoparticles (cores) by spray pyrolysis 2. Growth of luminescent :Gd 2 shell on the surface of the paramagnetic cores In solution after pulling out the solution Specific magnetisation, emu mg -1 4x10-3 3x10-3 2x10-3 1x x x x x10-3 Cores Core/shell Magnetic field, Oe VSM characterization - cores Magnetic separation from solution Characteristics of magnetic/luminescent core/shell particles From one to several primary Co:Nd:Fe 2 particles can be embedded in a single composite particle. Shell with thickness of 10 to 50 nm can be distinguished. 14

15 Characteristics of magnetic/luminescent core/shell particles In contrast to the ferromagnetic core characteristics, the composite particles showed paramagnetic behavior with magnetization at comparable levels to that of primary magnetic cores. Possible phase transformation during the second spray pyrolysis. Magnetic properties Luminescent properties were identical with that of pure :Gd 2 particles Other applications of nanophosphors Antibody labeled particles to image antigens on Raji lymphoma cells, showing polarization can be used for nanometer scale X-ray imaging with beam line from Advanced Light Source at LBL Magnetic phosphor particles can be used to apply forces to cellular structures while observing optically - probably needs Fe:Co 15

16 For lasing: Pump source Gain medium Optical cavity Optical Resonances Droplet lasing: Laser (Nd:YAG, Argon ion) Fluorescent dye, QD Microdroplet X= 2πr λ Computer Experimental Setup Controller CCD Camera Notch Filter Spectrometer Laser Beams Inkjet Droplet Generator Fluorescent Signal Delay Generator Function Generator Oscilloscope Collection Optics Nd:YAG or Argon Ion Filter + Focusing Optics He-Ne Laser (633 nm) Focusing Optics Filters + Photodiode Droplet Generator 16

17 E-Coli detection 20 µm droplets 10-5 M R6G in PBS with E. Coli KB FRET experiments in microdroplets 17

18 Droplets in a microchannel Conclusions Spray pyrolysis is a promising method for synthesis of nanoparticles with variety of properties including the valuable combination of magnetic and luminescent characteristics. Although magnetic properties of the synthesized particles can be further optimized, the particles are suitable for magnetic separation from water solution The combination of magnetic and luminescent properties within the same particle enables a new format of immunoassays to be developed based on ratiometric measurement with internal luminescent standard. A non-optimized immunoassay was demonstrated for detection of rabbit IgG to demonstrate the novel immunoassay format. 18

19 Acknowledgements National Science Foundation, Grant DBI , The Superfund Basic Research Program with Grant 5P42ES04699 from the National Institute of Environmental Health Sciences, NIH, U.S. Department of Agriculture, under Award No