Carbon-dot-hydrogel for enzyme-mediated bacterial detection

Article abstract of DOI:10.1039/c6ra25148j

A hybrid carbon-dot (C-dot)-hydrogel matrix was constructed and employed for detection of bacteria. The transduction mechanism is novel, based upon cleavage of ester bonds within the hydrogel scaffold by bacterially-secreted esterases; the ensuing fluidization of the hydrogel resulted in aggregation of the embedded C-dots and consequent quenching of their fluorescence. We show that the C-dot-hydrogel exhibits high sensitivity and can distinguish among bacterial species through modulation of the emitted fluorescence, depending upon their esterase secretions.

Full text of DOI:10.1039/c6ra25148j

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Carbon-dot–hydrogel for enzyme-mediated
bacterial detection†
Cite this: RSC Adv., 2017, 7, 588
a
a
ab
*
Sagarika Bhattacharya, Sukhendu Nandi‡ and Raz Jelinek
A hybrid carbon-dot (C-dot)–hydrogel matrix was constructed and employed for detection of bacteria. The
transduction mechanism is novel, based upon cleavage of ester bonds within the hydrogel scaffold by
bacterially-secreted esterases; the ensuing fluidization of the hydrogel resulted in aggregation of the
embedded C-dots and consequent quenching of their fluorescence. We show that the C-dot–hydrogel
exhibits high sensitivity and can distinguish among bacterial species through modulation of the emitted
fluorescence, depending upon their esterase secretions.
Received 12th October 2016
Accepted 1st November 2016
DOI: 10.1039/c6ra25148j
www.rsc.org/advances
physical state of the hydrogel as a sensing strategy could be, in
fact, a promising sensing avenue. Varied externally-added mole-
Introduction
cules were shown to affect the structural features and mechanical
properties of hydrogel matrixes.12–14 Hydrogel framework modu-
lation has been employed as a sensing platform.15,16
Hydrogels, three dimensional exible networks of water-soluble
polymers, are useful host materials enabling encapsulation of
high concentrations of varied guest molecules and nano-
particles.1–7 Hydrogels are generally classied according to two
categories: (i) thermally-irreversible hydrogels in which the gelator
molecules are crosslinked by covalent bonds (dened as chemical
gels),5 and (ii) thermally-reversible physical gels in which the
gelator molecules exhibit weak physical interactions.5 Hydrogels
have been used in varied applications including tissue engineering,
drug delivery and controlled-release, and medical implants.5
Hydrogel composites have been used as useful biosensing
platforms. Redox-based sensing schemes utilizing hydrogel-
encapsulated enzymes were reported.8,9 A peptide hydrogel
consisting of a Fmoc-diphenylalanine-encapsulating enzyme
receptor (e.g., glucose oxidase or horseradish peroxidase) and
semiconductor quantum dots (QDs) was reported for detection
of glucose and toxic phenolic compounds through quenching of
the QDs' uorescence.9 A hydrogel cross-linked with aptamers
was reported to detect very low amount of cocaine through
visual detection.10 Porous silica polyacrylamide hydrogel system
served as an optical bacterial sensor.11
Carbon dots (C-dots) are newly discovered uorescent
carbonaceous nanoparticles (<10 nm), and have attracted broad
interest in light of their intriguing luminescence properties,
particularly a broad range of excitation-dependent emission
range.17–24 C-dots are biocompatible and less cytotoxic than
semiconductor quantum dots, thus constituting promising
vehicles for biological studies.19,25,26 Modulation of C-dots'
uorescence has been used as a powerful transduction mech-
anism in varied sensing applications, for example targeting
metal ions,27,28 organic molecules,29,30 and radical species.31,32 C-
dots have been recently used as a platform for bacterial sensing
and imaging.33 Hybrid carbon-dot hydrogel systems were also
used for detection of heavy metal ions.34,35
Herein, we report construction of a new C-dot–hydrogel hybrid
material which was used for bacterial detection through modula-
tion of the uorescence emission of the hydrogel-embedded C-
dots. In particular, we found that cleavage of hydrogel scaffolds'
ester bonds by bacterially-secreted esterases and lipases resulted in
uidization of the hydrogel, consequent aggregation and
concomitant uorescence quenching of the embedded C-dots. The
C-dot–hydrogel constitutes a sensitive bacterial detection platform
since the transduction mechanism is based upon secreted
enzymes rather than the bacterial cells themselves. Furthermore,
the new assay can be employed for distinguishing bacterial species
based upon the extent of their esterase/lipase secretion.
The hydrogel-based sensors discussed above largely rely upon
molecular recognition and/or redox reactions involving embedded
enzymes as the components in the transduction mechanism. In
such systems, the hydrogel serves only as a host matrix for
immobilization of guest biomolecules. Exploiting changes in the
aDepartment of Chemistry, Ben-Gurion University of the Negev, Beer Sheva 84105,
Israel. E-mail: razj@bgu.ac.il; Fax: +972-8-6472943
bIlse Katz Institute for Nanotechnology, Ben-Gurion University of the Negev, Beer Sheva
Experimental
Materials
84105, Israel
† Electronic supplementary information (ESI) available: Photoluminescence
spectra of C-dots in gel and in solution (Fig. S1). See DOI: 10.1039/c6ra25148j
‡ Present address: Ruhr Universitat Bochum, Universitaetsstrasse 150, D-44780,
Bochum, Germany.
D-Glucose, sodium sulfate, pyridine, lauric acid and sodium
chloride were purchased from Sigma Aldrich, USA. L-(+)-Tartaric
588 | RSC Adv., 2017, 7, 588–594
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
acid was purchased from Alfa-Aesar, England. Lauroyl chloride and further heated for 2–3 min until the gelator compound was
was purchased from TCI, Japan. Chloroform and n-hexane were dissolved completely. Upon cooling to room temperature for
bought from Daejung chemicals, Korea. Dimethylformamide about 24 hours, the aqueous solution completely gelied, tested
(DMF) and acetone were purchased from Frutarom (Haifa, by the “stable to inversion” method.
Israel). Ethyl acetate and concentrated hydrochloric acid were
purchased from Bio-Lab Ltd (Jerusalem, Israel). Luria–Bertani
(LB) agar was purchased from Pronadisa (Spain). All chemicals
were used without further purication.
Bacterial growth
The bacteria used in the studies were Bacillus cereus, Bacillus
subtilis (stain Bacillus 3610), Pseudomonas aeruginosa PAO1 wild
type, and Staphylococcus aureus wild type.38 The bacteria were
grown aerobically at 37 ꢀC in sterilized solid LB medium
composed of 13.5% yeast extract, 27% peptone, 27% NaCl, and
32.5% agar at pH 7.4. Aer overnight growth, a colony from
each bacterial strain was taken and added to 10 mL sterilized LB
medium and incubated at 37 ꢀC. Bacterial growth was moni-
tored in the specic time points through measuring the
concentration of the bacteria by visible spectroscopy (108 cells
per mL when optical density at 600 nm was 1.0). Bacterial cells
were separated from the growth medium through centrifuga-
tion followed by washing with sterilized water several times and
resuspending in distilled water.
C-dot–hydrogel synthesis
The C-dots and hydrogel were prepared from 6-O-(O-O0-dilaur-
oyltartaryl)-D-glucose (compound 1, Fig. 1A) according to
a published procedure.36,37 Briey, we prepared 6-O-acylated
fatty acid ester of D-glucose by reacting it with O-O0-
dilauroyltartaric acid anhydride. In a Teon lm tightened,
septum-capped test tube 100 mg of 1 and 330 mL DI water were
ꢀ
placed and then heated in an oven to 125 C for 2.5 h. Upon
completion of the carbonization, the reaction mixture was
cooled to room temperature yielding a brown precipitate indi-
cating the formation of carbon dots and puried for further
work. For preparation of the C-dot–hydrogel, 5 mg of C-dots
were mixed with 50 mg of compound 1 and dissolved in 0.5
Confocal uorescence microscopy
ꢀ
mL DI water in a sealed vial by sonicating for 30 min at 50 C
Confocal microscopy images of the C-dot–hydrogel were
acquired on an UltraVIEW system (PerkinElmer Life Sciences,
Waltham, MA) equipped with an Axiovert-200M microscope
(Zeiss, Oberkochen, Germany) and a Plan-Neouar 63ꢁ/1.4 oil
objective. Excitation wavelengths of 405, 488 and 514 nm were
produced by an argon/krypton laser. For the experiment the C-
dot–hydrogel was added on a glass slide.
Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) images were recorded on
a Quanta 200 SEM. For the SEM study, the dilute solution of gel
materials was dried on a glass cover slip and gold coating was
carried out.
Fluorescence spectroscopy and detection of bacteria
A Varioskan 96-well plate reader was used for the uorescence
analysis. 50 mL of C-dot–hydrogel was placed in each well and
gelation was carried out. Aer 24 hours, uorescence
measurement and titration with different bacterial stains at 108
cells per mL bacterial concentrations were carried out using lex
¼ 375 nm at 37 ꢀC by addition of 15 mL aqueous bacterial
suspension. Quenching efficiencies (Q. E.) of the C-dot–hydro-
gel hybrid were calculated by the Stern–Volmer equation:
Fig. 1 The new carbon-dot (C-dot)–hydrogel construct and its use for
bacterial detection. (A) Preparation scheme; gel formation through
a hydrogen bond network among the amphiphilic glucose precursor
molecule 1. The hydrogen bonds are indicated in the broken pink lines.
The amphiphilic C-dots are dispersed within the hydrogel framework.
(B) Sensing scheme: bacterially-secreted enzymes cleave ester bonds
(some are indicated by the arrows), inducing collapse of the hydrogel
framework. The C-dots aggregate and their fluorescence is conse-
quently quenched. (C) Digital photographs depicting the bacterially-
induced hydrogel and fluorescence transformations. The left images
show the as-prepared gelatinous and fluorescent (lex: 365 nm) C-
dot–hydrogel. Photographs on the right depict the bacterially-
induced fluidization of the hydrogel and concomitant fluorescence
quenching.
I0 ꢂ I
Q: E: ¼
I
in which I0 and I refer to the uorescence intensities of the
system (lem ¼ 430 nm) in the absence and presence of bacterial
suspensions, respectively.
Thermal and chemical treatments on B. cereus
The aqueous suspensions of 108 cells per mL bacterial cells were
ꢀ
heated to 100 C and then cooled to room temperature. From
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 588–594 | 589

View Article Online
RSC Advances
Paper
this suspension, 15 mL bacteria were added to the C-dot– Fig. 1C depict the bacterially-induced transformation of the
hydrogel to observe the change in uorescence intensity hybrid C-dot–hydrogel. As shown in Fig. 1C, le, the as-
ꢀ
(430 nm emission wavelength at 37 C, where lex: 375 nm).
prepared dense gelatinous substance was uorescent (lex: 375
To determine the contribution of divalent metal ions to the nm) due to the embedded dispersed C-dots. Following incuba-
enzymatic activity of B. cereus, 15 mL of 5 mM EDTA solution was tion with a bacterial suspension, the gel material became more
added together with 15 mL of aqueous bacterial suspension to uid and non-uorescent (Fig. 1C, right).
the C-dot–hydrogel.
Fig. 2 presents microscopic characterization of the C-dot–
hydrogel composite. The representative scanning electron
microscopy (SEM) image in Fig. 2A reveals a highly porous sheet
morphology of the hydrogel displaying considerable internal
surface area. The uorescence microscopy data in Fig. 2B
conrm encapsulation of abundant C-dots within the hydrogel
matrix. Specically, the confocal uorescence microscopy
images in Fig. 2B reveal a highly uorescent hydrogel scaffold
due to the C-dots bound to the hydrogel surface. In particular,
the different colors of the hydrogel in Fig. 2B reect the
excitation-dependent uorescence emissions of the embedded
C-dots.21,22 The excitation-dependent emission spectra of the
hydrogel-embedded C-dots was distinctive from soluble C-dots
(see ESI, Fig. S2†), conrming their encapsulation within the
hydrogel scaffold.
We examined the effect of bacterial suspensions upon the
uorescence properties of the C-dot–hydrogel. Fig. 3 shows the
uorescence modulation (lex ¼ 375 nm) induced by addition of
Bacillus cereus (cell concentration 108 cells per mL) to the C-dot–
hydrogel. Fig. 3A reveals signicant reduction of the uores-
cence emission recorded 15 minutes aer incubating the
composite gel with the bacteria (concentration of 108 cells per
mL) at 37 ꢀC. The direct relationship between the degree of
uorescence quenching and bacterial concentration is depicted
in Fig. 3B. Indeed, the dose–response curve in Fig. 3B points to
a detection threshold of around 105 cells per mL reecting
excellent sensitivity of the C-dot–hydrogel platform.
Rheology measurements
Rheology experiments were carried out using an Advanced
Rheometer AR 2000 (TA Instruments) by cone and plate geom-
etry in a Peltier plate. The cone diameter was 40 mm, cone angle
4ꢀ, and truncation 104 mm. The rheoloꢀgy analysis was per-
formed by changing the shear rate (at 37 C).
NMR experiments
¨
NMR studies were carried out on a Bruker DPX 400 MHz spec-
trometer at 300 K. Compound concentrations were 10 mm in
methanol-d4. Chemical changes were monitored through
incubation of suspensions of Bacillus cereus with hydrogel
precursor 1 for 30 minutes. The reaction product was lyophi-
lized for 2 h, and 1H and 13C NMR experiments were carried out
without further purication.
FT-IR analyses
All the FT-IR measurements were performed on a Thermo
Scientic Nicolet 6700 spectrometer. The organic compound
before and aer addition of bacteria were identied by using
this technique. The extraction of the organic compound was
similar like the NMR experiments.
Results and discussion
While the data presented in Fig. 3A and B were recorded
Fig. 1 depicts the preparation scheme of the C-dot–hydrogel upon incubation of bacterial cell suspensions with the C-dot–
hybrid and the enzyme-mediated bacterial detection concept. hydrogels, we examined whether the uorescence quenching
The hybrid hydrogel was assembled through mixing the effect was related to molecules secreted by the bacteria. Indeed,
precursor molecule 6-O-(O-O0-dilauroyltartaryl)-D-glucose and Fig. 3C reveals that the uorescence quenching effect could be
amphiphilic C-dots in water (Fig. 1A). Electron microscopy effectively induced by the supernatant (i.e. aer discarding the
analysis conrmed the crystalline structure of the graphitic
cores of the C-dots (Fig. S1, ESI†). The C-dots/precursor solution
was gently heated until a homogeneous mixture was formed,
followed by gelation which occurred at room temperature.
Importantly, in the initial gel state prior to addition of bacterial
suspensions (Fig. 1B, le), the embedded amphiphilic C-dots
were dispersed within the hydrogel host matrix due to affinity
to amphiphilic and hydrophobic domains within the internal
porous surface. The hydrogel-embedded C-dots exhibited
enhanced uorescence as they were not aggregated, and thus
uorescence quenching was blocked.34,35
However, the hydrogel network was disrupted upon addition
of bacterial suspensions through ester bond cleavage by Fig. 2 Microscopic images of the C-dot–hydrogel: (A) scanning
electron microscopy (SEM) image of the C-dot–hydrogel. Scale bar
corresponds to 10 mm. (B) Confocal fluorescence microscopy images
of the C-dot–hydrogel. Bright field (i); and confocal fluorescence
bacterially-secreted esterases (enzymatic cleavage sites are
indicated by the arrows in Fig. 1B). The collapse of the hydrogel
network gave rise to aggregation of the embedded amphiphilic
microscopy images recorded using lex ¼ 405 nm, emission filter EM
C-dots and concomitant quenching of their uorescence emis-
445/60 (ii); lex ¼ 488 nm, emission filter EM 525/50 (iii); lex ¼ 514 nm,
sion (Fig. 1B, right). The visual and uorescence photographs in emission filter EM 525/50 (iv). Scale bar corresponds to 10 mm.
590 | RSC Adv., 2017, 7, 588–594
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
Fig. 4 Bacterially-induced changes to the rheological properties of
the C-dot–hydrogel. Angular frequency sweep experiments of C-
dot–hydrogel before and after addition of B. cereus (108 cells per mL)
at 37 ꢀC: (i) the storage modulus G0 before addition of bacterium
suspension; (ii) G0 after addition of bacterium suspension; (iii) the loss
modulus G00 before addition of bacterium suspension; (iv) G00 after
addition of bacterium suspension.
Fig. 3 Bacterially-induced quenching of the C-dot–hydrogel fluo-
rescence. (A) Fluorescence emission spectra (lex: 375 nm) of the C-
dot–hydrogel upon addition of B. cereus (108 cells per mL). Solid
spectrum: control sample (prior to supernatant addition); broken
spectrum: after addition of supernatant. (B) Bacterial dose response
curve (B. cereus) relating the extent of fluorescence quenching to
bacterial concentration; (C) fluorescence emission spectra (lex: 375
nm) of the C-dot–hydrogel upon addition of the bacterial supernatant
extracted from a B. cereus suspension containing 108 cells per mL.
release of glucose molecules. This is reected in the appearance
of a new downeld 13C peak at 63 ppm in the B. cereus-treated
sample (Fig. 6Aii) corresponding to the C6 atom of free glucose.
Similarly, 13C signals corresponding to C1a and C1b carbons of
free glucose appear at 94 and 98 ppm, respectively, following
incubation of the hydrogel precursor with the B. cereus
cell precipitate), indicating that molecules secreted by the suspension. While the 13C spectral analysis indicates cleavage
bacterial cells were responsible for uorescence modulation. No of the ester bonds linking the glucose moieties to the precursor
1
quenching was recorded upon mixing of bacteria and soluble C- hydrophobic backbone (Fig. 1), the H NMR experiments pre-
dots (data not shown), conrming that the quenching sented in Fig. 5B provide evidence for bacterially-induced
phenomenon was due to hydrogel disruption rather than direct hydrolysis of the ester bonds linking the lauryl side chains
interactions between the C-dots and bacterial cells.
(Fig. 1B). Indeed, inspection of Fig. 5B reveals generation of free
To account for the bacterially-induced modulation of lauric acid, reected in the appearance of the Ca-attached
hydrogel-embedded C-dots' uorescence we carried out experi- hydrogen at 2.25 ppm following addition of the bacteria.
ments aimed at characterization of the mechanical gel proper-
The uorescence data, mechanical, and spectroscopic anal-
ties before and aer addition of the B. cereus suspension (Fig. 4). yses in Fig. 3–5 suggest that ester-hydrolyzing enzymes, specif-
Specically, Fig. 4 depicts rheology analysis of the hydrogel ically esterases and lipases secreted by B. cereus are responsible
before and aer addition of B. cereus (108 cells per mL) at 37 ꢀC. for the structural and luminescent transformations observed in
The data in Fig. 4 present frequency sweep experiments in the C-dot–hydrogel system.40,41 Esterases constitute a diverse
which the storage modulus (G0) and loss modulus (G00) were enzyme family secreted by both mammalian and bacterial cells,
measured, reecting the gel-phase of the material in which G0 is which function through hydrolysis of ester bonds in target
greater than G00.39 The curves in Fig. 4ii and iv demonstrate that substrates.42 Notably, the hydrogel scaffold contains a multi-
both G0 and G00 signicantly decreased aer addition of the tude of ester bonds, both positioned in polar environments (e.g.
bacterial suspensions, reecting reduction of the hydrogel's ester bonds linking the glucose residues to the modied tartaric
visco-elasticity (e.g. greater uidity). The difference between G0 acid-based anhydride moieties, Fig. 1A) which serve as cleavage
and G00 was retained, however, pointing to retention of the sites for bacterially-secreted esterases, as well as ester bonds in
intrinsic gel organization of the C-dot–hydrogel in the non-polar regions of the hydrogel (e.g. between the acyl chains
frequency range measured.
and the central anhydride, Fig. 1A), constituting substrates for
Nuclear magnetic resonance (NMR) and Fourier transform lipases, considered as esterase subfamily which functions in
infrared (FTIR) spectroscopy experiments in Fig. 5 were non-polar lipidic environments.42
designed to elucidate the molecular factors responsible for the
Fig. 6 presents experiments designed to provide evidence for
bacterially-induced uidization of the C-dot–hydrogel. The 13C the assignment of bacterially-secreted esterases and lipases as
1
and H NMR data in Fig. 5A and B reveal that the molecules inducers for hydrogel reorganization and uorescence
secreted by B. cereus induced distinct chemical transformations quenching of the embedded C-dots. Fig. 6A highlights the
within the hydrogel scaffold. Specically, the 13C NMR spectra effects of heat and ethylenediaminetetraacetic-acid (EDTA)
in Fig. 5A demonstrate the occurrence of ester bond cleavage upon uorescence quenching. Specically, the uorescence
following addition of the B. cereus suspensions, resulting on emission data in Fig. 6A demonstrate that heating the B. cereus
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 588–594 | 591

View Article Online
RSC Advances
Paper
Fig. 6 Modulation of bacterially-induced fluorescence quenching by
physical and chemical treatments. (A) Fluorescence emission spec-
trum (lex: 375 nm) of C-dot–hydrogel (black solid line); after addition
of B. cereus (108 cells per mL; blue solid line); after addition of 15 mL
EDTA (5 mM) together with the bacterial suspension (dotted red
spectrum); after heating the bacterial supernatant to 100 ꢀC (broken
green spectrum). (B) Fluorescence emission spectrum of C-dot–
hydrogel (solid spectrum), and following addition of purified esterase
from B. subtilis at 10 U mLꢂ1 (short dash) and 20 U mLꢂ1 (long dash).
extracted from B. subtilis (B. subtilis belongs to the same
subgroup of Bacillus species as B. cereus).47 The uorescence
quenching apparent in Fig. 6B both conrms the role of ester-
ases in hydrogel disruption, as well as the fact that the
bacterially-induced quenching phenomena reported here
involve contributions from both esterases and lipases.
The C-dot–hydrogel uorescence quenching assay was
applied for analysis of several bacterial species (Fig. 7). Specif-
ically, the graph depicted in Fig. 7 illustrates the degree of
uorescence quenching induced by selected bacteria (108 cells
per mL) added to the C-dot–hydrogel. The signicant differ-
ences in quenching magnitudes highlighted in Fig. 7 likely
reect the variability of esterase/lipase secretions by the
bacteria tested.48,49 Importantly, while some esterase and lipase
enzymes secreted by bacteria have been identied, the
complexity and diversity of these enzyme families have been
recognized and manifested in many studies;40,49 in this context,
the C-dot–hydrogel uorescence assay provides a simple plat-
form for evaluating the overall secretion of these enzymes by
bacteria. Moreover, the distinct quenching degrees could be
employed for distinguishing and identication of different
bacteria.
Fig. 5 Spectroscopic evidence for bacterially-induced ester bond
cleavage. (A) 13C NMR spectra. (i) Precursor 1 before addition of
bacteria; (ii) after addition of B. cereus (108 cells per mL); (iii) free
glucose. The 13C spectral regions corresponding to C1a and C1b (left
rectangle) and C6 carbon atoms (right rectangle) are highlighted. A
1
scheme of the cleavage reaction and its products is indicated. (B) H
NMR spectra. (i) Precursor 1 before addition of bacteria; (ii) after
addition of B. cereus (108 cells per mL); (iii) free lauric acid. The 1H
spectral region corresponding to hydrogen atoms connected to Ca of
lauric acid is highlighted. Scheme of the cleavage reaction and its
products is indicated. (C) FT-IR of 1 before (black) and after (red)
addition of B. cereus: (i) carbonyl (–C]O) spectral region; (ii) ester and
carboxylic acid (–C–O) region.
supernatant to 100 ꢀC essentially cancelled the uorescence
quenching effect (long-dash spectrum in Fig. 6A). This result is
ascribed to heat-induced denaturation of bacterially-secreted
proteins, thereby eliminating enzymatic digestion of the
hydrogel scaffold.
Fig. 6A also shows that addition of EDTA to the bacterial
suspension reduced uorescence quenching (short-dash spec-
trum in Fig. 6A); EDTA binds divalent ions such as Ca2+, which
are cofactors that are necessary to lipase functioning,43,44 thus
inhibiting enzymatic cleavage of the hydrogel ester bonds. It
should be noted that esterases generally do not require metal
cofactors for activity thus the residual quenching still recorded
aer EDTA addition (Fig. 6A) might provide an indication as to
the relative contribution of esterases vs. lipases to the observed
C-dot–hydrogel uorescence quenching.45,46
Fig.
7 Quenching of C-dot–hydrogel fluorescence by different
bacterial stains. Degrees of fluorescence quenching (lex: 375 nm and
lem: 430 nm) of the C-dot–hydrogel upon addition of different
bacterial species (108 cells per mL). (i) C-dots–hydrogel (control); (ii)
Pseudomonas aeruginosa PAO1 wild type; (iii) Bacillus subtilis (stain
Fig. 6B depicts quenching of the C-dot–hydrogel uores-
cence following addition of commercially-obtained esterase Bacillus 3610); (iv) Staphylococcus aureus wild type; (v) Bacillus cereus.
592 | RSC Adv., 2017, 7, 588–594
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
19 S.-T. Yang, L. Cao, P. G. Luo, F. Lu, X. Wang, H. Wang,
M. J. Meziani, Y. Liu, G. Qi and Y.-P. Sun, J. Am. Chem.
Soc., 2009, 131, 11308–11309.
Conclusions
We have developed a new hybrid carbon-dot (C-dot)–hydrogel
system capable of detecting bacteria through quenching of the
C-dots uorescence. Bacterial sensing was demonstrated in
case of Bacillus and Staphylococcus strains, although the detec-
tion concept is generic and might be implemented for other
bacterial species. The sensing mechanism is based upon
cleavage of ester bonds within the hydrogel scaffold, induced by
esterases and lipases secreted by the bacteria. Cleavage of the
ester bonds disrupted the hydrogel framework, resulting in
uidization and aggregation and quenched uorescence of the
embedded C-dots. A series of physical and chemical experi-
ments provide evidence for the gel transformation and reaction
mechanism. The C-dot–hydrogel matrix enabled both high-
sensitivity detection of bacteria (B. cereus) as well as dis-
tinguishing among different bacteria through the extents of
uorescence quenching.
ˇ
20 A. B. Bourlinos, R. Zboril, J. Petr, A. Bakandritsos,
M. Krysmann and E. P. Giannelis, Chem. Mater., 2012, 24,
6–8.
21 L. Cao, M. J. Meziani, S. Sahu and Y.-P. Sun, Acc. Chem. Res.,
2013, 46, 171–180.
22 H. Nie, M. Li, Q. Li, S. Liang, Y. Tan, L. Sheng, W. Shi and
S. X.-A. Zhang, Chem. Mater., 2014, 26, 3104–3112.
23 S. Y. Lim, W. Shen and Z. Gao, Chem. Soc. Rev., 2015, 44, 362–
381.
24 L. Pan, S. Sun, A. Zhang, K. Jiang, L. Zhang, C. Dong,
Q. Huang, A. Wu and H. Lin, Adv. Mater., 2015, 27, 7782–
7787.
25 L. Cao, X. Wang, M. J. Meziani, F. Lu, H. Wang, P. G. Luo,
Y. Lin, B. A. Harruff, L. M. Veca, D. Murray, S.-Y. Xie and
Y.-P. Sun, J. Am. Chem. Soc., 2007, 129, 11318–11319.
26 S. K. Bhunia, A. R. Maity, S. Nandi, D. Stepensky and
R. Jelinek, ChemBioChem, 2016, 17, 614–619.
27 W. Wei, C. Xu, J. Ren, B. Xu and X. Qu, Chem. Commun.,
2012, 48, 1284–1286.
References
1 N. M. Sangeetha and U. Maitra, Chem. Soc. Rev., 2005, 34, 28 A. Zhu, Q. Qu, X. Shao, B. Kong and Y. Tian, Angew. Chem.,
821–836. Int. Ed., 2012, 51, 7185–7189.
2 R. V. Ulijn, N. Bibi, V. Jayawarna, P. D. Thornton, S. J. Todd, 29 A. Barati, M. Shamsipur and H. Abdollahi, Biosens.
R. J. Mart, A. M. Smith and J. E. Gough, Mater. Today, 2007,
10, 40–48.
3 T. R. Hoare and D. S. Kohane, Polymer, 2008, 49, 1993–2007.
Bioelectron., 2015, 71, 470–475.
30 W. Shi, Q. Wang, Y. Long, Z. Cheng, S. Chen, H. Zheng and
Y. Huang, Chem. Commun., 2011, 47, 6695–6697.
4 Z. Yang, G. Liang and B. Xu, Acc. Chem. Res., 2008, 41, 315– 31 E. Ju, Z. Liu, Y. Du, Y. Tao, J. Ren and X. Qu, ACS Nano, 2014,
326.
8, 6014–6023.
5 L. Yu and J. Ding, Chem. Soc. Rev., 2008, 37, 1473–1481.
6 A. Guiseppi-Elie, Biomaterials, 2010, 31, 2701–2716.
32 Y. Song, S. Zhu, S. Xiang, X. Zhao, J. Zhang, H. Zhang, Y. Fu
and B. Yang, Nanoscale, 2014, 6, 4676–4682.
´
7 C. Aime and T. Coradin, J. Polym. Sci., Part B: Polym. Phys., 33 S. Nandi, M. Ritenberg and R. Jelinek, Analyst, 2015, 140,
2012, 50, 669–680.
4232–4237.
8 P. P. Joshi, S. A. Merchant, Y. Wang and D. W. Schmidtke, 34 A. Cayuela, S. R. Kennedy, M. L. Soriano, C. D. Jones,
Anal. Chem., 2005, 77, 3183–3188.
M. Valcarcel and J. W. Steed, Chem. Sci., 2015, 6, 6139–6146.
9 J. H. Kim, S. Y. Lim, D. H. Nam, J. Ryu, S. H. Ku and 35 N. Gogoi, M. Barooah, G. Majumdar and D. Chowdhury, ACS
C. B. Park, Biosens. Bioelectron., 2011, 26, 1860–1865. Appl. Mater. Interfaces, 2015, 7, 3058–3067.
10 Z. Zhu, C. Wu, H. Liu, Y. Zou, X. Zhang, H. Kang, C. J. Yang 36 S. Nandi, H.-J. Altenbach, B. Jakob, K. Lange, R. Ihizane,
¨
¨
and W. Tan, Angew. Chem., 2010, 122, 1070–1074.
11 N. Massad-Ivanir, G. Shtenberg, T. Zeidman and E. Segal,
Adv. Funct. Mater., 2010, 20, 2269–2277.
12 I. Katakis and A. Heller, Anal. Chem., 1992, 64, 1008–1013.
13 A. F. Revzin, K. Sirkar, A. Simonian and M. V. Pishko, Sens.
Actuators, B, 2002, 81, 359–368.
M. P. Schneider, U. Gun and A. Mayer, Org. Lett., 2012, 14,
3826–3829.
37 S. Nandi, R. Malishev, K. Parambath Kootery, Y. Mirsky,
S. Kolusheva and R. Jelinek, Chem. Commun., 2014, 50,
10299–10302.
38 S. Melamed Yerushalmi, M. E. Buck, D.
M. Lynn,
14 R. M. Unruh, J. R. Roberts, S. P. Nichols, S. Gamsey,
N. A. Wisniewski and M. J. McShane, J. Diabetes Sci.
Technol., 2015, 9, 985–992.
N. G. Lemcoff and M. M. Meijler, Chem. Commun., 2013,
49, 5177–5179.
39 J. Nanda, A. Biswas and A. Banerjee, So Matter, 2013, 9,
15 K. Gawel, D. Barriet, M. Sletmoen and B. T. Stokke, Sensors,
2010, 10, 4381.
16 R. Wang and Y. Li, Biosens. Bioelectron., 2013, 42, 148–155.
4198–4208.
40 F. Hasan, A. A. Shah and A. Hameed, Biotechnol. Adv., 2009,
27, 782–798.
17 X. Xu, R. Ray, Y. Gu, H. J. Ploehn, L. Gearheart, K. Raker and 41 I. R. Montella, R. Schama and D. Valle, Mem. Inst. Oswaldo
W. A. Scrivens, J. Am. Chem. Soc., 2004, 126, 12736–12737.
Cruz, 2012, 107, 437–449.
18 Y.-P. Sun, B. Zhou, Y. Lin, W. Wang, K. A. S. Fernando, 42 J. Skjold-Jørgensen, J. Vind, A. Svendsen and M. J. Bjerrum,
P. Pathak, M. J. Meziani, B. A. Harruff, X. Wang, H. Wang, Biochemistry, 2016, 55, 146–156.
P. G. Luo, H. Yang, M. E. Kose, B. Chen, L. M. Veca and 43 I. Posner and A. Morales, J. Biol. Chem., 1972, 247, 2255–
S.-Y. Xie, J. Am. Chem. Soc., 2006, 128, 7756–7757.
2265.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 588–594 | 593

View Article Online
RSC Advances
Paper
¨
44 M. M. Momsen, M. Dahim and H. L. Brockman, 47 O. A. Økstad, I. Hegna, T. Lindback, A.-L. Rishovd and
Biochemistry, 1997, 36, 10073–10081.
A.-B. Kolstø, Microbiology, 1999, 145, 621–631.
48 K.-E. Jaeger and T. Eggert, Curr. Opin. Biotechnol., 2002, 13,
390–397.
49 R. Gupta, N. Gupta and P. Rathi, Appl. Microbiol. Biotechnol.,
2004, 64, 763–781.
45 U. T. Bornscheuer, FEMS Microbiol. Rev., 2002, 26, 73–81.
46 L. F. Godinho, C. R. Reis, P. G. Tepper, G. J. Poelarends and
W. J. Quax, Appl. Environ. Microbiol., 2011, 77, 6094–6099.
594 | RSC Adv., 2017, 7, 588–594
This journal is © The Royal Society of Chemistry 2017