Luminescent lanthanides
Evolution of reporter systems for immunoassays
iVik UNIVERSITY
W OFTURKU
Part I - Luminescent lanthanides and time-resolved fluorescence
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Immunoassays are used to quantify molecules
of interest based on specific recognition by antibodies
assay sensitivity, i.e. lower limit of detection, is defined by
- binding affinity of the labeled antibody
* Soukka, T. et a/. (2001) Anal. Chem. Anal Chem 73: 2254-2260
- detectability of the label attached to the antibody
- non-specifically bound fraction of the labeled antibody
separation
specific recognition
non-specific interaction
background
solid-phase
Jackson, TM and Ekins, RP (1986) J Immunol Methods 87: 13. https://doi.org/10.1016/0022-1759(86)90338-<j|^ ofVurku™
sandwich-type immunoassay
[Ab Ag Ab*]
specific recognition
solid-phase
[Ab*]
nsb
non-specific interaction |_
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co
CO
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<
ii
o
theoretical immunoassay limit of detection
SVT = specific activity rate/vol/time x vol x time MT = backround rate/time x time
[Ag]
[Ag]
tot
I od
Gorris, H.H. and Soukka, T. (2022). Anal Chem 94: 6073-6083. https://doi.org/10.1021/acs.analchem.1c05591
Fluorescent labels in immunoassays
enable rapid, accurate and quantitative detection
excitation emission
>■
CD I—
S;
s,
Detector
Excitation X,
So
Light source
Emission
JU
Excitation light
Emission light
Stokes' shift is the difference between X.i and A.-
fluorescent labels in immunoassays provide
- high specific activity
(number of detectable events per time unit per label)
- multiplexing capability
(using fluorescent labels with different spectral properties or spatial information on arrays)
Kricka, LJ. and Park, JY. (2014). Pathobiology of Human Disease, 3207-3221. doi:10.1016/b978-0-12-386456-7.06302-4
Autofluorescence
in immunoassays with conventional fluorophores
470 nm   520 nm
ex em
excitation emission
1000-
800-
AA
Stokes' shift = 40 nm
-1-1-1-1-1-1-■-1-'-1-1-1-1-1—
400       450       500       550       600       650       700 750
Wavelength (nm)
800 900
		—n
	1 1	
470 nm VW^S
background plastics     at > 470nm
autofluorescence originating from sample and plastics is major limitation in detectability
950      1000 1050
university of turku
Recognition of time-resolved fluorescence
for reduction of the autofluorescence background in immunoassays
EXCITATION
jj    , FLUORESCENCE WITH SHORT DECAY
L\
■ \ I \ \ ■	.FLUORESCENCE WITH LOtN	G DECAY
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DELAY TIME 10
COUNTING TIME At
Fig. 4. Dlapwn 01 tim©-f^solved fluoromotrk: maAs^emorits
h »m       ii—nmiii !■ mMii ii ■■! *»i ■«! i^ni^ft ptH ptm 1 «1 I
<cJ ha ctapMd. M| aMtfi flma »w *ort daor ttm* c— »^      k raduoad to i^m faro.
WaJluc. Wallac I .id was founded by Jnrma W'allasvaara in l'>5D in Turku. 'l*he company specialized in the production of laboratory instruments. 'ITie early product lines included radiometers, which were the coinp.im's mam product until (he lW's. In ihe l°"(ls, I->kki Sotni started to study tracer compounds that could replace radioisotopes. In 1974, the company began to study time-resolved fluorescence. In 1984, the company introduced a new product based on this technology, the immunological assay method, Dclfta. In the I Wis, this liecame the company's main product line.
CLIN. C+CM. 25/3,353-361(1979) Ruoroimmunoassay: Present Status and Key Problems
Erkkl Solnl1 and llkka Hemmlla3
Fluorescence immunoassay of biological fluids (for example, blood samples) is discussed. We attempt to chart present methods of assay as well as new possibilities. Different fluorescent probes, their detection limit, and methods for reduction of background are discussed: methods for separating the free and bound fraction are also m reviewed. Special consideration Is given to the possibilities
•! of enhancing sensitivity by developing both instruments
and chemical methods, and in particular to the possibilities inherent in time-resolved fluorometric applications and to the use of metal chelates in this application.
Soini, E. and Hemmila, I. (1979) Clin Chem. 25: 353-361
Background reduction in time-resolved fluorometry
1000
o c
<D Ü «
1—
o ■c
*j (0
■
E
1000
100
10
0.1
0.01
150
time-resolved fluorescence (200 ms delay)
850
-1:50 diluted whole blood, uv-excitation at 340 nm
300
500
700
K / wavelength
900
1100
Elimination of autofluorescence
using time-resolved fluorometer for lanthanide chelates
Lanthanide chelates and time-resolved fluorescence detection provide potentially very high detection sensitivity for the following reasons:
• Signal/photon emission ("specific activity") can be increased by a stronger excitation
• Background fluorescence from sample "blank" can be discriminated by using time-resolved detection
• Lanthanide concentrations in biological samples are normally negligible
• Lanthanide labels are biochemically inert (no interaction with the sample)
Soini, E. and Kojola, H. (1983) Clin Chem. 29: 65-68
Scattered encilaltün light
Wavelength t nm
Principle of time-resolved detection of lanthanide chelate luminescence. Luminescence is counted at lanthanide specific wavelength band is collected at delayed time-window.
Review:
Gudgin Dickson, E.F., et al. (1995) J. Photochem Photobioi B: Biol. 27: 3-19
Long lifetime luminescence emission
of rare earth metal complexes of organic light-harvesting ligand
first singlet exaled stale
triplet donor
acceptor
•rsystcm crossing
nlramclecuio' energy transfer
f*citot ion
Antenna ligand needed for excitation: direct ion absorption is low due to forbidden electric dipole f-f transitions.
Light absorbing organic ligand
Coordinated water (H20) is strong quencher of europium(lll).
Prediction of number of water molecules
in the first coordination sphere of a europium(lll).
ligand
Schematic diagram of the radiative processes of the chelate leading to Eu metal ion fluorescence.
q =1-11 [TÍb-r^-0.31] (Horrocks equation)
Supkowski, R.M. and Horrocks, Jr. W.D. (2002) Soini, E. and Hemmilä, I. (1979) Clin Chem. 25: 353-361     in0rg Chim Acta 340:44-48
university ofturku
35 r
Sm3,      Eu3,      Gd3,       Tb3' Dy3,
Principal emission lines of certain lanthanide ions.
The excitation and emission spectra of Eu3+ and Tb3+. The metal ion emits energy as narrow-band emission. The excitation band is typically broad and the Stokes shift more than 200 nm.
Soini, E. and Lovgren, T.. (1987). CRC Crit Rev Anal Chem 18: 105-154. doi: 10.1080/10408348708542802
I
c
LU
Anlenna
Ln
Si n a 01 01 s
to -* w ■ -*
o o w o o o
3 3 3 3 3 3
3 3 3 3 3 3
Eu"
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						---			
		■				♦			
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Emission characteristics of lanthanide chelates containing various lanthanidc ions			
Lanthanide	Approx. peak wavelength	Color of principal	Approx. luminescence
ion	of principal emission band	emission	lifetime
	(nm)		(MS)
Sm (111)	645	red	30-100
Eu (III)	615	orange	100-1000
Tb (III)	545	green	400-2000
Dy (III)	573	yellow	1-10
(Iff*
university of turku
ypical properties of most organic fluorophores, and nonspecific background signals, compared with lanlhanide chelates
Property	Organic fluorophores and nonspecific	Luminescent lanthanidc
	background signals	chelates
Wavelength of absorption and	Anywhere in	Absorption in ultraviolet,
I   i mission bands	ultraviolet/visible region	emission in visible
Width of absorption and emission	Both broad	Absorption broad, 1
htncH		emission several narrow bands of 1-20 nm each, separated by tens of nm j
1   Mokes' shift between	Small (0-50 nm)	Large (150-300 nm)
1   absorption and emission bands		
luminescence yield	Up to 100%	Usually lower, particularly in water
Luminescence lifetime	Fluorescence lifetimes on order of 1 to 100 ns	10     to 10 ms |
Sensitivity of luminescence to	Usually significant	Much less, within range of
variations in environment and		stable chelate formation
t •mpcraiure		
( hemical and photochemical	Usually chemically stable;	Ligand and chelator
^ability	may be photochemicalh/	binding to lanthanide ion
	unstable	often not strong; may be photochcmically unstable
Requirements of luminescent Ln(lll) chelates
as labels for protein conjugation and immunoassay application
EKitatMHt
Relative brightness R = 8 QY
Ugand
fluorescence
Ugand
phosphorescence
5D0
tu11 luminescence »©»-»'F, 595 nm 1 '(V+'F^ISnm £ »Do-r'F.eSSnm »0,-»7P«688nm
ligand ground state (SJ
Eu'" ground state
reactive group for bioconjugation to proteins
antenna with efficient light absorption (s) and triplet level suitable for energy-transfer to Ln3+ kinetically stable coordination of the antenna ligand to Ln3+
water molecules replaced from the coordinating sphere to improve QY (coordination number 9)
university of turku
Initially it was difficult to combine all the requirements in a single molecule
DELFIA label technology n n
dissociation enhanced lanthanide fluoroimmunoassay
was developed to circumvent the challenges
Excitation 320-340 nm
Enhance
Two ligands
u c
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1
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Europium-labeled antibody
T
Emission 615 nm
- (1) first to conjugate Eu3+ to antibodies
stable complex during assay - no antenna
- (2) second to form luminescent complex with Eu3+ in solution
highly efficient antenna
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DELFIA enhancement solution
- "non-luminescent" small EDTA-like N1-Eu-chelate is coupled to protein and Eu3+ is dissociated to enhancement solution before forming new "ideal" highly luminescent comple with 2-NTAin micellar structure with minimal interaction of water
OOunO Capture rnotecu* on grDcp
ITC-N1-Eu-chelate
Binding reaction* *a»h to remove aicatt unsound reagent*
r
( . M I
n
r n
(mo   COO COO
associative
Wjlion
luminesce nee
containing enn* nee Tien: reagent*
2-NTA
1-(2-naphtoyl)-3,3,3-trifluoroacetone
Non-fluorescent lanthanide chelate and enhancement
PRO
DELFIA label technology
dissociation enhanced lanthanide fluoroimmunoassay
-> spatial information is lost
-> unsuitable for direct measurement from solid surface
Hemmila I, et al. (1984) Anal Biochem 137: 335-343
Addition of enhancement solution
- low pH
-> ion dissociates
- excess of new ligand -> fluorescent complex
Highly sensitive detection of Eu3+with time-resolved fluorometry
- large Stake's shift
- narrow emission peaks
- long fluorescence lifetime (~ 1 ms)
excitation
emission
400 GOO Wiiv< Tl.tn^l\i l mu )
Mill
Soini E, Hemmilá i (1979) Clin Chem 25: 353-361
Background autofluorescence
800 1000 J
Delay time    Counting time Time (us)
UNIVERSITY OFTURKU
Principle of pulsed time-resolved fluorometer
Europium-chelate; emission at 615 nm, decay time - 0.7 ms - typically collection of integrated signal after 1000 Xe-flash pulses using 400 us delay and 400 us window.
11.11*
r-<gKtr
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lit M lf*T**<<l
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mi
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3F
A
rllKurtild itltln iui
to Kwn
I
fused silica/quartz optics
high power Xe-flash excitation (with afterglow up to 50 us)
UV-excitation filter
with efficient VIS/NIR blocking
narrow high-transmission band-pass filter for emission band
low dark count PMT and photon counting
special "yellow" microplates
with UV-quencherfor minimal background
Soini E, Kojola H (1983) Clin Chem 29: 65-68
UNIVERSITY OFTURKU
Time-resolved fluorometry
of lanthanide luminescence to suppress autofluorescence
pulsed ex
i4_ e^
•*-> "(/>
§
o
0.1
0
delayed
340 nm   61 5 nm
ex em
<^^yi Stokes'shift >250 nm
time-gated emission
t
100
200
300
400
500 600 Time (\is)
autofluorescence specific
Sim
700
800
980 nm
long living background
detectability is significantly improved, but careful material selection is required to avoid background
900
1000
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Development of intrinsically fluorescent lanthanide chelates
PROT
Takalo, ef a/. (1994) Bioconjug Chem 5: 278-282.
S
H II
PROT H    N\_/J
-» spatial information is preserved
-> direct measurement from (dry) solid surface (no need for enhancement)
n
I V
-> donors in fluorescence
resonance energy-transfer assays (FRET)
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u
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von Lode, et al. (2003) Anal Chem 75: 3193-3201. https://doi.org/10.1021/ac0340051
UNIVERSITY OFTURKU
Intrinsically fluorescent Eu, Tb, Sm and Dy chelates
Pulsed excitation at 320-340 nm; time-resolved fluorometry.
1200    -,_,
380 480 580 680
Karvinen J, et al. (2004J Anal Biochem. 325: 317-325. Wavelength (nm)
Correlation between the lowest triplet state energy level of the llgand and lanthanide(lll) luminescence quantum yield
FRET
Fluorescence resonance energy transfer
"energy-transfer excited"
excitation
FRET
sensitized acceptor emission
donor acceptor
r
distance
E=
1 + (r/R0)G
R0 = 50 % efficiency distance (~3 - 8 nm)
UNIVERSITY OFTURKU
FRET
excitation   /^\ emission
'—I-1-1-1-1--1-1-1-1-1-1-1-1-1-1-1-TT'V'l-1-1-'-1-'-1—
400       450       500       550       600       650       700       750       800 900       950      1000 1050
Wavelength (nm)
spectral overlapping
Conventional FRET
is excellent research tool, but has severe limitations
sensitized
excitation emission
- autofluorescence (background fluorescence)
- direct excitation of acceptor
- crosstalk of donor
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Autofluorescence background
in conventional FRET
1000 800
* 600
■2 400
200
♦ • " *•
ex
ex / *
♦em
*
V 0
donor
acceptor
• • ■ ■ *
*
*
no FRET
i—■—i—
400 450
i ■—n      ■-1     ' £ I
500       5*Q       600    ♦ 650
T-•-T
700 750 Wavelength (nm)
T^y^T 1 I 1 I 1 I
800 900       950      1000 1050
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Acceptor is excited directly
in conventional FRET
• ■ ■
ex
0
ex
em
ex
em
x
* ■ *
donor
acceptor
Crosstalk of donor emission
in conventional FRET
* * m
400
450
500
36Q       600    ♦"650       700 750 ^ • ■ ■ *     Wavelength (nm)
©
acceptor
no FRET
800 900       950      1000 1050
UNIVERSITY OFTURKU
ss
s.
Time-resolved FRET (TR-FRET)
using Ln3+ donor with conventional acceptor fluorophore provides significant advantages
g
Cl
a
IC
c ■v
S
o
C Ol
IÜCT
u c
Cli
1
o
JZ
Q.
CA
O
j=
ligand
K
I 5D
LA CT
donor
luminescence decay is
shortened
1
8 8
™ 2? o
3
i/, RET
2 1
0
excitation
delayed
sensitized emission
Tb3+ Donor
TMR Acceptor
FRET
delayed
sensitized emission
central ion (Eu3+)
acceptor
Blomberg, K. et al. (1999) Clin Chem 45:6.
UNIVERSITY OFTURKU
500
550 600 Wavelength (nm)
650
400 600
1DO0
- time-gating resolves autofluorescence and
short-living emission of direct excitation of acceptor
- no crosstalk of donor as
donor emission is narrow banded
excitation
delayed
sensitized emission
( A )
FRET
excitation
excitation
0
no emission
(at acceptor wavelength)
no delayed
emission
Multiparametric DELFIA label technology
Four luminescent lanthanides with minimally overlapping emission lines can be used simultaneous as labels
Eu/Sm + Tb/Dy with different enhancing ligands.
Tb 545 nm (100-1500 us, QY 40%) Eu 615 nm (500-730 us, QY 70%) Sm 642 nm (50 us, QY 2%) Dy 572 nm (1-20 us, QY 2%)
SS MO Ce ( l'mil ii	59 Uf Pr "rjMiKhiiiiiiiT	60 U4 Nd V'. :\\II III III	61 1« Pm hnllHitllLljn	62 Sm SlIII1.II 111 111	63 1?: Eu I uropiuffi	64 1^ Gd li.lt1>i|ltllUIII 4; ?N/if„:|	Tb Icrtmim ■j	66 163 Dy \}\ spir-iiiim	67 157 Ho lloliimim 4,1	68 i"" Er I i'.....	69 1« Tm 11 allium -i ■	7u r\ Yb \ ik'ihium ■i- -	71 m Lu 1   Hi-  1      ! II
				1 1									
Hemmia, i. and Mukkala, v-M. (2001) Crit Rev Clin Lab Sci 38:441-519.
Multiparamethc/multiplexed assay
= assay that measures more than one analyte simultaneously from the same aliquot of sample
in a single run/cycle of the assay
Multiplexed assay
to measure multiple analytes from single aliquot of sample
Separate assays
sample A result 1   sample A result 2   sample A result 3
Multiplexed assay
sample A result 1, result 2, result 3
Multiplexed assay
is more cost efficient to measure multiple analytes
Separate assays
3x consumables (3x reagents) 3x sample 3x work
Multiplexed assay
1x consumables (~1-3x reagents) 1x sample 1 x work
-> more economical
Multiplexed assay
is more accurate in ratiometric measurements
Separate assays
1,2,3
ratios =
result, x v1
result2 x v2
Multiplexed assay
v
ratiomp =
result, x
result, x
-> effects of common errors in analysis are eliminated
need in clinical diagnostics to measure multiple analytes
e.g. several infectious diseases share common basic symptoms, but the identification of the cause may be needed for selecting the proper treatment
t
t
t
result 1   result 2   result 3
vs.
ttt
results 1, 2, 3
Mode of multiplexing
to enable separate measurement of multiple analytes
Emission (spectral multiplexing) ^^ar    wavelength separation
result
1      2 3
band-pass filter
Mode of multiplexing
to enable separate measurement of multiple analytes
Emission (spectral multiplexing)
^^ar    wavelength separation ^ result
1      2 3
band-pass filter
Spot position
(spatial array)
& ooo imaging/scanning
X result
1       2 3
(too) (oto) (oot)
solid-phase area
amis*
Mode of multiplexing
to enable separate measurement of multiple analytes
Emission (spectral multiplexing)
V
f    wavelength separation
^ result
1      2 3
band-pass filter
Spot position
(spatial array)
ooo imaging/scanning V wL result
1       2 3
(too) (oio) (ooi) solid-phase area
Optical barcode
(suspension array)
rP
flow cytometry result
1        2 3 t t
M) (hi) (l.l optical tag readout
Dual-mode of multiplexing
combining two modes to measure multiple ana
Emission color and spot position
(spectral and spatial multiplexing)
J5% ooo ooo
V
dual-color imaging/scanning result
12 3
)00] (OOOJ tooo
4^     5 6
00) (000,) iOO
in
Q.
TO
solid-phase area
Protein array - biotinylated antibody spots
\ V *
Detection of intrinsically fluorescent europium chelate-labeled streptavidin with time-resolved microimager.
Scorilas A, Bjartell A, Lilja H, Moller C, Diamandis EP (2000) Clin Chem 46: 1450-1455
Multiparametric liquid-array on categorized microparticles
Assays with time-resolved microfluorometer.
Hakala H, et al. (1998) Nucleic Acids Res 26: 5581-5588 /*& UNIVERSITY
OFTURKU
Prostatus free/total PSA immunoassay
based on Sm3+ and Eu3+ dual-label DELFIA technology
100 M
80
ft
/ 60 f 50 I 40 * 30 M
in
Free/total PSA ratio in serum provides improved discrimination of cancer compared to total PSA.
-•-   PSA F/T PSA-total
A better speedily was reached a I all sensitivity levels
100    90    B0     70    60    50    *0    30    20    10 0 Specihty (S]
Sm-labeled antibody for total PSA and Eu-labeled antibody for free-PSA
Two-plex assay is more accurate than ratio of two separate assays.
Bare lanthanide ions are "non-nonluminescent"
Excitation through light-harvesting organic ligand
molar absorptivitity < 1 M_1 cm-1
quenched by
coordinated water molecules > practically no luminescence
- molar absorptivitity > 10000 M"1 cm-1
- high quantum yield
=^> highly luminescent (time-resolved detection enables low limit-of-detection)
Mixed-chelate complex formation
through biomolecular interactions
1111111111111
hV2
nv1
target
separation free -homogeneous assay
,Eu3;
		
111 111 111 I 1111 nil..........1		ill......M,,.
II I M I II M II I
o = carrier chelate
= antenna
Karhunen U et al. (2010) 4na/ Chem 82: 751-574
Switchable lanthanide luminescence
IOn Carrier Chelate Target oligonucleotide / pM
Chelate complementation
- fluorescent europium chelate divided to two label moieties
—» novel homogeneous reporter technology (very high degree of modulation)
Karhunen et al. (2001) Anal Chem 82: 751-754
UNIVERSITY OFTURKU
Summary
Luminescent lanthanides and time-resolved fluorescence
millisecond time-gated luminescence detection efficiently eliminates autofluorescence
low background optical material selection is needed for detection and consumables
organic light-harvesting antenna ligand is required for efficient excitation of lanthanides
lanthanide chelate-dyed nanoparticles provide extreme detectability
DELFIA technique resembles enzyme assays as it requires enhancement step
immunoassay sensitivity with lanthanide-chelate dyed nanoparticles is limited by non-specific interactions
nanoparticle based solid-phase assays are prone to steric limitations
most efficient luminescent lanthanides are Eu3+ and Tb^+ followed by Sm^+ and Dy^+ high-intensity UV-excitation and low emission intensity are challenges for detection
time-gated luminescence imaging requires special instrumentation ^university
ofturku
Mk UNIVERSITY OF TURKU