Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 188 (2018) 50–56

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Spectrochimica Acta Part A: Molecular and Biomolecular

Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Selective biosensing of Staphylococcus aureus using chitosan

quantum dots
Hani Nasser Abdelhamid a,b,c, Hui-Fen Wu a,b,d,e,f,⁎
a
Department of Chemistry, National Sun Yat-Sen University, Kaohsiung 804, Taiwan
b
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 800, Taiwan
c
Department of Chemistry, Assuit University, Assuit 71515, Egypt
d
Center for Nanoscience and Nanotechnology, National Sun Yat-Sen University, Kaohsiung 804, Taiwan
e
Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University and Academia Sinica, Kaohsiung 80424, Taiwan
f
Institue of Medical Science and Technology, National Sun Yat-Sen University, Kaohsiung 804, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: Selective biosensing of Staphylococcus aureus (S. aureus) using chitosan modified quantum dots (CTS@CdS QDs)
Received 4 December 2016 in the presence of hydrogen peroxide is reported. The method is based on the intrinsic positive catalase activity of
Received in revised form 29 May 2017 S. aureus. CTS@CdS quantum dots provide high dispersion in aqueous media with high fluorescence emission.
Accepted 30 June 2017
Staphylococcus aureus causes a selective quenching of the fluorescence emission of CTS@CdS QDs in the presence
Available online 5 July 2017
of H2O2 compared to other pathogens such as Escherichia coli and Pseudomonas aeruginosa. The intrinsic enzymat-
Keywords:
ic character of S. aureus (catalase positive) offers selective and fast biosensing. The present method is highly se-
Biosensing lective for positive catalase species and requires no expensive reagents such as antibodies, aptamers or
Quantum dots microbeads. It could be extended for other species that are positive catalase.
Pathogenic bacteria © 2017 Published by Elsevier B.V.
Catalase
Fluorescence
Staphylococcus aureus

1. Introduction
Pathogenic bacteria are serious threats for human being. They cause
several millions of deaths and hospitalizations each year according to
World Health Organization (WHO) [1]. The well-known methods such
as bacteria culture and colony counting based approach were time-con-
suming (typically 2–4 days) and required tedious procedure such as se-
rially diluted and re-culture of each suspension. Furthermore, the plate
counting is sometime misleading due to sensitivity of this approaches
for the cultivation conditions such as nutrient medium, temperature
and time. Thus, selective, sensitive and fast biosensor is highly required
with the raise of human morbidity, mortality and bioterrorism. The se-
lective detection of pathogenic bacteria was reported using many tech-
niques. Among these techniques, amperometric and optical techniques
were widely investigated during the last decades [2–4]. Pathogenic bac-
teria could be detected using affinity-based biosensors [5], electrochem-
ical [6], affinity-based magnetic nanoparticle [7–11] and others [12–17].
These techniques could be classified to biosensor technique for whole-
⁎ Corresponding author at: Department of Chemistry, National Sun Yat-Sen University,
Kaohsiung 804, Taiwan.
E-mail address: hwu@faculty.nsysu.edu.tw (H.-F. Wu).
cell or techniques that detect or biosensing a target analyte inside the
cell [18]. The first strategy is difficult as it required detection of large
size species (micrometer scale) than typical molecular analytes such
as cell biomolecules including protein, DNA, RNA and others. According
to our best of knowledge, there are no studies of the enzymatic charac-
ters of pathogenic bacteria. Recently (2016), Amine et al. reviewed en-
zymatic based biosensors [19]. They reported that enzymatic
biosensors are cost-effective, fast, can be miniaturized and are easy to
integrated for devices [19].
Staphylococcus aureus (S. aureus) is a common skin microorganism,
second major contaminating bacteria in food products and the most no-
torious microorganism that causes surgical death infections. It was con-
sidered as one of the top killing pathogenic bacteria [20]. According to
the economy research service of United States Department of Agricul-
ture (USDA), the infectious food borne illness caused by Staphylococcus
food poisoning was over 180,000 cases annually in US [21]. It was also
estimated that 20% of the human population are long-term carriers of
S. aureus [22]. Staphylococcus aureus (S. aureus) has been detected
using different biosensing technique [23,24]. U.S. Food and Drug Admin-
istration (FDA) stated culture based method as standard detection
methods for this microbial contamination [25]. However, this method
required incubation for long time, inoculation, as well as pretreatment

http://dx.doi.org/10.1016/j.saa.2017.06.047
1386-1425/© 2017 Published by Elsevier B.V.
 H.N. Abdelhamid, H.-F. Wu / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 188 (2018) 50–56
such as serially diluted. The other techniques such as antibodies based
technique require expensive antibodies or peptides, slow, and are com-
plicated [26–30]. We are in demand of rapid, selective and sensitive de-
tection of pathogenic bacteria in order to follow pathogen related
diseases and fight bioterrorism attacks [31].
Semiconductor quantum dots (QDs) are a fluorescent nanocrystal
with a particle size less than 10 nm [32,33]. QDs have exceptional opti-
cal properties such as long life time, large Stocks' shifts, high fluores-
cence emission, high photostability and they are flexible for further
surface modifications. QDs have been reported as probe for pH sensors
[34], cancer cell imaging [35], metals and hydrogen peroxide [36],
metallodrug detection [37,38], pathogenic bacteria [39], proteomics
[40,41], uric acid [42] and others [43–45]. Quantum dots advance live
cells, in Vivo imaging, and diagnostics [46,47] and offered many advan-
tages compared to other materials [48].
Herein, we reported a selective biosensor for S. aureus based on
their intrinsic enzymatic character as catalase positive strains.
Cadmium sulfide quantum dots modified chitosan (CTS@CdS QDs)
was synthesized and characterized using transmission electron
microscopy (TEM), UV–vis absorption and fluorescence emission.
CTS@CdS QDs was used as fluorescence nanoprobe for pathogenic
bacteria. S. aureus showed decomposition of hydrogen peroxide
that caused high quenching for the fluorescence emission of CTS@
CdS QDs. The specific catalase enzymatic bioactivity of S. aureus
offered high selectivity compared to other pathogens such as
Escherichia coli and Pseudomonas aeruginosa. The present strategy
is simple, selective, cheap, fast and provided a time dependent
biosensor for S. aureus.
2. Experimental Section
2.1. Materials and Methods
Chitosan (molecular weight (75,000 g mole− 1), and 75%
deacetylated), sodium sulfide, and glacial acetic acid, cadmium nitrate
(99.0%) were purchased from Sigma-Aldrich (Australia). The chemicals
were used without any purification. The solutions were mainly used
high distilled water from Milli-Q Plus water purification system
(Millipore, Bedford, MA, USA).
2.2. Instrumentation
The particle sizes and bacteria interactions with quantum dots mod-
ified with chitosan (CTS@CdS QDs) were imaged using transmission
electron microscope (TEM), JEOL TEM-3010 (JEOL, Japan). UV–vis ab-
sorption was carried out using Spectronic 20 (Germany). The fluores-
cence spectrometer (F-2700 FL, model: 4J1-0003, Hitachi) was used to
record the fluorescence emission at wavelength 593 nm.
2.3. Synthesis Chitosan Modified Quantum Dots (CTS@CdS QDs)
The synthesis procedure of CdS QDs modified with chitosan (CTS@
CdS) was carried out using the previous reported protocol with modifi-
cation [36,39]. Briefly, 11 mg of cadmium nitrate was dissolved in chito-
san solution (0.1 g, 20 mL, 1% acetic acid). The solution was stirred for
3 h. Then, a fresh solution of Na2S·9H2O (12 mg, 10 mL H2O) was
injected very fast to Cd2+@chitosan solution. The solution was stirred
at 35 °C for 5 h under nitrogen atmosphere to avoid oxidation of the
core of CdS QDs. The material was collected using centrifugation and
washed with water two times (2 × 20 mL). The precipitate was re-dis-
persed in water for biosensing application. The material was character-
ized using transmission electron microscopy (TEM), UV–vis absorption
and fluorescence spectroscopy.
51
2.4. Bacterial Culture
Staphylococcus aureus (BCRC 10451), Escherichia coli and Pseudomo-
nas aeruginosa (BCRC 10303) were purchased from Bioresource Collec-
tion and Research Center (BCRC, Hsin-Chu, Taiwan). The cells were
cultivated at 37 °C using DifcoTM Nutrient broth (Becton and Dickinson,
France, 8.0 g per 1.0 L) and Agar plates (1.5% wt, Gen Chain Scientific,
GCS, New York, USA). The bacteria suspension was prepared by
scratching few lines of the bacteria strain at the stationary phase from
the agar plate with sterilized needle. The harvested bacteria were dis-
persed in sterilized water for the biosensing application. The cell num-
ber was estimated using the standard plate count method and cell
numbers were counted after 24 h.
2.5. Procedure of the Pathogen Biosensing
The bacteria suspension (10 μL) were added to 1 mL of CTS@CdS
QDs. Then, 50 μL of hydrogen peroxide (35%) was added. For precaution,
open glass vials should be used to avoid the strong effervescence in case
of S. aureus. The other precautions of catalase test were followed; 1) the
test organisms should not be taken from blood agar culture, 2) culture
should be 18 to 24 h old, 3) hydrogen peroxide should be from a fresh
bottle, and 4) iron wire loop should be avoided. The measurements
were recorded at emission wavelength 593 nm.
2.6. Transmission Electron Microscope (TEM) Analysis
The interactions of the bacteria cells with chitosan modified quan-
tum dots (CTS@CdS QDs) were investigated using transmission electron
microscope (TEM). Quantum dots (100 μL) and hydrogen peroxide
were added to 1 mL of pathogenic bacteria. Then, 5 μL of dispersion (P.
aeruginosa and S. aureus with quantum dots) were spotted to
200 mesh copper grid for 1 min and the dispersion excess was then re-
moved using filter paper. The cells were fixed with 2.5% glutaraldehyde
in a phosphate buffer solution (0.1 M) at 4 °C for 2 h. The grid was rinsed
with phosphate buffer (3 times, 0.1 M), and the cells of the bacteria
were fixed with 1% osmium tetroxide in 0.1 M phosphate buffer. The
grid was rinsed with buffer solution and leaved for 2 h at 4 °C.
3. Results and Discussion
3.1. Material Characterization
Hydrothermal reaction of Cd2+@Chitosan (Cd2+@CTS) with a fresh
solution of Na2S leads to the nucleation and growth a tiny nanocrystals
of CTS@CdS quantum dots (QDs). The material was characterized using
TEM (Fig. 1a), UV–vis absorption and fluorescence emission (Fig. 1b).
Size and morphology of CTS@CdS were evaluated using TEM (Fig. 1a).
Data revealed that the average size is less than 6 nm. TEM image
shows almost a monodispersion size of CTS@CdS QDs (Fig. 1a). UV–vis
absorption of CTS@CdS shows a broad band absorbance in the range
of 325–500 nm (Fig. 1b). The size of the quantum dots can be calculated
form UV–vis absorbance using the empirical Eq. (1) [49]. The first exci-
ton absorbance is at 450 nm. Data shows that the average size is about
5.7 nm that agrees with the size that reported from TEM image. CTS@
CdS QDs shows emission at 593 nm (Fig. 1b).
 
D ¼ −6:6521  10−8 λ3
   
þ 1:9557  10−4 λ2 − 9:2352  10−2 λ þ ð13:29Þ ð1Þ
where, D (nm) is the size of a given nanocrystal sample, and λ (nm)
is the wavelength of the first excitonic absorption (λabs = 450 nm,
Fig. 1b).
52 H.N. Abdelhamid, H.-F. Wu / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 188 (2018) 50–56
Fig. 1. a) TEM image of CTS@CdS QDs, b) UV–vis absorption, inset image is the color of CTS@CdS QDs suspension and c) TEM image of CTS@CdS QDs after interactions with hydrogen
peroxide.
3.2. Selectivity of CTS@CdS QDs for Pathogenic Bacteria
Transmission electron microscopy (TEM) of CTS@CdS QDs after in-
teraction with hydrogen peroxide (H2O2) is shown in Fig. 1c. Data re-
veals that there are aggregations of the quantum dots after interaction
with hydrogen peroxide (Fig. 1c). Effect of H2O2 on CTS@CdS QDs
shows quenching of the luminescence emission [36]. Many mechanisms
were proposed to explain this effect. They are contradict and more in-
vestigations are required for real mechanism. It could be due to electron
removal or oxidative etching [50–52]. It was also reported that hydro-
gen peroxide caused surface passivation which promoting radiative re-
combination rate that significantly enhanced luminescence properties
(quantum efficiency ≅ 20%) [53]. We observed the same phenomena
for CTS@CdS QDs [36]. The material showed also emission enhancement
Fig. 2. a) Schematic representation of S. aureus catalase of H2O2 decomposition in presence
and b) color change of CTS@CdSwithout (left) and with (right) H2O2, image shows the
effervescences due to the decomposition of H2O2 using S. aureus.
upon the interactions with pathogenic bacteria such as Pseudomonas
aeruginosa and Staphylococcus aureus [39]. The interactions of the cell
membrane and chitosan backbone were the reasonable reasons for
these enhancements. It is worth to note that thioglycolic acid (TGA)
modified CdSe QDs showed also enhancement upon the interactions
with bacteria such as E. coli and S. aureus [54]. In other side, it was
well documented that S. aureus is catalase positive species. This charac-
ter mean that S. aureus enzymatically catalase hydrogen peroxide de-
composition [55]. This test is important in distinguishing Streptococci
(catalase-negative) from Staphylococci which are catalase positive.
Based on this unique feature of Staphylococcus aureus compared to
other pathogens, we proposed a new protocol as shown in Fig. 2a. Hy-
drogen peroxide decomposition is catalyzed by S. aureus. S. aureus
split hydrogen peroxide to water and oxygen. Thus, oxygen causes ag-
gregation of CTS@CdS as shown in Fig. 2b. For catalase-positive cultures
such as S. aureus, bubble due to hydrogen peroxide decomposition was
observed (Fig. 2b) [56].
When CTS@CdS interacts with P. aeruginosa in the presence of hy-
drogen peroxide, the fluorescence emission showed enhancement as
shown in Fig. 3a. This observation is consistence with our previous re-
sults [39]. Hydrogen peroxide causeno effect because of P.aeruginosais
catalase negative species. In contrast, S. aureus shows quenching of
CTS@CdSdue to its catalase behavior as shown in Fig. 3b. Thus, this strat-
egy offers high selectivity for S.aureus compared to P.aeruginosa and E.
coli as shown in Fig. 4. It is important to mention that E. coli is also cat-
alase positive species. Thus, it shows lower enhancement than P.
aeruginosa. The low ability of catalase of E.coli may be due to several rea-
sons. First, the catalase activity of E.coli is lower than S. aureus [57]. Sec-
ond, the enhancement of the luminescence emission due to the
interacts with CTS@CdS may higher than the quenching due to catalyze
decomposition of hydrogen peroxide [58]. Third, the locations of cata-
lase enzyme in E.coli may render the enzyme unable to decompose
H2O2 efficiently [57]. Fourth, the concentration of catalase and the bac-
teria strains and their efficiency of E.coli is lower than S. aureus. Finally,
the permeability of the cell wall to H2O2or CTS@CdS may be slightly
lower than in S. aureus.
Importantly, these changes are time dependent as shown in Fig. 5 for
the investigated species. Data shows a linearity in the range 3–10 min
(R2 = 0.99) and 3–30 min (R2 = 0.97) for P. aeruginosa and S. aureus,
 H.N. Abdelhamid, H.-F. Wu / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 188 (2018) 50–56
Fig. 3. Fluorescence emission of CTS@CdS at excitation wavelength 295 nm in presence of hydrogen peroxide for a) P. aeruginosa and b) S. aureus.
respectively. This observation indicates that the present approach is
very useful to follow the growth of bacteria cell.
3.3. Probe Interactions of Chitosan Quantum Dots (CTS@CdS) With Bacteria
Cells Using Transmission Electron Microscopy
Chitosan quantum dots (CTS@CdS) composed from a core (CdS QDs)
and surface capping i.e. chitosan. Chitosan quantum dots (CTS@CdS)
can bind to pathogenic bacteria cells through protein or carbohydrate
structures exposed on the surface of the bacterium. These binding
sites or species were called as adhesins. It is important to stress that
Fig. 4. Selectivity of CTS@CdS for pathogenic bacteria.
Fig. 5. Time-dependent luminescence emission of CTS@CdS for a) P. aeruginosa and b) S. aureus.
53
each bacterial strain has a very specific pattern of adhesins and there-
fore they could bind with different strength to chitosan quantum dots
through bacteria protein-chitosan interactions. Abdelhamid and Wu re-
ported the thermodynamic analysis of chitosan quantum dots (CTS@
CdS) interactions with bacteria cells [39]. They observed that the main
interactions were due to hydrogen bonds and electrostatic interactions.
In general, characterization of these interactions is a crucial challenge
and is very important to understand the interactions of bacteria cell
with nanoparticles. Thus, there are many techniques to probe or charac-
terize these interactions. Among the different technique, we used trans-
mission electron microscopy to probe these interactions as shown in
Fig. 6. We characterized the bacteria cell after the interaction with
CTS@CdS to track the interactions of bacteria cell with quantum dots.
The cell can be divided into three regions in general; nonpolar region,
subpolar and polar region. Polar region consisted of the cell poles and
comprised ~27% of the total cell surface area. Fig. 6a shows that CTS@
CdS are located outside and internalized the cell membrane of S. aureus.
Data shows also that CTS@CdS bind to the cell membrane of P.
aeruginosa (Fig. 6b). The polar groups of chitosan backbone in CTS@
CdS can bind to the polar region of the pathogenic bacteria. Further-
more, chitosan has also unpolar region where it can interact with
unpolar region of the cell membrane. It was reported that nanoparticle
(NP) modified with carboxylic groups (COOH) are attaching to emerg-
ing type IV pili [59]. It is important to mention that the region of type
IV pili surface elaboration in S. oneidensiswas localized to the subpolar
region [59]. Authors observed the opposite for Pseudomonas aeruginosa
in which the polar region is the site of type IV pili production [59]. Scan-
ning electron microscopy (SEM) was used to investigate the interac-
tions of nanoparticles with Staphylococcus aureus after centrifugation
54 H.N. Abdelhamid, H.-F. Wu / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 188 (2018) 50–56

features for real applications. Conventional detection methods such as

Fig. 6. Transmission electron microscopy (TEM) of CTS@CdS upon interactions with a) S.
aureus and b) P. aeruginosa.
and a comparison of colony forming unit (CFU) counting and optical
density (OD) measurements were reported [60]. Data revealed that
analysis using SEM depends on the concentrations of cells and NPs. It
is recommended to use low concentrations of both cells and NPs. Data
also showed that nanoparticles were centralized in the cell surface. Ac-
cording to TEM (Fig. 6), quantum dots modified with chitosan was local-
ized in the surface and also internalized inside the cell. The cell uptake of
QDs may be occur via endocytosis [61]or oxidative damage to the cell
membrane which is aided by light [62]. TEM image (Fig. 6) shows that
CTS@CdS bound strongly to the cell membrane of the bacteria and
was uptake inside the cell. It is important to mention that the amount
of the CTS@CdS bound to the bacterial surface was sufficient for the gen-
eration of detection signal for S. aureus and P. aeruginosa.
3.4. Comparison of the Current Approach and Other Techniques
There are many techniques that were investigated for the detection
of pathogenic bacteria. Among these techniques, matrix assisted laser
desorption/ionization mass spectrometry (MALDI-MS) [66–68], fluo-
rescence [69], and immunosorbent assay [70], showed a promising
enzyme-linked immunosorbent assay (ELISA) were lack of high sensi-
tivity. The detection limit of this technology is typically above 10-
4
cfu·mL−1due to the low infective dose. Conventional detection
method for pathogenic bacteria using plate culture required atleast
three days of bacterial cultivation and identification using specific
media. However, these methods require complicated procedures and
the need for skillful technicians. Thus, new approaches are required to
speed the detection of the pathogens. We compare between our ap-
proach and the same technique (fluorescence) and same nanoprobe
i.e. nanoparticle in quantum size. A fluorescence resonance energy
transfer (FRET) biosensor based on gold nanoparticles graphene quan-
tum dots (Au-GQDs) was reported for Staphylococcusaureus gene se-
quence detection [63]. This approach is sensitive and selective for
mecA gene sequence of Staphylococcusaureus (Table 1). However, it is
not suitable for the whole cell and required expensive chemicals. ZnO
quantum dots modified with bovine serum albumin (BSA), peptide
(PEP), near infrared dyes (hydrophilic indocyanine green (ICG) deriva-
tive (MPA)) and antibacterial agent (vancomycin) (ZnO@PEP-MPA-
Van) was reported for multifunctional applications such as detection
and as antibacterial agent for bacterial infections in vivo and in vitro
[64]. However, the material is toxic due to the presence of antibacterial
agents and required very long for imaging (Table 1). In contrast, our
strategy is simple as it requires no expensive gene or other biomole-
cules, and is highly selective for S. aureus as it depend on the intrinsic
catalase character of S. aureus, and time-dependent (Table 1). Recently
(2016), magnetic nanoparticles modified with Staphylococcus protein
A (SPA) was used for chemiluminescene detection of S. aureus using
the reagents luminol and H2O2 [65]. The method is sensitive, selective
and simple. However, it is important to keep in our mind that SPA is in-
terfered by Immunglobulin G (IgG). Furthermore, SPA is expensive. Sur-
face plasmon resonance (SPR) DNA array was used to probe S. aureus
[71]. The measurements take placed within several hours and required
a complicated sample pretreatment including cell disruption and
nucleic acid extraction.
In contrast with other reported methods (Table 1), the present ap-
proach is very simple, highly selective, and cost effective. Thus, it can
be easily developed for point-of-care test. However, the main challenge
is that the catalase activity is difficult to test clinical bacteria species col-
lected from agar blood. The test is invalid for blood agar because blood
itself will produce bubbles. Thus, we are planning in the near future to
separate the species from blood before we perform the test.
4. Conclusions
We successfully reported the intrinsic catalase enzymatic feature of
Staphylococcus aureus in the presence of hydrogen peroxide and chito-
san quantum dots for a selective biosensing of S. aureus. This new meth-
od is not only selective for S. aureus than already existing methods but it
is also a time dependent and sensitive approach. This method is cheap

Table 1
Comparison of the results obtained by the current strategy with other fluorescence detection using quantum dots for S. aureus.

Nanoprobe Principle LODa Linearitya Timeb Advantage Disadvantage Ref.

2
CTS@CdS Chitosan-bacteria interactions 150 1.5 × 10 – 1 • Sensitive • Lack of selectivity [39]
11 × 102
Au-GQDs Detection of mecA gene sequence of S. 1 • Selective • Expensive require purification of S. aureus gene [63]
aureus nM
Van@ZnO-PEP-MPA Fluorescence image 1× 600 • Multifunctional • Toxic for the cell [64]
104 • Lack of selectivity
SPA@MNPs Selective separation and 6.0 10× 109 50 • Selective • Expensive [65]
chemiluminescene using Luminol + H2O2 −1.0 × 109 • Interfered by IgG
CTS@CdS Catalase enzymatic behavior of hydrogen 200 2–30 • Highly selectivity • It cannot be used for bacteria that were collected Here
peroxide decomposition • Simple from blood agar, influential contaminated such as
• Cheap iron should be avoided
a
cfu/mL.
b
Min.
 H.N. Abdelhamid, H.-F. Wu / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 188 (2018) 50–56
as it requires inexpensive chemical. This new strategy showed great po-
tential for use in clinical diagnosis as well as research applications that
require high selective detection of S. aureus bacteria. This strategy can
be also extend to other catalase positive pathogens, such as Mycobacte-
rium tuberculosis, Legionella pneumophila, and Campylobacter jejuni.
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