Fluorescent Neuraminidase Assay Based on Supramolecular Dye Capture after Enzymatic Cleavage

Article abstract of DOI:10.1021/jacs.7b01628

A conceptually new type of enzymatic cleavage assay is reported that utilizes in situ supramolecular capture of the fluorescent product. A squaraine-derived substrate with large blocking groups at each end of its structure cannot be threaded by a tetralactam macrocycle until the blocking groups are removed by enzyme cleavage. A prototype design responds to viral neuraminidase, an indicator of influenza infection, and also measures susceptibility of the sample to neuraminidase inhibitor drugs. The substrate structure incorporates three key features: (a) a bis(4-amino-3-hydroxyphenyl)squaraine core with bright deep-red fluorescence and excellent photostability, (b) an N-methyl group at each end of the squaraine core that ensures fast macrocycle threading kinetics, and (c) sialic acid blocking groups that prevent macrocycle threading until they are removed by viral neuraminidase. The enzyme assay can be conducted in aqueous solution where dramatic colorimetric and fluorescence changes are easily observed by the naked eye. Alternatively, affinity capture beads coated with macrocycle can be used to immobilize the liberated squaraine and enable a range of heterogeneous analysis options. With further optimization, this new type of neuraminidase assay may be useful in a point of care clinic to rapidly diagnose influenza infection and also determine which of the approved antiviral inhibitor drugs is likely to be the most effective treatment for an individual patient. The assay design is generalizable and can be readily modified to monitor virtually any type of enzyme-catalyzed cleavage reaction.

Full text of DOI:10.1021/jacs.7b01628

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Article
Fluorescent Neuraminidase Assay Based on
Supramolecular Dye Capture After Enzymatic Cleavage
Wenqi Liu, César F. A. Gómez-Durán, and Bradley D. Smith
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 20 Apr 2017
Downloaded from http://pubs.acs.org on April 20, 2017
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Fluorescent Neuraminidase Assay Based on Supramolecular
Dye Capture After Enzymatic Cleavage
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Wenqi Liu, César F. A. GómezꢀDurán, Bradley D. Smith*
Department of Chemistry and Biochemistry, 236 Nieuwland Science Hall, University of Notre Dame, Notre Dame, Indiana
46556, USA
*smith.115@nd.edu
ABSTRACT: A conceptually new type of enzymatic cleavage assay is reported that utilizes inꢀsitu supramolecular capture of the
fluorescent product. A squaraineꢀderived substrate with large blocking groups at each end of its structure cannot be threaded by a
tetralactam macrocycle until the blocking groups are removed by enzyme cleavage. A prototype design responds to viral neuramꢀ
inidase, an indicator of influenza infection, and also measures susceptibility of the sample to neuraminidase inhibitor drugs. The
substrate structure incorporates three key features: (a) a bis(4ꢀaminoꢀ3ꢀhydroxyphenyl)squaraine core with bright deepꢀred fluoresꢀ
cence and excellent photostability, (b) an Nꢀmethyl group at each end of the squaraine core that ensures fast macrocycle threading
kinetics, and (c) sialic acid blocking groups that prevent macrocycle threading until they are removed by viral neuraminidase. The
enzyme assay can be conducted in aqueous solution where dramatic colorimetric and fluorescence changes are easily observed by
the naked eye. Alternatively, affinity capture beads coated with macrocycle can be used to immobilize the liberated squaraine and
enable a range of heterogeneous analysis options. With further optimization, this new type of neuraminidase assay may be useful in
a point of care clinic to rapidly diagnose influenza infection and also determine which of the approved antiviral inhibitor drugs is
likely to be the most effective treatment for an individual patient. The assay design is generalizable and can be readily modified to
monitor virtually any type of enzyme catalyzed cleavage reaction.
INTRODUCTION
have very high affinity for fluorescent dyes.23 To be clear,
there are enzyme assays that use dye displacement from metal
cations24 or container molecules such as cyclodextrin or cuꢀ
curbituril, but these systems do not immobilize the dye and
permit spatially localized detection.25–27
Enzyme assays are ubiquitous in modern society. They are
employed extensively as laboratory tools in basic biomedical
research, drug discovery, food technology, and pollution conꢀ
trol.1–3 In addition, a range of diagnostic devices are used to
monitor enzymes that affect public health, personal wellꢀ
being, and environmental protection.4ꢀ6 The high sensitivity of
fluorescence makes it an especially powerful technique, and
ongoing innovations in photonics engineering such as microꢀ
fluidic chips,7,8 CCD cameras for multiplex detection,9,10 and
bench top flow cytometers,11 offer new opportunities to greatly
enhance assay performance properties such as limit of detecꢀ
tion, spatiotemporal resolution, and sample throughput.12,13
Successful exploitation of the new instrumentation requires
concomitant improvements in fluorescence assay design.14ꢀ16
An enzyme is a catalyst, thus the fluorescence signal in a
cleavage assay continues to build up over time. But the cleavꢀ
age product diffuses throughout the entire volume of the assay
sample which dilutes the fluorescence signal. Moreover, the
signalꢀtoꢀbackground ratio is further lowered in turbid samples
due to scattering of the light. In principle, the sensitivity of a
substrate cleavage assay would be enhanced if the liberated
fluorescent product was sequestered by affinity capture
beads.17 Furthermore, product immobilization provides access
to a suite of automated heterogeneous analysis methods.18
Surprisingly, there has been very little exploration of assays
that incorporate inꢀsitu capture of the cleaved product,18ꢀ21 and
essentially no reports of a cleavage assay that captures and
concentrates a liberated dye.22 One reason for this circumꢀ
stance is the scarcity of supramolecular host molecules that
We have recently developed a highꢀaffinity selfꢀassembly
system that we call Synthavidin technology.23,28–30 In short, a
tetralactam macrocycle with anthracene sidewalls can be rapꢀ
idly threaded by squaraine dyes in water to form very stable
complexes with nanomolar dissociation constants. The threadꢀ
ed complexes are stabilized by four strong hydrogen bonds
with the two squaraine oxygen atoms and hydrophobic stackꢀ
ing of aromatic surfaces. Squaraine dyes exhibit very intense
deepꢀred absorption/emission bands and macrocycle threading
is indicated by three characteristic changes in photophysical
properties; (a) redꢀshifted squaraine absorption/emission
bands, (b) increased squaraine fluorescence quantum yield,
and (c) efficient energy transfer from the excited anthracene
sidewalls to the encapsulated squaraine dye. In addition, the
Nꢀsubstituents at each end of the dye structure have little imꢀ
pact on the association constant but greatly affect the threadꢀ
ing kinetics. When the dyes have Nꢀpropyl groups, the threadꢀ
ing kinetics are relatively slow and we have utilized this feaꢀ
ture to produce a preꢀassembly method that creates mechaniꢀ
cally stable threaded complexes for long term biological imagꢀ
ing.31,32 In this current study we demonstrate for the first time
how Synthavidin technology can be exploited for a very difꢀ
ferent purpose; that is, to create a new class of enzyme assays
that utilize inꢀsitu capture of the fluorescent cleavage product.
The basic concept is shown schematically in Scheme 1a. A
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group at each end of the squaraine core that allows rapid forꢀ
mation of M ⊃ SQ1’.
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The functional aim of this prototype study was to develop an
assay that responds to viral neuraminidase (VNA), an enzyme
indicator of influenza infection.35 Each year in the United
Sates, around 5ꢀ15 % of the population becomes infected with
seasonal influenza and there are approximately 25,000 influꢀ
enzaꢀassociated deaths.36,37 The morbidity and mortality inꢀ
creases dramatically during pandemics, such as the 2009 outꢀ
break of swine flu, placing great strain on the global health
care infrastructure. VNA cleaves Nꢀacetylneuraminic acid
(sialic acid) from the surface of host cells to release viral
progeny,38,39 and is considered a prominent biomarker of conꢀ
tamination in biomedical and environmental samples.35 Furꢀ
thermore, assays that measure VNA activity can provide direct
information about the likely efficacy of neuraminidase inhibiꢀ
tor drugs to treat the specific strain of influenza that infects an
individual patient. The efficacy window for VNA inhibitor
drugs is within 48 hours of the onset of influenza; thus, there
is a great need for lowꢀcost and portable methods to rapidly
confirm influenza infection. Most of the currently available
rapid response tests are based on nucleic acid amplification
tests or immunoassays directed at viral antigens.40 The former
requires sophisticated instruments or training while the latter
are often cost restricted. Other methods of neuraminidase deꢀ
tection include electrochemistry41 or chemiluminescence42
techniques that may be challenging to automate for highꢀ
throughput or multiplex testing. There are several neuraminiꢀ
dase cleavage assays that release a fluorescent dye,43,44 but
there is no example that captures and concentrates the liberatꢀ
ed dye on a bead for facilitated analysis.
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The project work flow was in three parts. First, the different
molecular building blocks had to be prepared and the selfꢀ
assembly processes evaluated in aqueous solution to confirm
that the supramolecular and photophysical properties were
appropriate. Then the enzymeꢀtriggered, dye capture process
in Scheme 1 had to be validated. Finally, affinity capture
beads coated with macrocycle M had to be prepared and used
in an assay that determines if a specific strain of neuraminiꢀ
dase enzyme is susceptible to inhibition by a clinically releꢀ
vant influenza drug.
Scheme 1. (a) Enzyme cleavage assay based on inꢀsitu dye
capture. A tetralactam macrocycle (red oval) encapsulates a
fluorescent squaraine dye (blue) after enzymatic cleavage of
the blocking groups (green). (b) Molecular structures used in
this prototype study for detection of neuraminidase. Selfꢀ
immolative cleavage of a sialic acid group releases a quinone
methide that is rapidly trapped by water and also liberates
SQ1’. (c) Structure of SQ2 used in preliminary binding studꢀ
ies.
RESULT AND DISCUSSION
Preliminary Demonstration of Self-Assembly
Solution state titration studies assessed the threading of M by
SQ2, a close structural analogue of SQ1’ whose easy preparaꢀ
tion on a large scale permitted an extensive number of supraꢀ
molecular measurements. We were pleased to observe strong
and very rapid threading to form M ⊃ SQ2 as indicated by
squaraine dye with large blocking groups at each end of its
structure cannot be threaded by a tetralactam macrocycle until
the blocking groups are removed by enzymatic cleavage.
Scheme 1b and 1c show the specific molecular structures used
in the current study. The key compound is squaraine substrate
SQ1 which has several supramolecular and photophysical
innovations. Guided by our previous experience, and some
molecular modelling (Scheme S5) we chose a bis(4ꢀaminoꢀ3ꢀ
hydroxyphenyl)squaraine as the fluorescent core which was
expected to be brighter and more photostable than the bis(2ꢀ
aminothiophene)squaraine system that we had used in our
earlier preꢀassembly studies.33,34 For reasons that are discussed
in the next paragraph, the blocking groups in SQ1 are sialic
acid derivatives that prevent threading of macrocycle M until
they are removed enzymatically. Selfꢀimmolative cleavage of
the sialic acid groups liberates SQ1’ which has an Nꢀmethyl
measured values of Ka = (5.8±1.3) x 107 Mꢀ1 and kon
=
(5.1±0.5) x 108 Mꢀ1 sꢀ1 (Figure 1). When the concentration of
both partners (M and SQ2) was each 50 nM, bimolecular asꢀ
sociation was essentially complete within 300 ms, suggesting
that squaraine threading by the macrocycle was not likely to
become the rate determining step in any putative enzyme
cleavage assay. The threading of SQ2 to form M ⊃ SQ2 proꢀ
duced very favorable changes in squaraine photophysical
properties (Table S1). Notably, there was a 39 nm redꢀshift in
the squaraine emission wavelength and an increase in fluoresꢀ
cence quantum yield from 0.08 to 0.19. The combination of
these changes meant that SQ2 complexation by M produced a
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Figure 1. (left) Titration isotherm showing change in fluoresꢀ
cence for SQ2 (250 nM) upon titration with M in water at 20 ˚C
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(ex: 680 nm, em: 695 nm). (right) Change in fluorescence over
time (stoppedꢀflow) for SQ2 (50 nM) after addition of M (50 nM)
in H2O at 20 ˚C (ex: 680 nm, em: 695 nm).
35ꢀfold enhancement in squaraine deepꢀred fluorescence inꢀ
tensity (ex:680 nm, em: 695 nm). The M ⊃ SQ2 complex
exhibited a Stokes shift of 34 nm, which is very large for
deepꢀred fluorescent dye.45 A large Stokes shift is a desirable
property because it minimizes dye selfꢀquenching when the
multiple copies are closely packed on the surface of an assay
bead. There was also efficient energy transfer from the excited
anthracene sidewalls in the surrounding macrocycle to the
encapsulated squaraine dye (Figure S2). Another favorable
property of SQ2 and threaded complex M ⊃ SQ2 is very high
photostability as judged by the lack of photobleaching after
long periods of constant irradiation (Figure S3). Taken togethꢀ
er, these favorable supramolecular and photophysical results
demonstrated the high feasibility of M and SQ2 (or related
analogues) as a selfꢀassembly pair for inꢀsitu capture and fluoꢀ
rescence signaling after enzymatic cleavage of suitably deꢀ
signed squaraine blocking groups.
Figure 2. Optical evidence for the neuraminidase triggered selfꢀ
assembly shown in Scheme 1. (a) Squaraine absorption band, (b)
squaraine fluorescence (ex: 610 nm), (c) anthracene fluorescence
(ex: 390 nm), (d) squaraine fluorescence after internal energy
transfer from excited M (ex: 390 nm); for samples of SQ1, SQ1 +
M, and SQ1 + M + BNA in water, where [SQ1] = [M] = 3 µM,
[BNA] = 0.03 U per nmolꢀ1 of substrate. Photographs of vials with
illumination by laboratory light or: (e) portable UV lamp (ex: 365
nm, indicated by +), (f) blue laser pointer (ex: 410 nm, indicated
by +). Each vial contained SQ1 (20 ꢁΜ) + M (40 µM) and the
presence of BNA (0.03 U nmolꢀ1 substrate) is indicated by +.
Solution State Studies of Neuraminidase Triggered
Formation of M ⊃ SQ1’
The sialic acid blocking groups on SQ1 had to be large
enough to prevent threading by M and also be cleaved by neuꢀ
raminidase within an acceptable analysis time frame. The
structure of SQ1 included selfꢀimmolative linkers with the
expectation that sialic acid hydrolysis would initiate a bond
breaking cascade that liberated squaraine SQ1’.8,46,47 This deꢀ
sign feature was confirmed by ESI mass spectrometry experiꢀ
ments that analyzed samples of SQ1 before and after treatment
with bacterial neuraminidase (BNA) from Clostridium
perfringens, a convenient neuraminidase for these preliminary
studies.48 As shown in Figure S4, the peak for SQ1 was conꢀ
sumed after adding BNA and a peak corresponding to SQ1’
was produced. Blocking group removal was also indicated by
changes in the squaraine absorption and fluorescence spectra
because the cleavage product SQ1’ selfꢀaggregates when it is
free in water leading to absorption band broadening and fluoꢀ
rescence quenching (Figure S5).
fluorescence emission, spectral changes that are highly conꢀ
sistent with BNA cleavage of the sialic acid blocking groups
followed by macrocycle threading to form M ⊃ SQ1’. The
most compelling evidence for macrocycle threading was the
enzymeꢀdependent enhancement in internal fluorescence enerꢀ
gy transfer when the sample was excited by UV light. As
shown in Figures 2cꢀd there was a significant decrease in the
macrocycle anthracene emission band and a corresponding
large increase in deepꢀred fluorescence from the encapsulated
squaraine. The fluorescence energy transfer effect was easily
observed by the naked eye as a dramatic change in sample
emission color from blue to red when the vials were Illuminatꢀ
ed with UV light from a handꢀheld lamp (Figure 2e) or a blue
laser pointer (Figure 2f).
Additional solutionꢀstate absorption and fluorescence studies
confirmed the other necessary supramolecular features shown
in Scheme 1. When M and SQ1 were mixed together there
were very small changes in the squaraine absorption and emisꢀ
sion spectra (Figures 2aꢀb) perhaps due to weak hydrophobic
association of the two molecules. Subsequent addition of BNA
to this mixture of M and SQ1 triggered the expected 18 nm
redꢀshift in the squaraine absorption maximum, along with
large enhancements in the strongly redꢀshifted squaraine
To gauge the relative reactivity of SQ1 as a substrate for
neuraminidase, the rate of enzymeꢀcatalyzed cleavage was
compared to 4ꢀmethylumbelliferylꢀNꢀacetylꢀαꢀDꢀneuraminic
acid (4ꢀMUNANA), a wellꢀknown fluorescent substrate that
releases a greenꢀemitting 4ꢀmethylumbelliferone (Scheme
S6).43 Experiments using BNA and the clinically relevant
VNA from H1N1 influenza virus showed that in each case the
enzyme reaction of SQ1 is about ten times slower than
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Scheme 2. Basis of neuraminidase drug susceptibility assay.
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Figure 4. (top) Neuraminidase detection and drug susceptibility
assay using Mꢀcoated beads to capture the SQ1’ produced by
enzymatic cleavage of SQ1. Each assay vial contained about
twenty Mꢀcoated capture beads covered by a citrate solution conꢀ
taining: a, SQ1; b, SQ1 + VNA; c, SQ1 + VNA + Oseltamivir; d,
SQ1 + BNA + Oseltamivir. (bottom left) Deepꢀred micrographs
(ex: 635 nmꢀ675 nm, em: 694 nm – 736 nm) showing a repreꢀ
sentative Mꢀcoated bead taken from the four separate assay vials.
(bottom right) Average values of mean pixel intensity (MPI) for
each set of ten Mꢀcoated beads taken from the four separate assay
vials (**** indicates p value < 0.0001).
Figure 3. Solutionꢀstate version of the drug susceptibility test.
(top) Photographs of four microwells containing aqueous soluꢀ
tions of: a, SQ1 + M; b, SQ1 + M + VNA; c, SQ1 + M + VNA +
Oseltamivir; d, SQ1 + M + BNA + Oseltamivir. (bottom) Quantiꢀ
tative comparison of fluorescence intensity (ex: 620 nm, em: 725
nm) for each microwell, N = 3. (*** indicates p value < 0.001).
The next step was to demonstrate the beadꢀcapture version of
the assay that is illustrated conceptually in Figure 4. In this
case, the cleavage product SQ1’ was captured by beads coated
with M and the individual beads subsequently interrogated
using deepꢀred fluorescence microscopy. The Mꢀcoated beads
were easily prepared by simply adsorbing the highly anionic
M onto the surface of cationic Amberlite IRAꢀ958 ion exꢀ
change beads (0.70 mm diameter). As shown in Figure S9 the
Mꢀcoated beads exhibited strong UV/blue anthracene fluoresꢀ
cence that was readily apparent to the naked eye. Control exꢀ
periments showed that the Mꢀcoated beads did not capture the
blocked squaraine SQ1 from aqueous solution but there was
obvious squaraine capture by the beads after SQ1 was conꢀ
verted to SQ1’. The Mꢀcoated beads rapidly became blue to
the naked eye, and fluorescence micrographs showed enꢀ
hanced deepꢀred fluorescence intensity. Strong evidence that
threaded M ⊃ SQ1’ had formed on the bead surface was the
observation of internal fluorescence energy transfer from exꢀ
cited M to SQ1’ (Figure S10).
4ꢀMUNANA (Figures S7 and S8). As expected from known
neuraminidase substrate structure/activity relationships,49 the
rate of VNAꢀcatalyzed cleavage was slower than the cleavage
catalyzed by BNA. Nonetheless, consumption of all SQ1 was
complete well within the designated one hour analysis period
using 0.1 unit (U) of VNA per nmol of SQ1.
Assay for Neuraminidase Detection and Drug Sus-
ceptibility
The basis of the neuraminidase detection and drug susceptiꢀ
bility assay is illustrated in Scheme 2. The presence of active
neuraminidase triggers cleavage of SQ1 and formation of
threaded M ⊃ SQ1’ along with diagnostic spectral changes.
These spectral changes are suppressed if the neuraminidase is
inhibited by a drug. Initial studies were conducted in aqueous
solution with the results shown in Figure 3. A microwell conꢀ
taining an admixture of M + SQ1 was blue to the naked eye
(microwell a) and changed to green after the addition of VNA
(microwell b). There was no color change when the VNA was
inhibited by the drug Oseltamivir, a known inhibitor of VNA
(microwell c). As expected, there was a color change to green
when the admixture of M + SQ1 + Oseltamivir was treated
with BNA (microwell d) which is not inhibited by Oseltamiꢀ
vir.50 These prototype results mimic the possible outcomes for
a patient who is infected with a strain of flu containing neuꢀ
raminidase that is either Oseltamivirꢀsensitive or Oseltamivirꢀ
resistant.
In the bottom left of Figure 4 are representative deepꢀred
fluorescence micrographs of four individual Mꢀcoated beads,
each one taken from a different assay vial. An individual assay
vial started with about twenty Mꢀcoated capture beads, and at
the end of the assay ten of these beads were removed for analꢀ
ysis using fluorescence microscopy (Figure S11). The bottom
right of Figure 4 shows a graphical comparison of average
fluorescence intensity for the four different sets of assay
beads. The bead assay results matched the solutionꢀstate assay
results above. That is, the presence of VNA produced a twoꢀ
fold increase in deepꢀred fluorescence from an Mꢀcoated bead
due to capture of the deepꢀred fluorescent cleavage product
SQ1’. The bead capture assay confirmed that Oseltamivir inꢀ
hibited the added VNA (indicated by no change bead deepꢀred
fluorescence) but did not inhibit the added BNA (twoꢀfold
increase in bead fluorescence).
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temperature for 30 min. SQ1 (33 µL, 600 µM) was added and
Conclusion
1
the resulting solution was diluted to 100 µL and incubated for
1 h at 37 ˚C. A 10 µL aliquot of each microwell solution was
transferred to 1 mL H2O and the single point fluorescence
intensity was measured (ex: 620 nm, em: 725 nm) and the
average intensity was plotted on a bar graph with error bars
corresponding to the standard deviation.
The results of this study are proof of concept for a novel fluoꢀ
rescent dye capture assay that detects enzymatic cleavage acꢀ
tivity. The prototype design responds to viral neuraminidase,
an indicator of influenza infection, and also measures suscepꢀ
tibility of the sample to neuraminidase inhibitor drugs. The
assay is enabled by the extremely rapid and high affinity
threading of tetralactam macrocycle M by squaraine dye SQ1’
that is liberated after enzymatic cleavage of blocking groups
from substrate SQ1 (Scheme 1). The enzyme assay can be
conducted in solution where the dramatic changes in optical
properties can be easily detected by the naked eye (Figures 2
and 3). Alternatively, the assay can incorporate affinity capꢀ
ture beads to localize the liberated SQ1’ which permits quantiꢀ
tative analysis by automated heterogeneous methods utilizing
fluorescence microscopy, microarrays, microfluidics, or flow
cytometry.7ꢀ13,18 These methods can rapidly analyze large
numbers of individual beads with great precision using cheap
cameras and detectors. In addition, bead capture can increase
detection sensitivity by removing the liberated dye from turbid
samples (which lowers the background signal), and concenꢀ
trating all of the dye within the interrogation volume of a foꢀ
cused excitation beam (which maximizes dye signal). With
further optimization, this new type of fluorescent neuraminiꢀ
dase assay may be useful in a point of care clinic to immediꢀ
ately determine which of the two currently approved antiviral
inhibitor drugs, Oseltamivir (Tamiflu) or Zanamivir (Relenza),
is likely to be the most effective treatment for an individual
patient. A portable and rapid test for the presence of influenza
would improve patient treatment and reduce overuse of antiviꢀ
rals. Another important clinical analysis goal is to differentiate
between the different classes of mammalian, bacterial and
viral neuraminidase, and it should be possible to fineꢀtune the
chemical structure of the sialic acid blocking groups in SQ1
and achieve high specificity for influenza VNA.49,50
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Preparation of M-coated beads
The macrocycle M (2 mg) and Amberlite IRAꢀ958(Clꢀ) ion
exchange beads (Alfa Aesar, average diameter 0.70 mm) (300
mg) were mixed together in H2O (1 mL) and the mixture shakꢀ
en at room temperature for 30 min. The beads were filtered by
centrifugation and washed with H2O (6 x 3 mL).
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Bead-capture assay for neuraminidase activity and
drug susceptibility
Appropriate aliquots were delivered to a vial to give 140 µL
solution with the following final concentrations: Sodium citꢀ
rate (143 mM), enzyme (VNA, 0.09 U/µL or BNA, 0.09
mU/µL), and Oseltamivir (3.6 ng/µL) and incubated at room
temperature for 30 minutes. SQ1 (60 µL, 1 mM) and about
twenty Mꢀcoated beads were added to the vials and the mixꢀ
ture incubated for 1 h at room temperature. The beads were
removed, washed with H2O (6 x 3 mL) and imaged by fluoresꢀ
cence microscopy at 10x magnification on a Zeiss Axiovert
100 TV epifluoresence microscope equipped with an Xꢀcite
120 fluorescence illumination system. Images of ten separate
beads taken from the same assay vial were acquired using an
Andor iXon3 EMCCD camera operating in CCD mode with
20 ms acquisition time and a Cy5.5 filter set (ex: 635 nmꢀ675
nm, em: 694 nm ꢀ 736 nm) or a UVꢀRed filter set (ex: 352 nm
ꢀ 402 nm, em: 694 nm ꢀ 736 nm). The fluorescence mean pixel
intensity (MPI) for each bead micrograph was measured after
using ImageJ software to draw a region of interest around the
bead image. The average MPI for each set of ten beads taken
from a specific assay vial was plotted on a bar graph with error
bars corresponding to the standard deviation.
While this study focused only on fluorescence detection, it is
worth emphasizing that squaraine dyes have structural features
that enable high sensitivity detection using other techniques
such as electrochemistry,51 electrogenerated chemiluminesꢀ
cence52 or Surface Enhanced Resonance Raman Spectroscopy
(SERRS).53 In addition, it should be straightforward to modify
the design of this inꢀsitu squaraine capture assay and monitor a
wide range of cleavage enzymes such as glycosidases, proteꢀ
ases, phosphatases, lipases, etc. Each case requires the enzyme
to remove a blocking group from a suitably designed
squaraine dye to allow release of SQ1’ (or structural analogue)
and subsequent solutionꢀstate capture by M or immobilization
by Mꢀcoated beads. The growing literature on enzymeꢀ
triggered selfꢀimmolative bond cleavage processes will guide
future efforts to create these new classes of squaraine subꢀ
strates.47,54
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on
the ACS Publications website at DOI: 10.1021/. Synthetic
procedures, compound characterization and spectral data,
experimental methods, and additional assay data.
AUTHOR INFORMATION
Corresponding Author
smith.115@nd.edu
EXPERIMENTAL SECTION
Detailed synthetic procedures and compound characterization
are provided in the Supporting Information.
ACKNOWLEDGEMENTS
This work was supported by grants from the NSF
(CHE1401783), the University of Notre Dame Center for
Advanced Diagnostics and Therapeutics, and the CONAꢀ
CyT (Mexico). We thank Scott Shaw for valuable assisꢀ
tance with fluorescence microscopy and analysis.
Solution-state assay for neuraminidase activity and
drug susceptibility
Appropriate aliquots were delivered to a microwell to give a
50 µL solution with the following concentrations: Oseltamivir
(10 ng/µL), neuraminidase (VNA, 0.24 U/µL or BNA, 0.24
mU/µL) and M (0.8 mM) and the mixture incubated at room
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(30) Smith, B. D. Beilstein J. Org. Chem. 2015, 11, 2540.
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