Photochemically amplified detection of molecular recognition events: An ultra-sensitive fluorescence turn-off binding assay

Article abstract of DOI:10.1039/c1ob05289f

Amplified fluorescence quenching methodology based on massive autocatalytic photo-unmasking of a dual function sensitizer-quencher is developed and adopted for photoassisted ultra-sensitive detection of molecular recognition events. The resulting binding

Full text of DOI:10.1039/c1ob05289f

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 4752
www.rsc.org/obc
COMMUNICATION
Photochemically amplified detection of molecular recognition events: an
ultra-sensitive fluorescence turn-off binding assay†
Tiffany P. Gustafson, Greg A. Metzel and Andrei G. Kutateladze*
Received 23rd February 2011, Accepted 9th May 2011
DOI: 10.1039/c1ob05289f
Amplified fluorescence quenching methodology based on
massive autocatalytic photo-unmasking of a dual function
sensitizer–quencher is developed and adopted for pho-
toassisted ultra-sensitive detection of molecular recognition
events. The resulting binding assay, based on a molecular
recognition-triggered photo-amplified cascade with concomi-
tant decrease of fluorescence is validated with the biotin–
avidin pair, achieving attomolar detection.
involving externally sensitized fragmentation in dithiane–ketone
7
adducts. Next a system was designed where a seed sensitizer trig-
8,9
gers a chain of fragmentation events. As fluorescence detection
is one of the most sensitive techniques, we have now developed a
methodology of conditional, i.e. molecular recognition-triggered,
photoamplified release with concomitant fluorescence quenching
(Scheme 1) based on benzophenone (BP) acting as both the
amplification chain carrier and the quencher for the reporter
fluorophore. Depending on the fluorophore, the emission turns
off either because of photophysical quenching by the amplified
BP, or an actual photochemical reaction with triplet excited BP
irreversibly bleaching the fluorophore.
Fluorescence-based screening for binding events on spatially
addressable chips has revolutionized biomedical research. Yet,
with all the remarkable advances in hardware and the surface
density of chips, it is premature to expect mass produced scanners
capable of single molecule detection levels any time soon. For
any given hardware detection limit the question then remains:
can one chemically pre-amplify the signal to a detectable level (or
alternatively turn-off the fluorescence from an easily detectable
level)? PCR is a powerful method for such pre-amplification, but
it is limited to genetic material. In a more general sense, beyond
1
classical enzymatic catalysis, amplification can be achieved with
Scheme 1 General concept for the massive amplified unmasking of
benzophenone resulting in fluorescence quenching (see text).
2
polymerization; yet the readout is less sensitive than fluorescence.
There are several powerful non-PCR methods, for example:
3
4
Mirkin’s biobarcode assays and PCR-like cascade reactions,
5
As shown in Scheme 1, masked BP is mixed with the reporter
fluorophore, which is chosen to have negligible absorption around
365 nm, i.e. the wavelength reserved for photoamplification.
Irradiation at 365 nm does not exert any effect until a seed sen-
sitizer is brought to the pool of masked sensitizers, for example, by
a molecular recognition event. At this point externally sensitized
photofragmentation of masked benzophenone is commenced,
unmasking more BP, which in turn induces more fragmentation,
thus carrying the amplification chain. The unmasked/amplified
BP turns the emission of the present fluorophore off, signifying a
positive “hit”. Unlike radical chain polymerization, the propaga-
tion of this photoamplified chain can be halted by interrupting
the UV source. This feature is critically important to prevent
Abbott’s liquid crystal reorientation, or a sensitive fluorescence
turn-off approach based on “superquenching” of conjugated
6
polymeric chromophores.
In this Communication we report a fundamentally different
methodology for amplified fluorescence quenching based on
massive autocatalytic photo-unmasking of a quencher, and its
implementation for ultra-sensitive detection of molecular recog-
nition events. We have validated this photoamplified fluorescence
turn-off concept with the biotin–avidin binding pair, which allows
the full range of physiologically relevant K values to be probed.
D
Utilizing a mass produced consumer CCD camera (no chip
cooling) we achieved a reproducible detection of 50 attomoles
of avidin.
We previously reported photoassisted detection of molecular
recognition events in solution based on a spatial proximity test
“overdevelopment” of the image and avoid false positives.
First we examined fluorophores with high-lying singlet states,
for example, polyphenyls, which are readily quenched with
amplified benzophenone via a bimolecular collisional energy
transfer mechanism with a very high Stern–Volmer constant. A
concentration series with quaterphenyl as a fluorophore is shown
in Fig. 1. The fluorophore-masked BP formulation is seeded with
progressively diluted BP, increasing the autocatalytic delay time.
Department of Chemistry and Biochemistry, University of Denver, Denver,
CO 80208, USA. E-mail: akutatel@du.edu; Fax: +1-303-871-2254; Tel: +1-
3
03-871-2995
†
Electronic supplementary information (ESI) available: Experimental
details and spectra. See DOI: 10.1039/c1ob05289f
4
752 | Org. Biomol. Chem., 2011, 9, 4752–4755
This journal is © The Royal Society of Chemistry 2011

View Article Online
a few emitting copies of the biological target to a certain spot on
the surface of the chip. The success of such an assay depends on
detecting a very limited number fluorophores, i.e. requires high
end X-Y fluorescence scanners.
We contend that in this general scheme the Z-direction is
underutilized and that without sacrificing the surface density of an
array one can utilize the depth of the screening chip (for example,
11
by using a now ubiquitous microcapillary array chip ), its pores
filled with enough fluorophore for fast detection/imaging with
inexpensive CCDs. Fig. 2 shows that for a typical focus depth of
an unsophisticated two lens imager (see SI for details) fluorescence
detection does benefit from the additional fluorophore stored
in the Z-direction: 0.86 mm ID capillaries were loaded with
increasing volumes of 10 M coumarin-6 in dichloromethane,
where 1 mL ª 1 mm in depth. A clearly discernible increase in
average pixel intensity was obtained by increasing the depth of the
fluorescing solution from 1 to 4–5 mm, at which point the intensity
levels off (corresponding to an approximate 7 : 1 ratio of the depth
to ID). For a microcapillary array chip with 200 mm pores this
translates into a useful depth of 1.4 mm and the corresponding
pore volume of >30 nL, containing near picomolar amounts of
fluorophore which is easily detectable with a simple imager.
Fig. 1 Quenching of quaterphenyl as a result of benzophenone (BP)
amplification (y-axis is the normalized emission at 360 nm).
-
5
At a certain dilution, under 10 nM, the resulting fluorescence
intensity is no longer different from the “no benzophenone
added” curve. This is the intrinsic detection limit, which is defined
by two factors, (i) the extent of non-sensitized fragmentation
of the masked sensitizer, i.e. its spontaneous monomolecular
fragmentation under direct absorption of light, generating enough
seed BP to mistrigger amplification (a false positive) and/or (ii)
the residual levels of free BP in the adduct, initiating amplification
chain even in the absence of a molecular recognition event.
The latter is rectified by a thorough purification of the masked
sensitizer from residual BP, while the former can potentially be
improved by further conditioning of the UV light used in the
amplification: a narrow UV source which selectively excites the
n→p* of benzophenone, while avoiding direct shorter wavelength
excitation of the deconjugated phenyl moieties in the masked
sensitizer.
The emission differences between the test samples and the
control increase and then decrease in time, as all masked sensitizer
is eventually released. As the system has a built-in off switch, where
discontinuing irradiation stops amplification, false positives are
prevented by optimizing the time it takes to obtain the maximum
fluorescence difference. For example, the 1 mM sample reaches a
Fig. 2 Effect of path length on fluorescence intensity as imaged by the
CCD camera. Each capillary shows an increase in average pixel intensity
(
1
graph on the left) which correlates with an increase in the depth of the
-5
0
M coumarin-6 solution.
Fig. 3 outlines the general concept for amplified detection in
1
: 15 emission ratio, as the vertical green line shows, whereas for
compartmentalized small volumes: (A) Microwells are uniformly
loaded with a fluorophore and masked sensitizer in organic
solvent or organogel. This formulation is generic and universally
applicable to any type of assayed molecules. The ligands, tethered
to an amphiphile, are printed on the surface of wells and therefore
displayed at the organic–aqueous interface. (B) A sensitizer-
tethered target protein is added in buffer and incubated to allow
for binding. A binding event brings the tethered sensitizer to
the solvent interface at which point irradiation commences. (C)
Initially the inserted sensitizer unmasks a few copies of BP in
its immediate vicinity. These benzophenones are free to diffuse
a smaller concentration of 100 nM it is about 1 : 4 (red line).
Fluorophores optimized for visible light microscopy are more
suitable for ready detection with mass-produced CCDs than
those emitting in the UV. Inherently, they have low-lying singlet
states, which are not quenched as effectively by benzophenone.
However, the excitation and intersystem crossing of the amplified
BP produces reactive triplet species, which can chemically destroy
the fluorophore achieving the same turn-off effect. We tested
several bright fluorophores and chose coumarin-6 (C6, structure
is shown in Fig. 4), which in the presence of triplet benzophenone
undergoes a cascade de-ethylation reaction with concomitant loss
10
of emission. This photochemical reaction partially depletes the
amplified benzophenone, but this depletion is negligible and does
not affect the amplification chain because the masked sensitizer
pool, 30–40 mM, is more than three orders of magnitude greater
than the total amount of fluorophore, present at 10–20 mM (see SI
for an NMR-monitoring experiment).
The current state-of-the-art in microarray screening for binding
takes advantage of (i) ligands modified chemically to immobilize
on spatially addressable chips and (ii) a biological target which is
chemically outfitted with a fluorophore. Binding events sequester
Fig. 3 Photoamplifed detection of a binding event (see text).
Org. Biomol. Chem., 2011, 9, 4752–4755 | 4753
This journal is © The Royal Society of Chemistry 2011

View Article Online
throughout the entire volume of the well, releasing and amplifying
BP en masse, which lead to complete quenching/bleaching of the
fluorophore. As a result, the whole well/pixel goes dark indicating
a “positive hit”.
We validated this concept using the biotin–avidin pair which
remains bound in a broad dynamic range of concentrations, and
thus allows for probing of the method’s intrinsic detection limit.
Commercial biotin-capped dipalmitoyl phosphoethanolamine or
its chain length variants, synthesized by tethering biotin to other
phosphatidyl ethanolamines, were used in this study. Avidin was
outfitted with xanthone as the seed sensitizer (for experimental
details refer to SI).
Fig. 5 (A) Bottom view pair wise comparison of the 0.86 mm i.d.
capillaries in the dilution series with (+) and without (-) avidin–xanthone
conjugates. The best contrast is achieved at around 30 min of irradiation.
(
B) a typical fluorescence turn-off outcome in an open ended 0.4 mm
i.d. capillary at 1 nM avidin–xanthone. Images are acquired with a 0.5 s
Biotin–avidin binding failed to initiate the amplification cascade
when the initiator’s tether was too short (C11) due to insufficient
lipid layer penetration. However, doubling the tether’s length to
exposure of a mass produced 1/4¢ CCD. (C) Images of capillaries with 4,
0.4, and 0.1 mL of 10 M C6 acquired with a cell phone camera.
-5
~
3 nm long, allowed for an efficient initiation of the photoampli-
lacking the avidin–xanthone conjugate “(-)” or a 5 mL aqueous
solution of the progressively dilute avidin–xanthone conjugate,
“(+)”. The capillaries were then subjected to gentle irradiation
with 365 nm 250 mW Nichia UV LED. All the necessary control
experiments were run in parallel (see SI).
fication chain.
Fig. 4 shows a “bulk” series where large 700mL fluorescence cells
were loaded with 300 mL solution of 30 mM dithiane-masked BP
(
structure in Scheme 1) and 10 mM fluorophore C6. At the organic–
aqueous interface either biotinylated lipid or phosphatidylcholine
POPS, control) was used. The avidin–xanthone conjugate, or
As follows from Fig. 5A the avidin containing spots and
spots lacking avidin exhibited the same emission level before
photoassisted amplification (top). Within approximately 30 min of
irradiation a reproducible fluorescence turn-off effect was achieved
in the capillaries containing avidin, while the control capillaries
lacking avidin remained brightly lit, offering an excellent contrast
ratio for detecting the “positive hits”. Photoamplification of
biotin–avidin binding events was thus reproducibly observed
down to 10 pM avidin which corresponds to a total of 50
attomoles – a remarkable achievement for a non-cooled CCD.
This is comparable to other binding assays, including Mirkin’s
(
blank PBS buffer as a control, was added in phosphate buffered
saline (PBS).
Upon irradiation, a 10-fold decrease of emission was detected
in the cell with both biotin and avidin present (Fig. 4A). Emission
in the cells lacking avidin (Fig. 4C,D) decreased negligibly. Of the
controls, fluorescence in the cell containing avidin–xanthone, but
not biotin, was reduced by approximately 35% (Fig. 4B), indicating
that, at 10 mM, a small amount of avidin is partially recruited to
the lipid interface by non-specific binding. However, this is still six
fold brighter than in (A), thus allowing for easy identification of a
12
bio-barcode assay which can detect ~10 attomoles of analyte,
and Bowman’s visible-light-polymerization which can detect 0.4
“
positive hit”.
13
attomoles of biotin with the help of a research grade microscope.
Although several successful runs were recorded for 1 pM avidin,
the reproducibility below 10 pM was poor. As biotin is still
expected to be mostly bound at this concentration, we interpret
this as the intrinsic detection limit for our photoamplified turn-
off assay. Remarkably, the observed detection limit of 10 pM was
considerably better than the amplification of untethered BP in a
bulk solution (Fig. 1). We rationalize it in terms of a local pre-
concentration effect due to recruitment of the avidin–sensitizer
conjugate to the biotinylated lipid interface.
The spatial resolution of mass-produced CCDs is fully adequate
for imaging high density arrays. However, their sensitivity of
detection leaves much to be desired, i.e. they simply cannot image
a few fluorophore molecules sequestered to a 2D spot on a surface
of a microarray chip. Utilizing the third – depth – dimension we
can image as little as 100 nanolitres of coumarin-6 with such a
ubiquitous imaging device as a cell phone camera (see Fig. 5C).
For a 2–3 mm thick microcapillary array chip this translates into
Fig. 4 A–D contain C6 (10-5 M) and masked sensitizer (30 mM) in
-5
1
,4-dichlorobutane. A, C biotin capped lipid (9.5 ¥ 10 M) is added; B, D
5
-
POPS (9.5 ¥ 10 M) is added. A, B were incubated with 0.01 M PBS pH
7
.5 containing the avidin–xanthone conjugate (10- M) while C, D were
5
4
incubated with 0.01 M PBS pH 7.5 as control, before irradiation. A is the
a surface density of pores exceeding 10 per square inch.
only sample containing both biotin and avidin.
As long as one can image the initial level of emission, the pho-
toamplified fluorescence turn-off assay can be successfully carried
out. This offers ultra-sensitive yes/no bioanalytical capabilities
which can be developed for situations when access to state-of-the-
art technology is limited.
We then emulated the micro-well environment with capillaries,
either 0.86 mm i.d. sealed from one end, or 0.4 mm open from both
ends (the latter loaded by capillary forces). Fig. 5A shows images
of the dilution series, where a 3 mL droplet of masked BP and C6 in
dichlorobutane was incubated with either a control of 5 mL PBS
For the high-end instrumentation, with sub-picolitre volumes
of fluorophores and scientific grade cooled CCD cameras our
4
754 | Org. Biomol. Chem., 2011, 9, 4752–4755
This journal is © The Royal Society of Chemistry 2011

View Article Online
photoassisted pre-amplification methodology could potentially
approach a single molecule detection limit. Again, to reiterate,
the uniform loading of the microwells with a fluorophore and
masked sensitizer makes this formulation generic and universally
applicable to any type of assayed molecules.
7 R. Kottani, R. A. Valiulin and A. G. Kutateladze, Proc. Natl. Acad.
Sci. U. S. A., 2006, 103, 13917.
8
R. R. Kottani, J. R. R. Majjigapu, A. N. Kurchan, K. Majjigapu, T. P.
Gustafson and A. G. Kutateladze, J. Am. Chem. Soc., 2006, 128, 14794.
This concept of unmasked sensitizer carrying the amplification chain
was later generalized by Shabat as the approach where released “free
reagents gain the chemical reactivity of the analyte of interest”: (a) E.
Sella and D. Shabat, J. Am. Chem. Soc., 2009, 131, 9934; (b) E. Sella,
A. Lubelski, J. Klafter and D. Shabat, J. Am. Chem. Soc., 2010, 132,
This work is supported by the NIH (NIBIB R21 EB008532).
3
945.
Notes and references
9
K. Majjigapu, J. R. R. Majjigapu and A. G. Kutateladze, Angew. Chem.,
Int. Ed., 2007, 46, 6137.
1
2
L. Zhu and E. V. Anslyn, Angew. Chem., Int. Ed., 2006, 45, 1190.
H. Sikes, C. Bowman, R. Hansen and R. Kuchta, PCT Int. Appl., 2006
WO 2006031248.
10 Oxydative de-ethylation in coumarins is precedented in the lit-
erature, see M. A. Kirpichenok, L. M. Mel’nikova, L. K.
Denisov and I. I. Grandberg, Khim. Geterotsikl. Soed., 1989,
460.
11 For example from Incom, Inc.; www.incomusa.com.
12 C. S. Thaxton, R. Elghanian, A. D. Thomas, S. I. Stoeva, J. S.
Lee, N. D. Smith, A. J. Schaeffer, W. H. Klocker, G. Bartsch and
C. A. Mirkin, C.A., Proc. Natl. Acad. Sci. U. S. A., 2009, 106,
18437.
3
J. M. Nam, C. S. Thaxton and C. A. Mirkin, Science, 2003, 301, 1884;
H. D. Hill and C. A. Mirkin, Nat. Protoc., 2006, 1, 324; S. I. Stoeva, J.
S. Lee, C. S. Thaxton and C. A. Mirkin, Angew. Chem., Int. Ed., 2006,
4
7
5, 3303; H. D. Hill, R. A. Vega and C. A. Mirkin, Anal. Chem., 2007,
9, 9218.
4
5
6
H. J. Yoon and C. A. Mirkin, J. Am. Chem. Soc., 2008, 130, 11590.
B. H. Clare and N. L. Abbott, Langmuir, 2005, 21, 6451.
S. W. Thomas III, G. D. Joly and T. M. Swager, Chem. Rev., 2007, 107,
1
13 H. D. Sikes, R. R. Hansen, L. M. Johnson, R. Jenison, J. W. Birks, K.
L. Rowlen and C. N. Bowman, Nat. Mater., 2008, 7, 52.
339.
This journal is © The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 4752–4755 | 4755