Efficient near infrared fluorescence detection of elastase enzyme using peptide-bound unsymmetrical squaraine dye

Article abstract of DOI:10.1016/j.bmcl.2017.07.057

Extended wavelength analyte-responsive fluorescent probes are highly desired for the imaging applications owing to their deep tissue penetration, and minimum interference from autofluorescence by biomolecules. Near infra-red (NIR) sensitive and self-quenching fluorescent probe based on the dye-peptide conjugate (SQ 1 PC) was designed and synthesized by facile and efficient one-pot synthetic route for the detection of Elastase activity. In the phosphate buffer solution, there was an efficient quenching of fluorescence of SQ 1 PC (86%) assisted by pronounced dye-dye interaction due to H-aggregate formation. Efficient and fast recovery of this quenched fluorescence of SQ 1 PC (> 50% in 30 s) was observed on hydrolysis of this peptide-dye conjugate by elastase enzyme. Presently designed NIR sensitive self-quenching substrate offers the potential application for the detection of diseases related to proteases by efficient and fast detection of their activities.

Full text of DOI:10.1016/j.bmcl.2017.07.057

Accepted Manuscript
Efficient near infrared fluorescence detection of elastase enzyme using peptide-
bound unsymmetrical squaraine dye
Maryala Saikiran, Daisuke Sato, Shyam S. Pandey, Shuzi Hayase, Tamaki Kato
PII:
S0960-894X(17)30764-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.07.057
BMCL 25170
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
16 May 2017
13 July 2017
21 July 2017
Please cite this article as: Saikiran, M., Sato, D., Pandey, S.S., Hayase, S., Kato, T., Efficient near infrared
fluorescence detection of elastase enzyme using peptide-bound unsymmetrical squaraine dye, Bioorganic &
Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.07.057
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Efficient near infrared fluorescence detection of elastase enzyme
using peptide-bound unsymmetrical squaraine dye
Maryala Saikiran, Daisuke Sato, Shyam S. Pandey, Shuzi Hayase and Tamaki Kato
Graduate School of Life Science and Systems Engineering, Kyushu Institute of
Technology, 2-4 Hibikino, Wakamatsu, Kitakyushu 808-0196, Japan
Email: msaikiran87@gmail.com, shyam@life.kyutech.ac.jp, tmkato@life.kyutech.ac.jp
Abstract
Extended wavelength analyte-responsive fluorescent probes are highly desired for the imaging
applications owing to their deep tissue penetration, minimum interference from autofluorescence by
biomolecules. Near infra-red (NIR) sensitive and self-quenching fluorescent probe based on the
dye-peptide conjugate (SQ 1 PC) was designed and synthesized by facile and efficient one-pot
synthetic route for the detection of Elastase activity. In the phosphate buffer solution, there was an
efficient quenching of fluorescence of SQ 1 PC (86 %) assisted by pronounced dye-dye interaction
due to H-aggregate formation. Efficient and fast recovery of this quenched fluorescence of SQ 1 PC
(> 50 % in 30 seconds) was observed on hydrolysis of this peptide-dye conjugate by elastase
enzyme. Presently designed NIR sensitive self-quenching substrate offers the potential application
for the detection of diseases related to proteases by efficient and fast detection of their activities.
Keywords: Far-red squaraine dye, aggregation, fluorescent peptide, self-quenching conjugate,
Elastase detection, bio imaging.
Growing demand for point-of-care testing (POCT)
screening of complex protein functions including
qualitative as well as quantitative estimation of
proteases [7]. Proteases are an important class of
physiological enzymes which hydrolyse the amide
bond at specific sites of the polypeptide chain thereby
playing a vital role in the regulation of a large
number of physiological processes such as cell
devices for home diagnostic and facile health care
monitoring, biosensors have attracted tremendous
attentions owing to utilization of small amount of
biological samples, ease of handling and user-
friendliness [1, 2]. Although, biosensors are one of
the strong contenders for POCT devices, due to the
existing challenges like utilization of single analyte at
a time, controlling the sensitivity in the presence of
interfering agents, prior analyte processing for higher
sensitivity and low throughput are needed to be
considered for the further development of more
efficient and versatile POCT devices. It is, therefore,
necessary to develop biosensors that are sensitive and
having the capability of high throughput detection
along with their compatibility with imaging
techniques for simultaneous multi-target analysis [3].
The advent of Microarray technology has led the
possibility of rapid profiling of huge number of
proteins in a single experiment [4, 5]. Working with
protein arrays for bio-sensing applications is
challenging and cumbersome to control polar and
ionic interactions, unspecific adsorption of proteins
and preservation of native form of protein along with
their spatial orientations onto the surface after
immobilization [6]. This is the reason why more
attentions are being paid for the development of
peptide arrays for rapid and high throughput
proliferation/differentiation,
apoptosis,
DNA
replication, haemostasis and immune responses [8, 9].
Human Neutrophil Elastase (HNE) belongs to serine
class of proteases and is a proteolytic enzyme stored
in the azurophilic granules of polymorphonuclear
cells [10]. Although the proteolytic destruction of
bacteria is one of the major roles of this enzyme, its
hyperactivity leads to the pathogenesis of acute and
chronic inflammations [11, 12]. Excess release of
HNE cleaves the cellular receptors, activates
protogenetic mediators and intrinsically involved in
the epithelial as well as well as endothelial membrane
damage [13, 14]. Therefore, knowledge of roles
played by different serine protease along with their
strict control and monitoring especially HNE activity
has a great importance for therapeutic viewpoints.
In spite of fast growth and development of
protease assay methods, methods based on
immunoassay consisted of specific binding to the
target protease with antibodies have been although

commercialized and able to estimate the proteases
quantitatively [15]. However, they are not much
suitable for mapping protease activity and stages of
disease which are associated with protease activity
rather than their quantity. In order to avoid these
issues of immunoassay-based detection of proteases,
several other methods such as utilization of suitable
appearance of the fluorescence [21]. Internally
quenched fluorescence-based fluorescent dendritic
peptides substrates have also been reported which
exhibit fluorescence ON sensing of chymotrypsin
enzyme due to increased fluorescence emission after
the enzymatic hydrolysis of the designed substrates
[22]. Squaraine dye belongs to a class of intensely
peptide
substrates
based
on
optical
colored
dyes
having
donor-acceptor-donor
(
absorbance/fluorescence) detection and appearance
zwitterionic molecular framework containing squaric
acid as central four-membered ring core surrounded
by electron-rich aromatic moieties. Incorporation
immensely available and judiciously selected
aromatic/heterocyclic systems with varying donor
strength it is easily possible to design a variety of
squaraine dyes with tunable light absorption and
fluorescence emission encompassing from visible to
IR wavelength region [23]. In this present work,
efforts have been directed to design an internally
or quenching of fluorescence emanating from Forster
resonance energy transfer (FRET) have been widely
used [16-18]. Interaction of proteases with these
chromogenic or fluorogenic substrates leads to the
selective proteolytic cleavage of the peptide bond
resulting in changes of their absorption or fluorescent
spectral behaviors which form the basis for the
detection of protease activity. In this context,
fluorogenic
substrates
are
preferred
over
chromogenic substrates owing to their enhanced
sensitivity and compatibility with the high throughput
fluorescence mapping on microarray-based platforms
quenched
homo-labelled
fluorescent-peptide
substrate utilizing a far-red sensitive unsymmetrical
squaraine dye coupled with a peptide sequence
susceptible to the pancreatic elastase enzyme. Figure
1 exhibits the structure of the main building blocks
[
19]. A perusal of the fluorogenic substrates used for
the protease assay reveals that the wide-spread use of
fluorescent tags working mainly in the visible or low
wavelength region. In this case, not only auto-
fluorescence resulting from the biological analyte
poses a limitation to attain good signal-to-noise (S/N)
ratio but also photo-damage to biological species
which cannot be avoided owing to the high energy
excitations. In this context, utilization of fluorophores
exhibiting emission in the near infra-red (NIR)
wavelength region not only imparts much higher
sensitivity owing to its highly diminished auto-
fluorescence from the biological samples but also are
capable of deep tissue penetration leading to efficient
bio-imaging [20]. On the other hand, the FRET-based
assay requires the logical development of suitable
fluorophore and quencher pair owing to their
synthetic complications due to the need of orthogonal
protecting groups while their coupling with peptide
substrate library.
like
direct
carboxy
ring-functionalized
unsymmetrical squaraine dye SQ-1 (1a), Elastase
enzyme specific peptide sequence β-Ala-Ala-Pro-
Ala-Lys-(OBzl) (1b) along with fluorescent peptide
substrate (1c). Figure 1(d) represents the MM2
energy minimized three-dimensional structure of (1b)
and led to the proposal of a possible structure of
peptide-dye conjugate as shown in the Fig. 1(c).
The design of peptide substrates tagged with
fluorescent moieties having the capability of self-
quenching and re-appearance of the fluorescence
after enzymatic hydrolysis has also been simplified
for the solution of the relatively complicated
fluorescence-peptide-quencher molecular system.
This was demonstrated by designing the substrates
after the incorporation of a multiple number of
identical fluorophores leading to concentration-
dependent quenching. Sato et al have demonstrated
the concentration dependent fluorescence quenching
for their fluorescein isothiocyanates (Fluorescent
moiety) tagged peptide substrate for Trypsin enzyme
and proteolytic cleave of the peptide bond led to the
Figure 1. Chemical structure of unsymmetrical
squaraine dye (a), elastase enzyme specific peptide
(
b), peptide-dye conjugate (c) and MM2 energy
minimized 3D structure of the peptide (d).
It can be clearly seen from the structure of
our newly proposed peptide-dye conjugate that two
dye molecules are connected to one peptide at either
end of the terminals. SQ-1 was selected as model dye
not only due to its NIR fluorescence capability but

also an appreciably good interaction of squaraine
dyes with most commonly used model proteins like
bovine serum albumin and human serum albumin [24,
Scheme 1. Synthetic strategy of dye-peptide
conjugate used for present investigation.
2
5]. It has been reported that pancreatic serine
Electronic absorption spectrum of SQ-1 in
dimethylformamide (DMF) solution exhibits a strong
electronic absorption maximum (λmax) at 672 nm
along with the feeble vibronic shoulder around 6105
nm and molar extinction coefficient of 1.02 x 10
protease elastase cleaves the substrate at peptide
bonds on the carboxyl side of amino acid residues
bearing small alkyl side chain which has led us to
design Ala-Pro-Ala as target peptide for elastase
enzyme [26]. β-Ala and Lys-containing free amino
groups have not only been used as a spacer but also
for assisting the easy binding of the SQ-1 dye with its
free –COOH group in a single one pot reaction. A
perusal of this newly designed substrate as shown in
Fig. 1(c) reveals dense packing of the SQ-1, which is
expected to bring the internal self-quenching of the
fluorescence. At the same time, the flat molecular
structure of squaraine dyes is also expected to
promote the dye aggregation especially in the buffer
solution and aggregated dye molecules have been
reported to exhibit the quenched fluorescence [27].
Internally fluorescence quenched peptide-dye
conjugate was synthesized by one-pot reaction of
NHS-ester activated unsymmetrical squaraine SQ-1
and peptide H-β-Ala-A-P-A-K-OBzl as per the
scheme-1. Detailed synthesis and characterization of
dye SQ-1, peptide and dye-peptide conjugate along
with various intermediates have been provided in the
supporting information.
3
-1
-1
dm .mol .cm
which is associated with π-π*
electronic transition which is a typical characteristic
of the squaraine dyes. The λmax absorption and
emission values, molar extinction coefficient and
stoke shifts of SQ 1 dye and SQ 1 PC in both DMF
and PBS solutions were shown respectively in Table
1.
Merging of the vibronic shoulder with the
main absorption for SQ-1 in DMF solution indicates
the existence of dye aggregated species. It has been
reported that in the case of H-aggregate formation by
squaraine dye, sometimes shoulder becomes even
more pronounced as compared to monomeric dye
absorption peak. Prevention of this dye aggregation
by using aggregation preventing species like
chenodeoxycholic acid leads to decrease in the
absorption corresponding to this shoulder verifying
the suppression of dye aggregation [28]. On the other
hand, its emission spectrum shows fluorescence
emission peak at 754 nm with relatively large Stokes
shift of 82 nm. Typically, squaraine dyes exhibit a
Stokes shift of 20-30 nm due to its rigid molecular
structure and indicate the nearly similar molecular
configuration of dyes in both of the ground as well as
excited states [29]. This large Stokes shift in the case
of SQ-1 in DMF solution indicates rather diminished
conformational stability of SQ-1 in an excited state as
compared to typical squaraine dyes. On the other
hand, the electronic absorption spectrum of a dye-
peptide conjugate (SQ 1 PC) exhibits relatively
broader optical absorption having a blue-shifted λmax
at 664 nm and fluorescence emission maximum at
7
36 nm with a stoke shift of 72 nm as shown in the
Fig. 2(b). This blue-shift in the absorption and
emission maximum could be attributed to the dye
aggregate formation. It has been reported that
squaraine dyes form the blue-shifted H-aggregates
and red-shifted J-aggregates depending on their
structure, molecular environment owing to their
relatively flat molecular structure [30].

Table 1. Spectral properties of dye and dye-conjugate in DMF and 0.1 M PBS solution at pH 7.4.
DMF Solution
PBS Solution
λ (max)
Absorption
λ (max)
Emission
Stoke
Shift
(ε)
λ (max)
Absorption
λ (max)
Emission
Stoke
Shift
(ε)
3
-1
3
-1
(dm M
(dm M
-
1
-1
cm )
cm )
5
5
SQ 1 Dye
SQ 1 PC
672 nm
664 nm
754 nm
736 nm
82 nm
72 nm
1.02×105
644 nm
605 nm
700 nm
658 nm
56 nm
53 nm
0.66×105
3.7×10
0.98×10
considering the large stoke shift and small spectral
overlap contribution from FRET-based quenching
seem to be small. At the same time, blue shifted
absorption and emission maxima which are highly
pronounced in the case of phosphate buffer solution
(
PBS) as shown in the Fig. 2 (c, d) clearly
corroborates the dominance of the second mechanism
based on static concentration dependent quenching.
Thus considering the peak fluorescence intensities of
dye alone (SQ-1) and dye-peptide conjugate (SQ 1
PC), fluorescence quenching efficiency was
estimated to be 73 % and 86 % in the DMF and PBS,
respectively.
Figure 2. Electronic absorption and fluorescence
emission spectra of (a) unsymmetrical squaraine dye
SQ-1 and (b) peptide-dye conjugate (SQ 1 PC) in the
DMF solution of concentration 5 μM. Figures (c) and
Fairly
good
internal
self-quenching
fluorescence of dye SQ-1 after coupling with target
peptide was observed. This property of conjugate
enabled us to explore the application of dye-peptide
conjugate as enzyme activity detection based on
fluorescence ON bio-sensing. As discussed earlier,
Elastase enzyme recognition probe Ala-Pro-Ala
amino acid residue was chemically bound with the
free –COOH group of SQ-1 by simple one pot
reaction. It can be seen from the structure of SQ 1
PC that two terminal amino acids β-Alanine and
Lysine have been introduced to solve the two
purposes. Firstly, they function as a spacer to
promote the access of Elastase enzyme towards the
target tripeptide Ala-Pro-Ala. Secondly, availability
of free amines allows dense incorporation of SQ-1
dye promoting the internal self-quenching. To
demonstrate this, 10 μM solution of this target dye-
peptide conjugate SQ 1 PC was subjected to
enzymatic hydrolysis using Elastase enzyme in 0.1M
phosphate buffer at pH 7.4 and fluorescence emission
spectra were measured at different time intervals as
shown in the Fig. 3 (a). A perusal of this figure
clearly reveals that there was a pronounced recovery
of quenched fluorescence of the dye within 30
seconds and reached to saturation after 30 min
leading to about fivefold enhancement in the
fluorescence signal compared to that in the absence
of Elastase. This enhancement in the fluorescence
after enzymatic hydrolysis could be attributed to the
(
d) represents the same spectral profile for SQ-1 and
SQ 1 (PC) in 0.1 M phosphate buffer at pH 7.4 and
concentration of 10 μM, dotted line represents the
emission spectra corresponding solutions in all of the
cases.
It is interesting to note here that there is a drastic
decrease in the fluorescence intensity associated with
SQ-1 for the dye-peptide conjugate as compared to
the pure dye SQ-1 having a similar concentration (5
μM) and the same solvent (DMF). This indicates that
SQ-1 undergoes the fluorescence quenching upon its
conjugation with peptide under investigation (SQ 1
PC). In general two basic mechanisms have been
proposed for the fluorescence quenching. First is
related to quenching due to fluorescence resonance
energy transfer (FRET) while the second one is
related to the concentration dependent static internal
quenching. In the homo-dye labeled probes FERT
based quenching generally occurs when stoke shift is
very small allowing the sufficient overlapping
between the absorption and emission spectra of the
dye [31]. On the other hand, in the case of static
quenching, there is a change in the spectral shape
generally blue-shift in the absorption maximum
facilitated by H-aggregate formation [32]. Although
both of the mechanism seems to be applicable to
explain the fluorescence quenching of SQ 1 PC

fact that dye-aggregate formation is promoted after
conjugation of SQ-1 with peptide in phosphate buffer
leading to the H-aggregate assisted efficient
fluorescence quenching. After the enzymatic
hydrolysis of the peptide bond, multiple copies of
dyes are released and attain the special separation
leading to the re-appearance of the dye fluorescence.
there was completely no change in the case of control
that is in the absence of enzyme.
Overall we have demonstrated the NIR
fluorescence quenching promoted by enhanced dye
aggregation of dye-peptide conjugate and disruption
of this aggregation in the presence of enzyme led to
the facile detection of proteases based on
fluorescence-ON biosensing. Although detection of
Elastase activity has been successfully demonstrated
in this work, such design and concept can be easily
implemented to any target protease by judicious
selection of suitable central peptide sequence
susceptible to the target enzyme under investigation.
Use of single fluorophore and its one-pot coupling
with target peptide sequence provide simplicity and
rapid profiling of various proteases. Such a concept
of fluorescence-ON biosensing based on aggregation-
induced fluorescence quenching and re-appearance of
fluorescence after enzyme hydrolysis bears good
potential in medical diagnosis based on the
qualitative and quantitative estimation of enzymes.
Integration of such dye-peptide conjugates with
microarray technology is expected to tremendously
enhance the throughput of analysis. Utilization of
such dye-peptide conjugate for microarray
application has been schematically shown in the Fig.
Figure 3. Fluorescence emission spectra of the 0.1M
phosphate buffer solution of dye-peptide conjugate
(
10 μM) at pH 7.4 as a function of time after the
addition of 21 nmol of elastase enzyme. Right hand
side figure (b) represents the change in fluorescence
intensity as a function of time with different
concentrations elastase for a fixed concentration of
dye-peptide conjugate (10 μM).
4
. Amine functionalization of glass surface has been
widely studied along with the report of very high-
density amine functionalization using 3-amino
propyltriethoxysilane (APTS) by Beyer et. al [33]. It
can be easily seen from the Fig. 4 (b) that free –
COOH group in our proposed dye-peptide conjugate
can be easily obtained by hydrogenation of O-benzyl
Considering the fluorescence intensities before
(
control) and after enzymatic hydrolysis of SQ 1 PC,
quenching efficiency was estimated to be 81 %. This
indicates such a simple and homo-labelled dye-
peptide conjugate based on internal quenching is
capable to efficiently detect the enzymatic activity
based on fluorescence ON bio-sensing. Efforts were
also directed to test this designed dye-peptide
conjugate by changing the amount of Elastase
enzyme (3 nmol to 21 nmol) for a fixed concentration
of this dye-peptide conjugate (10 μM). Change in the
group using H , Pd/C, which is now ready to couple
2
with the free amine group of amine functionalized
glass surface (a) making glass surface-bound dye-
peptide conjugate (c). Thus we can attach various
kinds of such molecular probes on a single glass
surface in the microarray format. At the same time,
use of wavelength tunable squaraine dyes in such
dye-peptide conjugate system not only imparts the
widespread design of molecular probes but also
provide the capability of efficient and simultaneous
multi-target protease analysis facilitated by multi-
wavelength fluorescence-ON biosensing.
fluorescence intensity as
a function of time,
enzymatic hydrolysis was monitored for different
enzyme concentration and has been shown in the Fig.
3
(b). A perusal of this figure also indicates that rate
of change in fluorescence as function of
a
concentration is not much significant. On the other
hand, saturation fluorescence intensity after complete
hydrolysis exhibits clear and distinguishing change
for different concentrations of the Elastase. Therefore,
consideration of fluorescence intensity at saturation
seems to be more plausible for the quantitative
aspects of enzyme activity estimations using such
dye-peptide conjugate by the fluorescence-ON
biosensing. It can be seen that in all of the cases the
fluorescence intensities were reaching saturation after
about 25 min. of the enzymatic hydrolysis, while

6
7
.
.
Templin MF, Stoll D, Schrenk M, Traub PC,
Vöhringer CF, Joos TO. Protein microarray
technology.
Drug
discovery
today.
2
002;7(15): 815-822.
Legutki JB, Zhao Z-G, Greving M,
Woodbury N, Johnston SA, Stafford P.
Scalable high-density peptide arrays for
comprehensive health monitoring. Nature
communications. 2014;5.
8
9
.
.
Turk B. Targeting proteases: successes,
failures and future prospects. Nature reviews
Drug discovery. 2006;5(9): 785-799.
Rakash S, Rana F, Rafiq S, Masood A,
Amin S. Role of proteases in cancer: A
review. Biotechnology and Molecular
Biology Reviews. 2012;7(4): 90-101.
1
1
0. Korkmaz B, Moreau T, Gauthier F.
Neutrophil elastase, proteinase and
3
cathepsin G: physicochemical properties,
activity and physiopathological functions.
Biochimie. 2008;90(2): 227-242.
1. Avlonitis N, Debunne M, Aslam T, et al.
Highly specific, multi-branched fluorescent
reporters for analysis of human neutrophil
elastase. Organic & biomolecular chemistry.
Figure 4. Schematic representation for application of
proposed dye-peptide conjugate in peptide
microarray for enzyme detection based on
2
013;11(26): 4414-4418.
1
1
1
2. Doring G. The role of neutrophil elastase in
chronic inflammation. American journal of
respiratory and critical care medicine.
fluoresecence-ON
biosensing.
(a)
Amine
functionalized glass surface after treatment of glass
with APTS (b) dye-peptide conjugate and (c) glass-
surface modified with dye-peptide conjugate.
1
994;150(6): S114.
3. Shapiro SD. Neutrophil Elastase: Path
Clearer, Path ogen Killer, or Just Path
ologic? American journal of respiratory cell
and molecular biology. 2002;26(3): 266-268.
4. Abe T, Usui A, Oshima H, Akita T, Ueda Y.
A pilot randomized study of the neutrophil
elastase inhibitor, Sivelestat, in patients
undergoing cardiac surgery. Interactive
cardiovascular and thoracic surgery.
References:
1
2
3
.
.
.
Mashamba-Thompson TP, Sartorius B,
Drain PK. Point-of-care diagnostics for
improving maternal health in South Africa.
Diagnostics. 2016;6(3): 31.
Solanki PR, Kaushik A, Agrawal VV,
Malhotra BD. Nanostructured metal oxide-
based biosensors. NPG Asia Materials.
2
009;9(2): 236-240.
1
1
5. Lee Y-m, Jeong Y, Kang HJ, Chung SJ,
Chung BH. Cascade enzyme-linked
immunosorbent assay (CELISA). Biosensors
and Bioelectronics. 2009;25(2): 332-337.
6. Lesner A, Brzozowski K, Kupryszewski G,
Rolka K. Design, chemical synthesis and
kinetic studies of trypsin chromogenic
substrates based on the proteinase binding
loop of Cucurbita maxima trypsin inhibitor
2
011;3(1): 17-24.
Zeng Y, Wang L, Wu S-Y, et al. High-
throughput imaging surface plasmon
resonance biosensing based on an adaptive
spectral-dip tracking scheme. Optics
Express. 2016;24(25): 28303-28311.
4
5
.
.
Cretich M, Damin F, Pirri G, Chiari M.
Protein and peptide arrays: recent trends and
new directions. Biomolecular engineering.
(
CMTI-III). Biochemical and biophysical
research communications. 2000;269(1): 81-
4.
2
006;23(2): 77-88.
Foong YM, Fu J, Yao SQ, Uttamchandani
M. Current advances in peptide and small
molecule microarray technologies. Current
opinion in chemical biology. 2012;16(1):
8
1
7. Harris JL, Backes BJ, Leonetti F, Mahrus S,
Ellman JA, Craik CS. Rapid and general
profiling of protease specificity by using
combinatorial fluorogenic substrate libraries.
2
34-242.

Proceedings of the National Academy of
Sciences. 2000;97(14): 7754-7759.
The Journal of Physical Chemistry B.
2010;114(17): 5912-5919.
1
1
2
8. Sun H, Panicker RC, Yao SQ. Activity
based fingerprinting of proteases using
26. ꢀefꢁowitz ꢂꢃ, ꢄchmid-ꢄchꢅnbein ꢆꢇ,
Heller MJ. Whole blood assay for elastase,
chymotrypsin, matrix metalloproteinase-2,
and matrix metalloproteinase-9 activity.
Analytical chemistry. 2010;82(19): 8251-
8258.
27. Galande AK, Hilderbrand SA, Weissleder R,
Tung C-H. Enzyme-targeted fluorescent
imaging probes on a multiple antigenic
peptide core. Journal of medicinal chemistry.
2006;49(15): 4715-4720.
28. Yum J, Moon S, Humphry-Baker R, et al.
Effect of coadsorbent on the photovoltaic
performance of squaraine sensitized
nanocrystalline solar cells. Nanotechnology.
2008;19(42): 424005.
29. Marchena MaJ, de Miguel G, Cohen B, et al.
Real-time photodynamics of squaraine-
based dye-sensitized solar cells with iodide
and cobalt electrolytes. The Journal of
Physical Chemistry C. 2013;117(23):
11906-11919.
FRET
peptides.
Peptide
Science.
2
007;88(2): 141-149.
9. Tomizaki Ky, Usui K, Mihara H.
Protein‐Detecting Microarrays: Current
Accomplishments
and
Requirements.
ChemBioChem. 2005;6(5): 782-799.
0. Gong Y-J, Zhang X-B, Mao G-J, et al. A
unique approach toward near-infrared
fluorescent probes for bioimaging with
remarkably enhanced contrast. Chemical
Science. 2016;7(3): 2275-2285.
1. Sato D, Kato T. Novel fluorescent substrates
for detection of trypsin activity and inhibitor
screening by self-quenching. Bioorganic &
Medicinal Chemistry Letters. 2016;26(23):
2
2
5
736-5740.
2. Ellard JM, Zollitsch T, Cummins WJ,
Hamilton AL, Bradley M. Fluorescence
enhancement through enzymatic cleavage of
internally quenched dendritic peptides: A
sensitive assay for the AspN endoproteinase.
Angewandte Chemie International Edition.
30. Hu L, Yan Z, Xu H. Advances in synthesis
and application of near-infrared absorbing
squaraine dyes. RSC Advances. 2013;3(21):
7667-7676.
2
002;41(17): 3233-3236.
2
2
2
3. Pandey SS, Watanabe R, Fujikawa N, et al.
Effect of extended π-conjugation on
photovoltaic performance of dye sensitized
solar cells based on unsymmetrical
squaraine dyes. Tetrahedron. 2013;69(12):
31. Ternon M, Dꢈaz-Mochón JJ, Belsom A,
ꢉ
Bradley M. Dendrimers and combinatorial
chemistry—tools
enhancement
for
protease
fluorescent
assays.
in
2
633-2639.
Tetrahedron. 2004;60(39): 8721-8728.
32. Packard BZ, Toptygin DD, Komoriya A,
Brand L. [2] Design of profluorescent
protease substrates guided by exciton theory.
Methods in enzymology. 1997;278: 15-23.
33. Beyer M, Felgenhauer T, Bischoff FR,
Breitling F, Stadler V. A novel glass slide-
based peptide array support with high
functionality resisting non-specific protein
adsorption. Biomaterials. 2006;27(18):
3505-3514.
4. Sai kiran M, Pandey SS, Hayase S, Kato T.
Photophysical characterization and BSA
interaction of direct ring carboxy
functionalized symmetrical squaraine dyes.
Journal of Physics: Conference Series. (In
Press)
5. Jisha VS, Arun KT, Hariharan M, Ramaiah
D. Site-selective interactions: squaraine
dye− serum albumin complexes with
enhanced fluorescence and triplet yields.

Highlights:
1
) Novel carboxy functionalized squaraine dye (SQ-1) have been used as NIR fluorescent
probe.
2
3
4
) One-pot Dye-Peptide conjugate (SQ 1 PC) was synthesized by facile synthetic route.
) Efficient fluorescence quenching of SQ 1 PC (81%) assisted by dye aggregation.
) Demonstration of fluorescence ON biosensing after Elastase hydrolysis.