Upconverting Labels in Biology

1
Upconverting Labels in Biology
Where are we now and where are we going?
C.G. Morgan, S. Dad and A.C. Mitchell Biomedical
Sciences Research Institute University of
Salford UK
2
Fluorescence/Luminescence is used in
Biology for- Ultrasensitive Detection
Sensing with environmentally responsive labels
Sensitivity almost always depends on ability to
reject background signals
Background- Scattered light (Rayleigh and
Raman) Ambient light Fluorescence from
contaminants
3
Upconverting Materials can be excited with
low-intensity IR and emit UV/Visible radiation
No excitation of background fluorescence
Exciting radiation easily filtered out Raman
scattering not a problem Relatively long decay
time with pulsed excitation allows use when
ambient light is present
We need surface-functionalised water-stable
nanoparticles for biological labelling
4
Upconverting Phosphors (1)
Electron Trapping Phosphors are charged by
exposure to UV/Visible light or radiation
Emission is stimulated by IR light
SrS/Sm/Eu
Laser pointer is 980nm
IR-Quenched phosphors are also known (typically
doped II-VI material)
ZnS/Cu/Pb
5
Optically Quenched Phosphors have scarcely been
investigated since WW2
incident IR light
Doped ZnS sample excited by near UV (Sample
courtesy of Phosphor Technology Ltd (UK))
6
Electron Trapping (Storage) Phosphors
Basically two
types Visible/UV activated, IR stimulated
emission (examples, SrSSmEu, CaSSmEu,
ZnSCuPb) X-Ray/Radiation activated, IR
stimulated emission (example, Eu2-activated
BaFBr)
7
Any use as biological labels?
For this we need nanoparticles
SNAGS SrS, CaS (but not
ZnS) are immediately hydrolysed in water and
need surface protection as nanoparticles.
Emission spectra can be broad and this limits
uses Particles must be charged before use
BUT Sensitivity to IR can be high and is
linear with intensity Stimulation possible in
tissue window (650-900nm) Red/Far red
emission also penetrates tissues well
(Upconverting or Optically-Quenched QDs?)
8
Upconversion Phosphors (2) Multiphoton Excitation
(photographed through prism)
Upconversion in Yb3 /Er3 glass ceramic
coverslip excited at 980nm
9
2H11/2
GREEN
4S3/2
Sensitised Upconversion
4F9/2
RED
2F5/2
4I11/2
4I15/2
2F7/2
Yb3
Er3
10
Why bother with upconverting labels?
Improved background rejection...hence better
sensitivity of detection Compatibility with
highly fluorescent samples without
interference Improved multiplexing
capability Wider dynamic range for
proximity-based immunoassays and hybridisation
assays New types of binding assay for
detection of large molecules, viruses etc.
11
Binding Assays in Biology
Many bioassays depend on the detection
of proximity between molecules e.g.-
Immunoassays where antibodies bind to targets
Nucleic acid hybridisation for genomics
Detection of enzyme activity (cleaving a target)
One popular format is the sandwich assay
12
FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET)
IMMUNOASSAY
FRET from donor to acceptor is only efficient if
the two are in very close proximity (within about
5nm usually)
blue light excites green fluorescent label
radiationless energy transfer
red fluorescent label excited indirectly by
resonance energy transfer and gives red emission
13
PROBLEM WITH FRET ASSAY RELIES ON NOT
DIRECTLY EXCITING ACCEPTOR - BUT SOME LEVEL OF
EXCITATION IS HARD TO AVOID. THIS LIMITS
DYNAMIC RANGE OF ASSAY BECAUSE FREE ACCEPTOR IS
EXCITED ALONG WITH BOUND LABEL
blue light also excites red fluorescent label (
Bound and free!)
blue light excites green fluorescent label
radiationless energy transfer
14
Fluorescein
An example of a widely used FRET donor-acceptor
pair The light that excites fluorescein also
excites rhodamine to some extent
Rhodamine
15
Another Problem with FRET Some overlap between
emission of the donor and acceptor
Applies to all FRET assays including
time-resolved assays
16
A further problem with FRET
Real-world samples often have lots of
fluorescent contaminants. These are also
excited and their emission gives a
strong BACKGROUND signal
This is a particular nuisance because FRET assays
are often designed to be 'mix-and-measure' to
avoid time-consuming separation and washing steps
and background plagues this type of measurement
17
Can Upconverting Nanoparticles be used for FRET?
Potential Advantages No background from
impurities! No Direct Acceptor Excitation!
No interference from narrow UCP emission in
spectral region where acceptor fluorescence
is measured!
FRET
fluorescent label
analyte
IR
But!
Must use small nanoparticles for efficient
FRET Need low phonon host for efficiency
'Surface Engineering' is critical for UC particle
18
Very low emission between erbiums green and red
emission gives an ideal window for FRET
measurements of sensitized acceptor
fluorescence
1 x107
Emission Intensity (counts/sec.)
8 x106
Emission Intensity (counts/sec.)
6 x106
4 x106
Wavelength (nm)
19
Possible Snag!
'core-shell' upconverting nanoparticle
Typical biospecific ligands are quite large and
increase the distance between label and
upconverter
20
UC FRET now demonstrated?
50nm NaYF4/Er/Yb
avidin
7nm gold colloid
sandwich of polyanion and biotinylated polycation
Claims gt60 quenching of upconverted
luminescence presumed by FRET to gold acceptor!
Wang, L.Y. et al, Angewandte Chem. Int. Ed.
44(37) 6054, 2005
21
FRET to a fluorescent Acceptor has also been
achieved recently!
Ball-milled commercial phosphor c. 300nm
particle Avidin/biotin chemistry Surface
bound phycoerythrin as label
SNAG- Most of the upconverter is out of range
of FRET Reabsorption gives some background
signal
Kuningas, K. et al Anal. Chem. (2005), 77,
7348-7355
22
A Word of Caution!
Commercial PTIR 550 NaYF4YbEr phosphor
Green Filter
Red Filter
Manufacturing process for commercial
phosphors can result in inhomogeneous samples.
This can be a problem for interpretation of
spectroscopic data
23
Er 3 emission depends on the power density of
incident excitation
Red emission increases more rapidly than green
emission for focused excitation of Er 3 and a
violet peak emerges
Emission Intensity
High Power Density
Low Power Density
400
450
500
550
600
650
700
Wavelength (nm)
24
Upconverted Emission from Ytterbium/Erbium
doped Heavy Metal Fluoride Glass Excited at 980nm
Laser Diode c. 10mW C.W.
Tightly focused
Defocused
25
Photobleaching is seen for some upconverting
samples. The mechanism isnt yet clear, but this
might have implications for bioassay development
Ex. 980nm Em. 550nm c.16mW focused spot
26
Is FRET the answer for UC-based bioassays? Fine
for small analytes but what about
viruses, spores, bacteria etc that are much
bigger! If we use FRET from an upconverting
label to an extended fluorescent surface (e.g. a
microsphere) the distance dependence is less
steep so bigger analytes can be studied
For even larger analytes we suggest a new
format NEAR-FIELD REABSORPTION BIOASSAY
27
Hypothetical far-field assay Fluorescence
from a Donor is absorbed by a fluorescent
Acceptor within a microsphere Useless with
conventional fluorescent donor because of direct
acceptor excitation
28
For Once Physics is on our side!
Spatially averaged radiation pattern from a
fluorescent label in the near field close to a
high index microsphere Emission couples
preferentially into the higher index medium
29
Microsphere Resonator with surface-bound label
If the microsphere is undoped part of the light
is totally internally reflected. The
microsphere becomes an optical cavity
If the microsphere is doped with an absorbing
fluorophore the 'Q' of the cavity is
reduced The dye is excited efficiently
30
Novel ideas for upconverting phosphors in bioassay
Upconverting Glasses can have high refractive
indices- this suggests that they might be used
for surface-selective excitation of fluorescent
labels using EVANESCENT WAVE excitation
31
Penetration of evanescent wave into air
Upconverting glass ceramic excited with 980nm at
low power density gives primarily green
luminescence emission, much of which is totally
internally reflected and exits at the edge. An
evanescent wave is present at the surface. This
can excite surface-bound fluorophores
radiationlessly.
32
Surface monolayers of labels are very easily
detected on upconverting TIRF substrate
Potential applications for - Microarray
detection for genomics etc. Multicolour TIRF
microscopy without special geometry or
equipment (C.G. Morgan and A.C.
Mitchell, Biosensors and Bioelectronics,
in press)
33
Multiple Internal Reflection in an Upconverting
Coating
Where the optical path is an integral number of
wavelengths Whispering Gallery standing waves
are established
34
How Can We Use Evanescent Wave for Homogeneous
Assay?
Upconverting Coatings on Micro-resonators-
Whispering-Gallery Modes enhance
evanescent-wave coupling to surface bound labels
35
Challenge!
Can we coat microspheres with efficient UC
layers? Sol-gel on silica/glass?
Sputtering, laser ablation on polymer? Seeded
nanoparticle growth on polymer?
Then coat with high index, low phonon barrier
layer and functionalise surface. Use hollow
microspheres for neutral buoyancy in aqueous
suspension?
36
SUMMARY
We dont really yet know just how well
the upconverting particles will work
for FRET but it looks good!
Nanoparticles are presently reported to be c.
100-fold less efficient than bulk
material for upconversion This probably
doesnt matter too much...still easily
detected with very cheap IR lasers
Core-shell particles offer possible higher
IR-VIS conversion efficiency but maybe
lower FRET efficiency. Increased Q.Y.
would however increase Ro somewhat.
Core-Shell no matter if a reabsorption
format or FRET to an extended surface
is used! There are also good
opportunities for assays and sensors
using evanescent wave coupling to upconverting
surfaces!
37
Thanks are due to BBSRC for support in areas
related to upconverting waveguides through the
Consortium for Postgenomic Research. We also
wish to acknowledge generous recent support of
time-resolved imaging for proteomics from BBSRC
and support for development of novel optical
sensing technology from EPSRC