J Nanopart Res (2016) 18:111
DOI 10.1007/s11051-016-3414-1
RESEARCH PAPER
Detection of Salmonella typhi utilizing bioconjugated
fluorescent polymeric nanoparticles
Swati Jain . Sruti Chattopadhyay .
Richa Jackeray . Zainul Abid . Harpal Singh
Received: 29 January 2016 / Accepted: 5 April 2016 / Published online: 16 April 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract Present work demonstrates effective utiliza-
tion of functionalized polymeric fluorescent nanoparti-
cles as biosensing probe for the detection of Salmonella
typhi bacteria on modified polycarbonate (PC) filters in
about 3 h. Antibody modified-PC membranes were
incubated with contaminated bacterial water for selec-
tive capturing which were detected by synthesized novel
bioconjugate probe. Core–shell architecture of poly-
meric nanoparticles endows them with aqueous stabi-
lization and keto-enolic functionalities making them
usable for covalently linking S. typhi antibodies without
any crosslinker or activator. Bradford analysis revealed
that one nanoparticle has an average of 3.51 9 10-19 g
or 21 9 104 bound S. typhi Ab molecules. Analysis of
the regions of interest (ROI) in fluorescent micrographs
of modified fluoroimmunoassay showed higher detec-
tion sensitivity of 5 9 102 cells/mL due to signal
amplification unlike conventional naked dye FITC-Ab
Electronic supplementary material The online version of
this article (doi:10.1007/s11051-016-3414-1) contains supple-
mentary material, which is available to authorized users.
S. Jain (&)  S. Chattopadhyay  R. Jackeray 
Z. Abid  H. Singh
Centre for Biomedical Engineering, Indian Institute of
Technology-Delhi, New Delhi 110016, India
e-mail: swatijain.iitd@gmail.com
S. Chattopadhyay
e-mail: sruticiitd@gmail.com
H. Singh
e-mail: harpal2000@yahoo.com
conjugate. Fluorescence of pyrene dye remained same
on immobilization of biomolecules and nanoparticles
showed stable fluorescent intensity under prolong expo-
sure to laser owing to protective polymeric layer
allowing accurate identification of bacteria. Surface-
functionalized PC matrix and fluorescent label NPs
permit covalent interactions among biomolecules
enhancing signal acquisitions showing higher detection
efficiency as compared to conventional microtiter plate-
based system. Our novel immunoassay has the potential
to be explored as rapid detection method for identifying
S. typhi contaminations in water.
Keywords Fluoroimmunoassay  Fluorescent
nanoparticles  Salmonella typhi  ELISA  Detection 
Bioconjugation  Health safety
Abbreviations
FPNP Fluorescent polymeric nanoparticles
NPs Nanoparticles
AAEM Acetoacetoxy ethyl methacrylate
S. typhi Salmonella typhi
Ab Antibody
Fl Fluorescent
Introduction
Biolabeling with fluorescent dyes has many biomed-
ical applications like fluorescent immunoassays for
diagnostics and imaging in commercial and
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institutional setups. Fluorescent immunoassays (FIA)
increase the high throughput efficiency and sensitivity
(Wang and Mazza 2002; Yang et al. 2004). The
traditionally used organic fluorophores like FITC
suffer from lack of photostability in addition to being
relatively less fluorescent. The new improved Alexa
series though have high intensity but have disadvan-
tages of low labeling efficiency, and photostability.
Advances in nanotechnology have facilitated the
engineering of nanoassemblies for various analytical
and biological applications. Last decade witnessed the
emergence of silica (Si) nanoparticles (NPs), gold
(Au) nanoparticles quantum dots (QDs), and florescent
polymeric nanoparticles as the new generation mark-
ers (Harma et al. 2003). Various QDs have been
utilized to image many cancer cell lines such as human
mammary epithelial tumor, human breast cancer,
human prostate cancer, and lung tumor (Santra et al.
2005a, b). However, QDs are difficult to synthesize
requiring extreme temperature–pressure conditions
along with water free environment. Furthermore, the
utilization of QDs is restricted owing to their cyto-
toxicity and water incompatibility. Surface modifica-
tions are also necessary to make them water
dispersible (Wang et al. 2008a, b). Extensive research
work is going on dye-doped silica nanoparticles in bio-
imaging and bio-analysis applications for the detec-
tion of various analytes including tumor necrosis
factor-a, enterotoxins, bacteria, and cancer cells (Hun
and Zhang 2007a, b; Santra et al. 2005a, b). Zhao et al.
have developed a rapid test for the analysis of bacterial
cell E. Coli O157:H7 using bioconjugated Si nanopar-
ticles (Zhao et al. 2004). An indirect homogenous
solution-based immunofluorescence microscopic
method was developed for the detection of Mycobac-
terium tuberculosis using Si NPs (Qui et al. 2007). But
reports suggest that the dye molecules doped inside the
Si shell have high chances of leaching because of
porous nature of Si. They also require multistep
reactions for the generation of functionality for the
attachment of biological moieties (Bagwe et al. 2006;
Wang 2008a, b).
‘‘Tailor’’-made Polymeric fluorescent nanoparti-
cles having in-built surface functionality are being
developed for various biomedical applications. Poly-
meric nanoparticles have special characteristics of
high surface to volume ratio, size controllability, and
can be easily functionalized during synthesis (Kumari
et al. 2010; Vakurov et al. 2009). Size-controlled
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J Nanopart Res (2016) 18:111
polymeric nanoparticles were synthesized by Pich
et al. with styrene and acetoacetoxyethyl methacrylate
for the immobilization of enzyme (Pich et al. 2006).
Europium-doped gadolinium oxide nanoparticles
were modified with Poly L-Lysine by Nichkova et al.
(2005). They had developed a microimmunoassay to
detect phenoxybenzoic acid, a generic biomarker of
human exposure to insecticides. Holzapfel et al.
(2005) have developed fluorescent carboxy and
amine-terminated polystyrene nanoparticles by mini-
emulsion polymerization as the Hela cell marker. Hun
et al. have prepared unique polymeric fluorescent
particles of poly(methyl methacrylate) with butyl
rhodamine B dye as marker for the bio-imaging of
ovarion cancer by immobilizing Her-2 antibody on the
NP surface (Hun et al. 2008). Polymeric nanoparticles
doped with fluorescent molecules show promising
potential as label for the detection of cells including
bacteria. In most of the developed FIA for the
detection of various analytes, antibodies are physically
immobilized on the polystyrene microtiter plates
which affect the sensitivity of the assay since
biomolecules leach out on extensive washing (Cheng
et al. 2010). Park et al. (2004) synthesized 2-methacry-
loyloxyethyl phosphorylcholine (MPC)-polymeric
nanoparticles (MPC-PNP) having active ester groups
for bioconjugation on the side chains where anti-
C-reactive protein (CRP) monoclonal antibodies were
immobilized on the MPC-PNP using the active ester
group, while the remaining active ester groups were
thoroughly reacted with glycine. A detection limit
about serum-free CRP in the calibration curve was
shown from 0.01 to 10 mg/dL.
Supply of safe drinking water globally is a
challenge; United Nations regularly tracks and mon-
itors access to safe water and sanitation in collabora-
tion with many individual nations. Salmonella typhi
the causative organism for typhoid fever is the
pathogenic bacteria primarily responsible for almost
21 million deaths worldwide (Zhou and Pollard 2010).
Failure to detect bacterial adulterations in water
rapidly is the most egregious consequence since the
culture plate method yields results in 24–48 h after
several steps of enrichment and isolation. Rapid
biosensing approaches are currently being investi-
gated including PCR, surface Plasmon resonance
(SPR), quartz crystal microbalance (QCM), and
piezoelectric electronic noses. However, these meth-
ods are at explorative stage also need high amount of
J Nanopart Res (2016) 18:111
capital investments which deters their applications in
rural set ups (Ivnitski et al. 1999). Enzyme-linked
immunosorbent assays (ELISAs) are the chief bioan-
alytical testing technique for environmental samples
but suffer from low sensitivity (Abdel-Hamid et al.
1998). Therefore, sensitive, rapid, and prompt detec-
tion of bacteria is highly obligatory especially in
endemic areas.
Integration of nanomaterials with ELISA design
could lead to enhanced sensitivity and the experimen-
tal setup could be economical, ultrasensitive, and easy
to configure. The paper outlines the utilization of
functionalized polymeric nanoparticles as bioconju-
gated fluorescent labels. We have used surface-
modified polycarbonate membranes as solid matrix
for capturing S. typhi and capitulated on sandwich
fluorescent immunosorbent assay (FIA) with anti-
body-conjugated fluorescent probe.
Materials and methods
TM
13 mm black colored Isopore Polycarbonate filters
(size: 0.6 9 0.3 lm) with 0.22 lm pore size were
purchased from Millipore Inc. (New York, USA).
Monomers styrene (St), methyl methacrylate (MMA),
acetoacetoxyethyl methacrylate (AAEM), crosslinker
ethylene glycol diacrylate (EGDA), fluorescent dye,
pyrene along with glutaraldehyde, and Tween-20 were
purchased from Sigma Aldrich (St. Louise, USA).
Surfactant, sodium lauryl sulfate (SLS), and initiators,
ammonium persulfate (APS) and sodium peroxydisul-
phate (SPDS) of AR grade quality were obtained from
CDH chemicals (Mumbai, India). Styrene and MMA
were washed with dilute sodium hydroxide solution in
a separating funnel to remove inhibitor and stored over
fused calcium chloride and finally distilled under
vacuum.
Immunoreagents Heat killed culture of ‘‘O’’ antigen
of pathogenic bacteria Salmonella typhi of 6 9 108 -
cells/mL concentration were received as gift from
Rockland Hospital, New Delhi, India and were used
strictly under isolated conditions after proper heat
inactivation. Affinity-purified polyvalent somatic ‘‘O’’
type immunoglobulins (1 mg/mL aliquots) against
whole cell antigen of S. typhi (generated in New
Zealand Wistar rats) were generously gifted by
National Institute for Health and Family Welfare
(NIHFW), New Delhi, India. S. typhi-HRP conjugate,
Page 3 of 11 111
substrate 3, 30 , 5, 50 tetramethylbenzidine-hydrogen
peroxide (TMB/H2O2), and fluorophore-tagged anti-
body Goat anti-Rabbit- fluorescein isothiocyanate
(GAR-FITC) were received from Bangalore Genie
(India). All solvents and reagents were purchased from
commercial sources and were used as-received unless
noted otherwise. Milli-Q water (Millipore Corp.,
USA) was used for the preparation of 1X-phosphate-
buffered saline (PBS) and double-distilled water was
used for polymerization.
Surface modification of polycarbonate membrane
and immobilization of S. typhi antibodies
Sequential surface modification of black colored PC
membranes (area 0.66 cm2) was done according to
protocol optimized in our lab. Briefly, nitration and
reduction with 30 % conc. nitric acid (for 30 min at
60–70 °C) and 30 % solution of sodium borohydride
(prepared in 1X-PBS, for 2 h at room temperature)
respectively, were done to generate amino groups.
This modified membrane designated as mPC were
immersed in a mild solution of 5 % glutaraldehyde
and stirred for 2 h at room temperature for activation
and were further washed with 1X-PBS thrice. Glu-
taraldehyde-activated mPC membranes (PC-NH2-
Glu) were immobilized with 400 lL of 10 lg/mL of
S. typhi-IgG for 16 h at 4 °C and were stored after
washing with Tween/PBS and 1X PBS till further use.
Synthesis of fluorescent polymeric nanoparticles
Polymeric nanoparticles of styrene, MMA, and
AAEM were synthesized by free radical mini-emul-
sion polymerization technique in the presence of
fluorescent dye pyrene (Jain et al. 2013). Briefly,
15 mg of fluorescent dye pyrene along with homoge-
nous monomer mixture of MMA (3.5 mL), styrene
(1 mL), AAEM (1 mL), and crosslinker EGDA
(0.280 mL) was added to oxygen-free 170 mL dis-
tilled water with 0.1 g surfactant sodium lauryl
sulphate (SLS) and heated to 60 °C for 15 min.
Initiator SPDS (0.15 g in 3 mL) was added, temper-
ature was raised to 80 °C, and the reaction was
continued for 2 h. Further addition of monomers—
MMA (3.5 mL), Styrene (1.25 mL), AAEM (0.5 mL)
along with mixed initiator solution of SPDS (0.15 g in
3 mL), and APS (0.06 g in 1 mL), was done after
decreasing the temperature to 40 °C which was once
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again increased to 80 °C and stirring was continued
for 8 h to complete the conversion of monomers into
polymers.
Characterization of FPNP-S. typhi Ab conjugate
Functional group analysis of S. typhi Ab-conjugated
FPNP was done on Perkin Elmer Spectrum One ATR-
FTIR spectrometer. FPNP-S. typhi Ab were examined
through ZEISS EVO series scanning electron micro-
scope Model EVO 50 for any morphological changes.
Immobilized FPNP sample was dried to remove water
and mounted on the metallic stub using double-sided
tape. Silver paint was applied on the samples to make
them conducting for better resolution while gold
sputtering was done on the sample for conduction using
the sputtering instrument (BioRad Polaron Sputter,
Model 50X). The morphology of FPNP-S. typhi Ab
conjugate was also assessed by transmission electron
microscope (Phillips, CM12) at an acceleration voltage
of 100 kV at magnification of 110 KX. 3 lL of dilute
FPNP-Ab conjugate solution was placed on the
200-mesh carbon-coated grid and dried for 20–25 min
at room temperature. The grid was fed in the instrument
in the external sample chamber at 27 °C.
It is important to measure the fluorescence param-
eters of nanoparticles for the development of assay.
100 lL of washed poly(St-MMA-AAEM) fluorescent
nanoparticles was incubated with 200 lL of different
concentrations of S. typhi Ab (10–80 lg/mL) as done
previously. After washing with 1X PBS, the FPNPs
were re-suspended in 3 mL of 1X-PBS and fluorescent
intensity was determined on LS55 PerkinElmer Spec-
trofluorometer (USA).
Different concentrations of nanoparticles immobi-
lized with fixed S. typhi Ab were also subjected to
florescent spectroscopic measurements. 200 lL of
FPNP solution (0.232 9 10-4 mol pyrene/Kg of latex)
immobilized with 200 lL of 20 lg/mL of S. typhi Ab
was diluted in 3 mL PBS for the Fl analysis.
100 lL of washed FPNP immobilized with different
concentrations of S. typhi Ab was subjected to Bradford
assay to determine the amount of antibody attached on
them. The amount of Ab immobilized on FPNP was
calculated approximately as according to Eq. 1:
Q ¼ ½ðCi  Ct ÞV =m ð1Þ
where Q is the amount of S. typhi Ab immobilized on
to a unit mass of the nanoparticles (mg/mg); Ci and Ct
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J Nanopart Res (2016) 18:111
are the initial concentration of Ab and in supernatant
after the immobilization reaction, respectively, as
determined by Bradford method. V is the volume of
aqueous phase (mL) and m is the amount of nanopar-
ticles (mg). The surface coverage of S. typhi Ab on
single nanoparticle was quantified by utilizing the
mass of one particle. The intensity of the colored
solutions was read at 595 nm wavelength in Biorad
ELISA plate reader. Immobilization efficiency (IE %)
for immobilization of different concentrations of Ab
on nanoparticles was estimated using Eq. 2:
Absorbance of immobilized Ab
IEð%Þ ¼
Absorbance of Ab in initial solution
 100 ð2Þ
Development of fluorescence-based assay using
mPC and FPNPs
Washed 100 lL of FPNPs was incubated with 200 lL
of different concentrations of S. typhi antibody
(10–50 lg/mL) for 16 h at 4 °C. After washing, NPs
were re-suspended in 500 lL of 1X-PBS for further
analysis.
Modified mPC membranes were used to capture S.
typhi bacteria and FPNP-S. typhi Ab were utilized as
the fluorescent label in the detection method. Glu-
taraldehyde-activated mPC was immobilized with
10 lg/mL of S. typhi-IgG. Unbound sites of the
membranes were blocked with 6 % skimmed milk
for 1.5 h at 37 °C. These Ab immobilized mPC
membranes were washed with 1X-PBS and incubated
with whole cell antigen of S. typhi (105 cells/mL) for
1.5 h at 37 °C. After washing with PBS, they were
incubated with 200 lL of S. typhi Ab immobilized
fluorescent nanoparticles probe (FPNP-S. typhi Ab) for
1.5 h at 37 °C. Afterwards, the probe was decanted and
the membranes were washed thoroughly with PBS.
mPC was then subjected to confocal laser microscopic
analysis for evaluating the formation of immuno-
complex (mPC-S. typhi Ab-Ag-Ab-FPNP). Along with
the test samples, negative control experiments were
also performed. Immobilized mPC membranes were
incubated with 1X-PBS instead of S. typhi Ag.
Sensitivity of the developed fluorescence-based
immunoassay was also determined for the detection of
S. typhi. 10 lg/mL antibody-immobilized mPC mem-
branes were incubated with different concentrations of
the bacteria S. typhi (107–102 cells/mL) and were
evaluated by the above-mentioned protocol.
J Nanopart Res (2016) 18:111
Specificity of modified mPC assay was measured
by incubating S. typhi Ab immobilized membranes
with cross-reactants of S. typhi—Pseudomonas aergi-
nosum, Escherichia coli, Klebsiella pneumoniae with
104cells/mL concentration which were detected by S.
typhi-FPNP probe. A region of interest was located on
mPC, and fluorescent intensity was recorded for
graphical presentation.
Results and discussions
The amino groups of the antibody react with the
diketone groups of the AAEM functionality of
Poly(St-MMA-AAEM) fluorescent nanoparticles
forming the covalent linkage between the two moieties
and the reaction is schematically represented in
Fig. 1a. The keto-enolic forms of diketone moieties
facilitate conjugation with biomolecules via poly-
condensation reaction with amino groups. This bio-
conjugated probe was lyophilized and stored at 4 °C
for further use. Chemically derivatized PC membranes
with approximately 0.1689 mmol NH2 groups/cm2
equivalent to 1.01 9 1020 NH2 groups/cm2 were used
to immobilize 4.65 lg/cm2 of S. typhi-IgG as deter-
mined by Bradford assay.
Surface functionalization and biomolecules attach-
ment on FPNP were assessed by FTIR spectroscopy
and the spectrum is given in supplementary data as Fig
S1. The spectra showed characteristic peaks of C–H
(CH2) at 2930 and 2893 cm-1 and distorted vibration
peaks at 1450 cm-1. The stretching vibration bands
centered at 1723 cm-1 could be attributed to stretch-
ing vibration peak of C=O in the keto group.
Absorption band at 2506 cm-1 was different from
that of nanoparticles, which correspond to the spec-
trum of antibody. A band around 2506 cm-1 appeared
which may be attributed to the O–H stretching of
carboxyl group present in the Fc region of Ab (Allmer
et al. 1989). The IR spectral analysis confirmed the
attachment of Ab on nanoparticles.
Morphology analysis
The morphology of bioconjuagte fluorescent nanopar-
ticles (FPNP-S.typhi Ab) is important to illustrate their
feasibility as fluorescent markers indicating if the
particles have aggregated to form large-sized clumps.
Scanning and transmission electron micrographs of
Page 5 of 11 111
Poly(St-MMA-AAEM) nanoparticles and S. typhi Ab
immobilized FPNP are given in Fig. 2. As seen from
micrographs, the antibody-conjugated FPNPs remain
monodispersed and the circular shape of nanoparticles
is conserved. It was also visualized in TEM analysis
that the nanoparticles were well dispersed after their
conjugation with antibody indicating immobilization
without any aggregation.
Spectroscopic characterization of bioconjugate
probe
The synthesized nanoparticles have polycyclic aro-
matic hydrocarbon pyrene as the fluorescent dye
encapsulated inside a polymeric sheath composed of
styrene, methyl methacrylate, and AAEM. The probe
attains core–shell morphology owing to different
polarities of monomers and dye where more hydropho-
bic components constituted by St, MMA, and dye,
form the core while hydrophilic AAEM resides at the
periphery of nanosystem oriented toward water
molecules. Pyrene is a spatially sensitive probe with
ensemble of monomeric emission peaks at 393, 383,
and 370 nm when excited with 350 nm wavelength.
Owing to hydrophobicity, the molecules are primarily
localized in the core of FPNP while the surface
topology is governed by hydrophilic groups. The core–
shell structural analogy is achieved thereby protecting
the fluorescence of pyrene dye from degrading envi-
ronment of water, salts, and oxygen [Jain et al. 2013]
making NPs viable for biological analysis.
Before the utilization of bioconjugated FPNP probe
for the detection of bacteria, their fluorescent charac-
teristics were studied and are presented in Fig. 3. The
influence of antibody on the Fl properties of pyrene-
loaded FPNP was investigated with different concen-
trations of S. typhi Ab (10–80 lg/mL) immobilized on
FPNP. No discernible loss in fluorescence was
observed and no appreciable change in the spectra
appeared for the encapsulated pyrene molecules after
immobilization with biomolecules. There was no
effect of concentration of antibody on the fluorescent
spectra when fixed 100 lL (5.46 mg) of FPNP was
incubated with Ab. Thus, nanoparticles offer excellent
Fl properties without loss of antibody function on
covalent binding as compared to Cy5 (Gruber et al.
2000).
It was also observed that the fluorescence intensity
is proportional to the concentration of bioconjugate
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Fig. 1 a Covalent linkage between FPNP and S. typhiAb
forming bioconjugate probe and b Generation of immuno-
complex for the formation of mPC fluoroimmunoassay
FPNP (Fig. 3b). A direct correlation is followed by the
Fl intensity with the FPNP concentration. The same
pattern was followed by the bioconjugate probe where
with the increase in concentration of FPNP-S. typhi Ab
from 40 to 120 lg/mL also resulted in the increase in
Fl intensity of the all the three emission peaks.
Quantification of S. typhi Ab-immobilized FPNP
The surface coverage of Abs on FPNP was estimated
using Bradford method. Different concentrations of S.
typhi antibody (10–40 lg/mL) were immobilized on
100 lL of washed FPNP. It was observed that immo-
bilization efficiency (IE %) increased with the increas-
ing S. typhi Ab concentration from 10 to 40 lg/mL for
the constant amount of FPNP which leveled at
20 lg/mL of Ab (Fig. 4). This may be attributed to
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J Nanopart Res (2016) 18:111
involving addition of (i) nitric acid (ii) sodium borohydride
(iii) glutaraldehyde, antibody (Ab), antigen (Ag), and biocon-
jugate probe (FPNP-Ab)
the limitation of the available functional groups on
nanoparticles or the steric hindrance at higher Ab
concentration. Maximum I E % of 50 % was observed
when 20 lg/mL of S. typhi was immobilized on FPNPs
and was taken as optimum antibody concentration
immobilized on FPNP for the detection of S. typhi
antigen. Thus, the diketone functional groups on FPNP
exhibit high reactivity and can rapidly combine with
Ab with high immobilization efficiency. Engineered
nanoparticles with distinct core–shell morphology
utilize surface modification process for functionaliza-
tion and bioconjugation adding more complexity to the
physico-chemical properties. The results demonstrated
that when the initial amount of Ab was 4 lg, 2.175 lg
of Ab were immobilized on 5.46 mg of FPNP. The
amount of Ab immobilized on NPs was calculated as
0.398 lg/mg of FPNP and based on viscosity/light
J Nanopart Res (2016) 18:111 Page 7 of 11 111

Fig. 2 Morphological analysis of bare and immobilized nanoparticles, (1) scanning and (2) transmission electron micrographs of
(a) S1 FPNP and (b) FPNP-S. typhi Ab conjugate

Fig. 3 a Effect of
concentration of S. typhi-
IgG on fluorescence of
pyrene loaded in FPNP.
b Fluorescent intensity as a
function of concentration of
FPNP-S. typhi Ab conjugate

scattering method one particle has an average of It has been proven by Soukka et al. (2001) that the
3.51 9 10-19 g or 21 9 104 S. typhi Ab molecules binding affinity constant for antibody–nanoparticle
bound to them. This was the optimal binding of the bioconjugates is approximately eight times stronger
FPNP-S. typhi Ab conjugate for the detection protocol. than intrinsic affinity of free Ab.

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Fig. 4 Effect of Ab concentration on immobilization efficiency
(IE %) on FPNP
Fig. 5 A CLSM image of a bacteria captured on the edge of
mPC membrane labeled with fluorescent probe at high
magnification, b negative control, fields (1) CLSM (2) DIC
images (B) Superimposed view of DIC and fluorescent CLSM
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J Nanopart Res (2016) 18:111
Determination of S. typhi antigen using mPC
membrane and bioconjugated fluorescent probe
A method of fluorescent nanoparticle-based sandwich
immunofluorescence microscopy was developed for
the detection of S. typhi and the principal of solid
phase fluoroimmunoassay is depicted in Fig. 1b.
Antigen is first recognized by the antibody-bound
mPC surface and the nanoparticle–antibody conju-
gates creating suitable immuno-complex were used to
generate fluorescent signal. The binding events were
observed through confocal laser scanning microscope
and the images are shown in Fig. 5A and B. Bright
blue fluorescence of pyrene dye was observed for the
images of FPNP-S. typhi Ab labeled different antigen concen-
trations captured on mPC membrane a 106, b 104, and
c 102 cells/mL
J Nanopart Res (2016) 18:111 Page 9 of 11 111

Table 1 Comparison between different bacteria detection methods as reported in literature

NP type Method Targeted bacteria Analyte Detection limit Reference

Cadmium selenide Fluorescence Mycobacteria Buffer solution 104 bacteria/mL Liandris et al.
quantum dots (2011)
QDs
QDs Fluorescence E. coli and Saline 1.95 9 103 and Yang and Li
Salmonella 3.35 9 104 CFU/ (2006)
Typhimurium mL
Multi-wall carbon Electrochemical immunoassay Salmonella spp. Milk \103 CFU/mL Chumyim et al.
nanotubes (2014)
(MWNTs)
Magnetic beads Immunomagnetic separation E. coli O157 and Foods and 1000–2000 bacteria Yu and Bruno
(IMS) and electro- Salmonella fomites per mL in food (1996)
chemiluminescence (ECL) typhimurium samples
Fe3O4, magnetic Superparamagnetic separation S. aureus Buffer solution 0.5 9 103 CFU/mL Chen and
core, and silica Zhang (2012)
(SiO2) shell
Streptavidin- Fluorescence Salmonella Buffer solution 103 CFU/mL Yaohua et al.
coated QDs typhimurium (2014)
Liposomal Immunoassay E. coli and TBS 1.5 9 106 and Chen and Durst
nanovesicles Salmonella spp. 5 9 104 CFU/mL (2006)
Direct Sandwich enzyme-linked Salmonella typhi Food and water 104–105 CFU/mL Kumar et al.
immunosorbent assay samples without (2008)
(sELISA) enrichment

bacteria bound with FPNP antibody conjugate as blue
spheres. In order to demonstrate that the binding of
FPNP on modified mPC was through specific antigen–
antibody reaction and not by some other non-specific
interactions, control experiment was also conducted.
In negative control, no fluorescence was observed in
CLSM when S. typhi antigen was replaced with PBS
indicating that specific interaction occurs between the
biological moieties leading to the detection of bacteria
(Qui et al. 2007). It was observed that number of
fluorescent field decreased with the decrease of
bacterial concentration from 106 to 102 cells/mL
incubated with mPC membrane immobilized with Ab
as seen from Fig. 5B. Since antibody–antigen binding
is the most significant and widely used strategy for
biofunctionalization, the high Ab attachments facili-
tate high bio-recognition events.
The fluorescent immunoassay on mPC membrane
with FPNP as the label was able to detect approxi-
mately 5 9 102 cells/mL of S. typhi antigen. The
synthesized FPNP-S.typhi Ab conjugates give ampli-
fied detection signal because of encapsulation of lager
number of pyrene molecules in the protective
polymeric shell. This results in low detection limit as
compared to dye-based fluoroimmunoassays which
also suffer from photobleaching of organic molecules.
The designed mPC-based ELISA is comparable with
other biosensing system and has high sensitivity and
specificity while the fluorescent nanoparticles are easy
to handle. Different methods are compared with our
system in Table 1 highlighting the scope and rele-
vance of current approach. Covalent bioconjugation of
biomolecules on nanomaterials is the core idea for
monitoring bio-recognition events operating at nanos-
cale. Bound antibodies facilitate multiple contacts
between nanomaterials and whole cell bacterial anti-
gen displaying higher binding affinity and sensitivity
of the system.
Analysis of mPC fluoroimmunoassay with different
bacterial cross-reactants was done and measured by
confocal laser scanning microscope as a function of
fluorescent intensity. No fluorescence was observed
with S. typhi Ab-FPNP conjugates on mPC-Ab-
immobilized membrane when incubated with cross-
reactant bacteria as analytes (Fig. 6). This indicated
that S. typhi-FPNP conjugates were removed during

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Fig. 6 Evaluation of specificity of developed fluoroimmunoas-
say with different cross-reactants as a function of fluorescent
activity
final washing step and have not participated in the
formation of immuno-complex. The selected ROI
showed negligible intensity as compared with specific
Ab–Ag combination. Due to amplification of fluores-
cent signal of pyrene dye inside the protective
polymeric layer, the designed immunoassay can easily
sense 500 cells/mL of bacteria detecting lower bacte-
ria count than the infection level of typhoid fever
(105 CFU/mL). Thus, the polymeric bioconjugated
probe has the potential to be effectively used as a
fluorescent label for the detection of bacteria.
Conclusion
In the present study, water dispersible polymeric
nanoparticles having keto-enolic functional groups on
the outer shell layer were bioconjugated with S. typhi-
specific IgGs without using any activator or cross-
linking agent. Pyrene-loaded nanoparticles were suc-
cessfully used as a bioconjugated probe for rapid and
sensitive detection of Salmonella typhi on amino-
functionalized polycarbonate membrane in a sand-
wich format. Confocal laser scanning microscopic
studies revealed very low bacterial concentration;
500 cells/mL was easily detected owing to amplifica-
tion of fluorescent signal of pyrene dye inside the
protective polymeric layer. FPNP bioconjugates were
sensitive and specific probes detecting bacterial count
below the infection level of typhoid fever.
Acknowledgments This work was supported by Department
of Biotechnology (DBT), Govt. of India; authors duly
123
J Nanopart Res (2016) 18:111
acknowledge their generous financial support. The authors are
also thankful to CSIR-UGC and ICMR, India for their research
fellowships.
References
Abdel-Hamid I, Ghindilis AL, Atanasov P, Wilkins E (1998)
Development of a flow-through immunoassay system. Sens
Actuat B 49:202–210
Allmer K, Huly A, Runby B (1989) Surface modification of
polymers. III. Grafting of stabilizers onto polymer films.
J Polym Sci Part A 27:3405–3417
Bagwe RP, Hilliard LR, Tan W (2006) Surface modification of
silica nanoparticles to reduce aggregation and non-specific
binding. Langmuir 25:4357–4362
Chen CS, Durst RA (2006) Simultaneous detection of Escher-
ichia Coli and O157:H7, Salmonella spp. and Listeria
monocytogenes with an array-based immunosorbent assay
using universal protein G liposomal nanovesicles. Talanta
69:232–238
Chen L, Zhang J (2012) Bioconjugated magnetic nanoparticles
for rapid capture of gram-positive Bacteria. J Biosens
Bioelectrons 11:005
Cheng CM, Martinez A, Gong J, Mace CR, Phillips ST, Carrilho
E, Mirica K, Whitesides G (2010) Paper-based ELISA.
Angew Chem Int Ed 49:4771–4774
Chumyim P, Rijiravanich P, Somasundrum M, Surareungchai
W (2014) Detection of Salmonella enterica serovar
Typhimurium in milk sample using electrochemical
immunoassay and enzyme amplified labeling. Int Conf
Agric Environ Biol Sci 24–25 Phuke
Gruber HJ, Hahn CD, Kada G, Riener CK, Harms GS, Ahrer W,
Dax TG, Knaus HG (2000) Anomalous fluorescence
enhancement of Cy3 and Cy3.5 versus anomalous fluo-
rescence loss of Cy5 and Cy7 upon covalent linking to IgG
and noncovalent binding to avidin. Bioconjugate Chem
11:696–704
Harma H, Pelkkikangas A, Soukka T, Huhtinen P, Huopalahti S,
Lovgern T (2003) Sensitive miniature single-particle
immunoassay of prostate-specific antigen using time-re-
solved fluorescence. Anal Chim Acta 482:157–164
Holzapfel V, Musyanovych A, Landfaster K, Lorenz MR,
Mailander V (2005) Preparation of fluorescent carboxyl
and amino functionalized polystyrene particles by
miniemulsion polymerization as markers for cells.
Macromol Chem Phys 206:2440–2449
Hun X, Zhang Z (2007a) A novel sensitive staphylococcal
enterotoxin C1 fluoroimmunoassay based on functional-
ized fluorescent core-shell nanoparticle labels. Food Chem
105:1623–1629
Hun X, Zhang Z (2007b) Fluoroimmunoassay for tumor
necrosis factor- a in human serum using Ru(bpy)3Cl2-
doped fluorescent silica nanoparticles as labels. Talanta
73:366–371
Hun X, Zhang Z, Tiao L (2008) Anti-Her-2 monoclonal anti-
body conjugated polymer fluorescent nanoparticles probe
for ovarian cancer imaging. Anal Chim Acta 625:201–206
Ivnitski D, Abdel-Hamid I, Atanasov P, Wilkins E (1999)
Biosensors for detection of pathogenic bacteria. Biosens
Bioelectron 14:599–624
J Nanopart Res (2016) 18:111
Jain S, Chattopadhyay S, Jackeray R, Abid Z, Singh H (2013)
Novel functionalized fluorescent polymeric nanoparticles:
synthesis, characterization and immobilization of biomo-
lecules. Nanoscale 5:6883–6892
Kumar S, Balakrishna K, Batra HV (2008) Enrichment-ELISA
for detection of Salmonella typhi from food and water
samples. Biomed Environ Sci 21(2):137–143
Kumari A, Yadav SK, Yadav SC (2010) Biodegradable poly-
meric nanoparticles based drug delivery systems. Colloids
Surf B 75:1–18
Liandris E, Gazouli M, Andreadou M, Sechi LA, Rosu V, Iko-
nomopoulos J (2011) Detection of pathogenic mycobac-
teria based on functionalized quantum dots coupled with
immunomagnetic separation. PLoS One 6:e20026–e20032
Nichkova M, Dosev D, Gee SJ, Hammock BD, Kennedy IM
(2005) Microarray immunoassay for phenoxybenzoic acid
using polymer encapsulated Eu:Gd2O3 nanoparticles as
fluorescent labels. Anal Chem 77:6864–6873
Park J, Kurosawa S, Watanabe J, Ishihara K (2004) Evaluation
of 2-methacryloyloxyethyl phosphorylcholine polymeric
nanoparticle for immunoassay of C-reactive protein
detection. Anal Chem 76(9):2649–2655
Pich A, Bhattacharya S, Hans-Juergen Adler P, Wage T,
Taubenberger A, Li Z, Pee K, Böhmer U, Bley T (2006)
Composite magnetic particles as carriers for laccase from
Trametes versicolor. Macromol Biosci 6:301–310
Qui D, He X, Wang K, Zhao X, Tan W, Chen J (2007) Fluo-
rescent nanoparticle-based indirect immunofluorescence
microscopy for detection of Mycobacterium tuberculosis.
J Biomed Biotechnol 7:89364–89370
Santra S, Dutta D, Moudgil BM (2005a) Functional dye-doped
silica nanoparticles for bioimaging, diagnostics and ther-
apeutics. Food Bioprod Process 83:136–140
Santra S, Dutta D, Walter G, Moudgil B (2005b) Fluorescent
nanoparticle probes for cancer imaging. Technol Cancer
Res Treat 4:593–602
Soukka T, Halrmal H, Paukkunen J, Lolvgren T (2001)
Utilization of kinetically enhanced monovalent binding
Page 11 of 11 111
affinity by immunoassays based on multivalent nanopar-
ticle-antibody bioconjugates. Anal Chem 73:2254–2260
Vakurov A, Pchelintsev NA, Forde J, Fagain CO, Gibson T,
Millner P (2009) The preparation of size-controlled func-
tionalized polymeric nanoparticles in micelles. Nanotech-
nology 20:295605–295612
Wang J, Mazza G (2002) Effects of anthocyanins and other
phenolic compounds on the production of tumor necrosis
factor a in LPS/IFN-c-activated raw 264.7 macrophages.
J Agric Food Chem 50:4183–4191
Wang L, Zhao W, Tan W (2008a) Bioconjugated silica
nanoparticles: development and applications. Nano Res
1:99–115
Wang X, Wu J, Li F, Li H (2008b) Synthesis of water-soluble
CdSe quantum dots by ligand exchange with p-sulfonato-
calix(n)arene (n = 4, 6) as fluorescent probes for amino
acids. Nanotechnology 19:205501–205509
Yang L, Li Y (2006) Simultaneous detection of Escherichia coli
O157:H7 and Salmonella Typhimurium using quantum
dots as fluorescence labels. Analyst 131:394–401
Yang W, Zhang CG, Qu HY, Wang HH, Xu JG (2004) Novel
fluorescent silica nanoparticle probe for ultrasensitive
immunoassays. Anal Chim Acta 503:163–169
Yaohua H, Chengcheng W, Bing B, Mintong L, Wang R, Li Y
(2014) Detection of Staphylococcus aureus using quantum
dots as fluorescence labels. Int J Agric Biol Eng 7:77–81
Yu H, Bruno JG (1996) Immunomagnetic-electrochemilumi-
nescent detection of Escherichia coli O157 and Salmonella
typhimurium in foods and environmental water samples.
Appl Environ Microbiol 62:587–592
Zhao X, Hillard LR, Mechery S, Wang Y, Bagwe RP, Jin S
(2004) A rapid bioassay for single bacterial cell quantita-
tion using bioconjugated nanoparticles. PNAS
101:15027–15032
Zhou L, Pollard AJ (2010) A fast and highly sensitive blood
culture pcr method for clinical detection of Salmonella
enterica serovar typhi. Clin Microbiol Antimicrob 9:14–18
123