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- conjugate. Fluorescence of pyrene dye remained same
tion of functionalized polymeric fluorescent nanoparti- on immobilization of biomolecules and nanoparticles
cles as biosensing probe for the detection of Salmonella showed stable fluorescent intensity under prolong expo-
typhi bacteria on modified polycarbonate (PC) filters in sure to laser owing to protective polymeric layer
about 3 h. Antibody modified-PC membranes were allowing accurate identification of bacteria. Surface-
incubated with contaminated bacterial water for selec- functionalized PC matrix and fluorescent label NPs
tive capturing which were detected by synthesized novel permit covalent interactions among biomolecules
bioconjugate probe. Core–shell architecture of poly- enhancing signal acquisitions showing higher detection
meric nanoparticles endows them with aqueous stabi- efficiency as compared to conventional microtiter plate-
lization and keto-enolic functionalities making them based system. Our novel immunoassay has the potential
usable for covalently linking S. typhi antibodies without to be explored as rapid detection method for identifying
any crosslinker or activator. Bradford analysis revealed S. typhi contaminations in water.
that one nanoparticle has an average of 3.51 9 10-19 g
or 21 9 104 bound S. typhi Ab molecules. Analysis of Keywords Fluoroimmunoassay  Fluorescent
the regions of interest (ROI) in fluorescent micrographs nanoparticles  Salmonella typhi  ELISA  Detection 
of modified fluoroimmunoassay showed higher detec- Bioconjugation  Health safety
tion sensitivity of 5 9 102 cells/mL due to signal
amplification unlike conventional naked dye FITC-Ab
Abbreviations
FPNP Fluorescent polymeric nanoparticles
Electronic supplementary material The online version of NPs Nanoparticles
this article (doi:10.1007/s11051-016-3414-1) contains supple- AAEM Acetoacetoxy ethyl methacrylate
mentary material, which is available to authorized users. S. typhi Salmonella typhi
S. Jain (&)  S. Chattopadhyay  R. Jackeray  Ab Antibody
Z. Abid  H. Singh Fl Fluorescent
Centre for Biomedical Engineering, Indian Institute of
Technology-Delhi, New Delhi 110016, India
e-mail: swatijain.iitd@gmail.com Introduction
S. Chattopadhyay
e-mail: sruticiitd@gmail.com Biolabeling with fluorescent dyes has many biomed-
H. Singh ical applications like fluorescent immunoassays for
e-mail: harpal2000@yahoo.com diagnostics and imaging in commercial and

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institutional setups. Fluorescent immunoassays (FIA) polymeric nanoparticles were synthesized by Pich
increase the high throughput efficiency and sensitivity et al. with styrene and acetoacetoxyethyl methacrylate
(Wang and Mazza 2002; Yang et al. 2004). The for the immobilization of enzyme (Pich et al. 2006).
traditionally used organic fluorophores like FITC Europium-doped gadolinium oxide nanoparticles
suffer from lack of photostability in addition to being were modified with Poly L-Lysine by Nichkova et al.
relatively less fluorescent. The new improved Alexa (2005). They had developed a microimmunoassay to
series though have high intensity but have disadvan- detect phenoxybenzoic acid, a generic biomarker of
tages of low labeling efficiency, and photostability. human exposure to insecticides. Holzapfel et al.
Advances in nanotechnology have facilitated the (2005) have developed fluorescent carboxy and
engineering of nanoassemblies for various analytical amine-terminated polystyrene nanoparticles by mini-
and biological applications. Last decade witnessed the emulsion polymerization as the Hela cell marker. Hun
emergence of silica (Si) nanoparticles (NPs), gold et al. have prepared unique polymeric fluorescent
(Au) nanoparticles quantum dots (QDs), and florescent particles of poly(methyl methacrylate) with butyl
polymeric nanoparticles as the new generation mark- rhodamine B dye as marker for the bio-imaging of
ers (Harma et al. 2003). Various QDs have been ovarion cancer by immobilizing Her-2 antibody on the
utilized to image many cancer cell lines such as human NP surface (Hun et al. 2008). Polymeric nanoparticles
mammary epithelial tumor, human breast cancer, doped with fluorescent molecules show promising
human prostate cancer, and lung tumor (Santra et al. potential as label for the detection of cells including
2005a, b). However, QDs are difficult to synthesize bacteria. In most of the developed FIA for the
requiring extreme temperature–pressure conditions detection of various analytes, antibodies are physically
along with water free environment. Furthermore, the immobilized on the polystyrene microtiter plates
utilization of QDs is restricted owing to their cyto- which affect the sensitivity of the assay since
toxicity and water incompatibility. Surface modifica- biomolecules leach out on extensive washing (Cheng
tions are also necessary to make them water et al. 2010). Park et al. (2004) synthesized 2-methacry-
dispersible (Wang et al. 2008a, b). Extensive research loyloxyethyl phosphorylcholine (MPC)-polymeric
work is going on dye-doped silica nanoparticles in bio- nanoparticles (MPC-PNP) having active ester groups
imaging and bio-analysis applications for the detec- for bioconjugation on the side chains where anti-
tion of various analytes including tumor necrosis C-reactive protein (CRP) monoclonal antibodies were
factor-a, enterotoxins, bacteria, and cancer cells (Hun immobilized on the MPC-PNP using the active ester
and Zhang 2007a, b; Santra et al. 2005a, b). Zhao et al. group, while the remaining active ester groups were
have developed a rapid test for the analysis of bacterial thoroughly reacted with glycine. A detection limit
cell E. Coli O157:H7 using bioconjugated Si nanopar- about serum-free CRP in the calibration curve was
ticles (Zhao et al. 2004). An indirect homogenous shown from 0.01 to 10 mg/dL.
solution-based immunofluorescence microscopic Supply of safe drinking water globally is a
method was developed for the detection of Mycobac- challenge; United Nations regularly tracks and mon-
terium tuberculosis using Si NPs (Qui et al. 2007). But itors access to safe water and sanitation in collabora-
reports suggest that the dye molecules doped inside the tion with many individual nations. Salmonella typhi
Si shell have high chances of leaching because of the causative organism for typhoid fever is the
porous nature of Si. They also require multistep pathogenic bacteria primarily responsible for almost
reactions for the generation of functionality for the 21 million deaths worldwide (Zhou and Pollard 2010).
attachment of biological moieties (Bagwe et al. 2006; Failure to detect bacterial adulterations in water
Wang 2008a, b). rapidly is the most egregious consequence since the
‘‘Tailor’’-made Polymeric fluorescent nanoparti- culture plate method yields results in 24–48 h after
cles having in-built surface functionality are being several steps of enrichment and isolation. Rapid
developed for various biomedical applications. Poly- biosensing approaches are currently being investi-
meric nanoparticles have special characteristics of gated including PCR, surface Plasmon resonance
high surface to volume ratio, size controllability, and (SPR), quartz crystal microbalance (QCM), and
can be easily functionalized during synthesis (Kumari piezoelectric electronic noses. However, these meth-
et al. 2010; Vakurov et al. 2009). Size-controlled ods are at explorative stage also need high amount of

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capital investments which deters their applications in substrate 3, 30 , 5, 50 tetramethylbenzidine-hydrogen

rural set ups (Ivnitski et al. 1999). Enzyme-linked peroxide (TMB/H2O2), and fluorophore-tagged anti-
immunosorbent assays (ELISAs) are the chief bioan- body Goat anti-Rabbit- fluorescein isothiocyanate
alytical testing technique for environmental samples (GAR-FITC) were received from Bangalore Genie
but suffer from low sensitivity (Abdel-Hamid et al. (India). All solvents and reagents were purchased from
1998). Therefore, sensitive, rapid, and prompt detec- commercial sources and were used as-received unless
tion of bacteria is highly obligatory especially in noted otherwise. Milli-Q water (Millipore Corp.,
endemic areas. USA) was used for the preparation of 1X-phosphate-
Integration of nanomaterials with ELISA design buffered saline (PBS) and double-distilled water was
could lead to enhanced sensitivity and the experimen- used for polymerization.
tal setup could be economical, ultrasensitive, and easy
to configure. The paper outlines the utilization of Surface modification of polycarbonate membrane
functionalized polymeric nanoparticles as bioconju- and immobilization of S. typhi antibodies
gated fluorescent labels. We have used surface-
modified polycarbonate membranes as solid matrix Sequential surface modification of black colored PC
for capturing S. typhi and capitulated on sandwich membranes (area 0.66 cm2) was done according to
fluorescent immunosorbent assay (FIA) with anti- protocol optimized in our lab. Briefly, nitration and
body-conjugated fluorescent probe. 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)
Materials and methods respectively, were done to generate amino groups.
This modified membrane designated as mPC were
TM
13 mm black colored Isopore Polycarbonate filters immersed in a mild solution of 5 % glutaraldehyde
(size: 0.6 9 0.3 lm) with 0.22 lm pore size were and stirred for 2 h at room temperature for activation
purchased from Millipore Inc. (New York, USA). and were further washed with 1X-PBS thrice. Glu-
Monomers styrene (St), methyl methacrylate (MMA), taraldehyde-activated mPC membranes (PC-NH2-
acetoacetoxyethyl methacrylate (AAEM), crosslinker Glu) were immobilized with 400 lL of 10 lg/mL of
ethylene glycol diacrylate (EGDA), fluorescent dye, S. typhi-IgG for 16 h at 4 °C and were stored after
pyrene along with glutaraldehyde, and Tween-20 were washing with Tween/PBS and 1X PBS till further use.
purchased from Sigma Aldrich (St. Louise, USA).
Surfactant, sodium lauryl sulfate (SLS), and initiators, Synthesis of fluorescent polymeric nanoparticles
ammonium persulfate (APS) and sodium peroxydisul-
phate (SPDS) of AR grade quality were obtained from Polymeric nanoparticles of styrene, MMA, and
CDH chemicals (Mumbai, India). Styrene and MMA AAEM were synthesized by free radical mini-emul-
were washed with dilute sodium hydroxide solution in sion polymerization technique in the presence of
a separating funnel to remove inhibitor and stored over fluorescent dye pyrene (Jain et al. 2013). Briefly,
fused calcium chloride and finally distilled under 15 mg of fluorescent dye pyrene along with homoge-
vacuum. nous monomer mixture of MMA (3.5 mL), styrene
Immunoreagents Heat killed culture of ‘‘O’’ antigen (1 mL), AAEM (1 mL), and crosslinker EGDA
of pathogenic bacteria Salmonella typhi of 6 9 108 - (0.280 mL) was added to oxygen-free 170 mL dis-
cells/mL concentration were received as gift from tilled water with 0.1 g surfactant sodium lauryl
Rockland Hospital, New Delhi, India and were used sulphate (SLS) and heated to 60 °C for 15 min.
strictly under isolated conditions after proper heat Initiator SPDS (0.15 g in 3 mL) was added, temper-
inactivation. Affinity-purified polyvalent somatic ‘‘O’’ ature was raised to 80 °C, and the reaction was
type immunoglobulins (1 mg/mL aliquots) against continued for 2 h. Further addition of monomers—
whole cell antigen of S. typhi (generated in New MMA (3.5 mL), Styrene (1.25 mL), AAEM (0.5 mL)
Zealand Wistar rats) were generously gifted by along with mixed initiator solution of SPDS (0.15 g in
National Institute for Health and Family Welfare 3 mL), and APS (0.06 g in 1 mL), was done after
(NIHFW), New Delhi, India. S. typhi-HRP conjugate, decreasing the temperature to 40 °C which was once

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again increased to 80 °C and stirring was continued are the initial concentration of Ab and in supernatant
for 8 h to complete the conversion of monomers into after the immobilization reaction, respectively, as
polymers. determined by Bradford method. V is the volume of
aqueous phase (mL) and m is the amount of nanopar-
Characterization of FPNP-S. typhi Ab conjugate ticles (mg). The surface coverage of S. typhi Ab on
single nanoparticle was quantified by utilizing the
Functional group analysis of S. typhi Ab-conjugated mass of one particle. The intensity of the colored
FPNP was done on Perkin Elmer Spectrum One ATR- solutions was read at 595 nm wavelength in Biorad
FTIR spectrometer. FPNP-S. typhi Ab were examined ELISA plate reader. Immobilization efficiency (IE %)
through ZEISS EVO series scanning electron micro- for immobilization of different concentrations of Ab
scope Model EVO 50 for any morphological changes. on nanoparticles was estimated using Eq. 2:
Immobilized FPNP sample was dried to remove water
and mounted on the metallic stub using double-sided Absorbance of immobilized Ab
IEð%Þ ¼
tape. Silver paint was applied on the samples to make Absorbance of Ab in initial solution
them conducting for better resolution while gold  100 ð2Þ
sputtering was done on the sample for conduction using Development of fluorescence-based assay using
the sputtering instrument (BioRad Polaron Sputter, mPC and FPNPs
Model 50X). The morphology of FPNP-S. typhi Ab
conjugate was also assessed by transmission electron Washed 100 lL of FPNPs was incubated with 200 lL
microscope (Phillips, CM12) at an acceleration voltage of different concentrations of S. typhi antibody
of 100 kV at magnification of 110 KX. 3 lL of dilute (10–50 lg/mL) for 16 h at 4 °C. After washing, NPs
FPNP-Ab conjugate solution was placed on the were re-suspended in 500 lL of 1X-PBS for further
200-mesh carbon-coated grid and dried for 20–25 min analysis.
at room temperature. The grid was fed in the instrument Modified mPC membranes were used to capture S.
in the external sample chamber at 27 °C. typhi bacteria and FPNP-S. typhi Ab were utilized as
It is important to measure the fluorescence param- the fluorescent label in the detection method. Glu-
eters of nanoparticles for the development of assay. taraldehyde-activated mPC was immobilized with
100 lL of washed poly(St-MMA-AAEM) fluorescent 10 lg/mL of S. typhi-IgG. Unbound sites of the
nanoparticles was incubated with 200 lL of different membranes were blocked with 6 % skimmed milk
concentrations of S. typhi Ab (10–80 lg/mL) as done for 1.5 h at 37 °C. These Ab immobilized mPC
previously. After washing with 1X PBS, the FPNPs membranes were washed with 1X-PBS and incubated
were re-suspended in 3 mL of 1X-PBS and fluorescent with whole cell antigen of S. typhi (105 cells/mL) for
intensity was determined on LS55 PerkinElmer Spec- 1.5 h at 37 °C. After washing with PBS, they were
trofluorometer (USA). incubated with 200 lL of S. typhi Ab immobilized
Different concentrations of nanoparticles immobi- fluorescent nanoparticles probe (FPNP-S. typhi Ab) for
lized with fixed S. typhi Ab were also subjected to 1.5 h at 37 °C. Afterwards, the probe was decanted and
florescent spectroscopic measurements. 200 lL of the membranes were washed thoroughly with PBS.
FPNP solution (0.232 9 10-4 mol pyrene/Kg of latex) mPC was then subjected to confocal laser microscopic
immobilized with 200 lL of 20 lg/mL of S. typhi Ab analysis for evaluating the formation of immuno-
was diluted in 3 mL PBS for the Fl analysis. complex (mPC-S. typhi Ab-Ag-Ab-FPNP). Along with
100 lL of washed FPNP immobilized with different the test samples, negative control experiments were
concentrations of S. typhi Ab was subjected to Bradford also performed. Immobilized mPC membranes were
assay to determine the amount of antibody attached on incubated with 1X-PBS instead of S. typhi Ag.
them. The amount of Ab immobilized on FPNP was Sensitivity of the developed fluorescence-based
calculated approximately as according to Eq. 1: immunoassay was also determined for the detection of
S. typhi. 10 lg/mL antibody-immobilized mPC mem-
Q ¼ ½ðCi  Ct ÞV =m ð1Þ
branes were incubated with different concentrations of
where Q is the amount of S. typhi Ab immobilized on the bacteria S. typhi (107–102 cells/mL) and were
to a unit mass of the nanoparticles (mg/mg); Ci and Ct evaluated by the above-mentioned protocol.

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Specificity of modified mPC assay was measured Poly(St-MMA-AAEM) nanoparticles and S. typhi Ab
by incubating S. typhi Ab immobilized membranes immobilized FPNP are given in Fig. 2. As seen from
with cross-reactants of S. typhi—Pseudomonas aergi- micrographs, the antibody-conjugated FPNPs remain
nosum, Escherichia coli, Klebsiella pneumoniae with monodispersed and the circular shape of nanoparticles
104cells/mL concentration which were detected by S. is conserved. It was also visualized in TEM analysis
typhi-FPNP probe. A region of interest was located on that the nanoparticles were well dispersed after their
mPC, and fluorescent intensity was recorded for conjugation with antibody indicating immobilization
graphical presentation. without any aggregation.

Spectroscopic characterization of bioconjugate

Results and discussions probe

The amino groups of the antibody react with the The synthesized nanoparticles have polycyclic aro-
diketone groups of the AAEM functionality of matic hydrocarbon pyrene as the fluorescent dye
Poly(St-MMA-AAEM) fluorescent nanoparticles encapsulated inside a polymeric sheath composed of
forming the covalent linkage between the two moieties styrene, methyl methacrylate, and AAEM. The probe
and the reaction is schematically represented in attains core–shell morphology owing to different
Fig. 1a. The keto-enolic forms of diketone moieties polarities of monomers and dye where more hydropho-
facilitate conjugation with biomolecules via poly- bic components constituted by St, MMA, and dye,
condensation reaction with amino groups. This bio- form the core while hydrophilic AAEM resides at the
conjugated probe was lyophilized and stored at 4 °C periphery of nanosystem oriented toward water
for further use. Chemically derivatized PC membranes molecules. Pyrene is a spatially sensitive probe with
with approximately 0.1689 mmol NH2 groups/cm2 ensemble of monomeric emission peaks at 393, 383,
equivalent to 1.01 9 1020 NH2 groups/cm2 were used and 370 nm when excited with 350 nm wavelength.
to immobilize 4.65 lg/cm2 of S. typhi-IgG as deter- Owing to hydrophobicity, the molecules are primarily
mined by Bradford assay. localized in the core of FPNP while the surface
Surface functionalization and biomolecules attach- topology is governed by hydrophilic groups. The core–
ment on FPNP were assessed by FTIR spectroscopy shell structural analogy is achieved thereby protecting
and the spectrum is given in supplementary data as Fig the fluorescence of pyrene dye from degrading envi-
S1. The spectra showed characteristic peaks of C–H ronment of water, salts, and oxygen [Jain et al. 2013]
(CH2) at 2930 and 2893 cm-1 and distorted vibration making NPs viable for biological analysis.
peaks at 1450 cm-1. The stretching vibration bands Before the utilization of bioconjugated FPNP probe
centered at 1723 cm-1 could be attributed to stretch- for the detection of bacteria, their fluorescent charac-
ing vibration peak of C=O in the keto group. teristics were studied and are presented in Fig. 3. The
Absorption band at 2506 cm-1 was different from influence of antibody on the Fl properties of pyrene-
that of nanoparticles, which correspond to the spec- loaded FPNP was investigated with different concen-
trum of antibody. A band around 2506 cm-1 appeared trations of S. typhi Ab (10–80 lg/mL) immobilized on
which may be attributed to the O–H stretching of FPNP. No discernible loss in fluorescence was
carboxyl group present in the Fc region of Ab (Allmer observed and no appreciable change in the spectra
et al. 1989). The IR spectral analysis confirmed the appeared for the encapsulated pyrene molecules after
attachment of Ab on nanoparticles. immobilization with biomolecules. There was no
effect of concentration of antibody on the fluorescent
Morphology analysis spectra when fixed 100 lL (5.46 mg) of FPNP was
incubated with Ab. Thus, nanoparticles offer excellent
The morphology of bioconjuagte fluorescent nanopar- Fl properties without loss of antibody function on
ticles (FPNP-S.typhi Ab) is important to illustrate their covalent binding as compared to Cy5 (Gruber et al.
feasibility as fluorescent markers indicating if the 2000).
particles have aggregated to form large-sized clumps. It was also observed that the fluorescence intensity
Scanning and transmission electron micrographs of is proportional to the concentration of bioconjugate

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Fig. 1 a Covalent linkage between FPNP and S. typhiAb involving addition of (i) nitric acid (ii) sodium borohydride
forming bioconjugate probe and b Generation of immuno- (iii) glutaraldehyde, antibody (Ab), antigen (Ag), and biocon-
complex for the formation of mPC fluoroimmunoassay jugate probe (FPNP-Ab)

FPNP (Fig. 3b). A direct correlation is followed by the the limitation of the available functional groups on
Fl intensity with the FPNP concentration. The same nanoparticles or the steric hindrance at higher Ab
pattern was followed by the bioconjugate probe where concentration. Maximum I E % of 50 % was observed
with the increase in concentration of FPNP-S. typhi Ab when 20 lg/mL of S. typhi was immobilized on FPNPs
from 40 to 120 lg/mL also resulted in the increase in and was taken as optimum antibody concentration
Fl intensity of the all the three emission peaks. immobilized on FPNP for the detection of S. typhi
antigen. Thus, the diketone functional groups on FPNP
Quantification of S. typhi Ab-immobilized FPNP exhibit high reactivity and can rapidly combine with
Ab with high immobilization efficiency. Engineered
The surface coverage of Abs on FPNP was estimated nanoparticles with distinct core–shell morphology
using Bradford method. Different concentrations of S. utilize surface modification process for functionaliza-
typhi antibody (10–40 lg/mL) were immobilized on tion and bioconjugation adding more complexity to the
100 lL of washed FPNP. It was observed that immo- physico-chemical properties. The results demonstrated
bilization efficiency (IE %) increased with the increas- that when the initial amount of Ab was 4 lg, 2.175 lg
ing S. typhi Ab concentration from 10 to 40 lg/mL for of Ab were immobilized on 5.46 mg of FPNP. The
the constant amount of FPNP which leveled at amount of Ab immobilized on NPs was calculated as
20 lg/mL of Ab (Fig. 4). This may be attributed to 0.398 lg/mg of FPNP and based on viscosity/light

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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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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
Fig. 4 Effect of Ab concentration on immobilization efficiency
(IE %) on FPNP blue fluorescence of pyrene dye was observed for the

Fig. 5 A CLSM image of a bacteria captured on the edge of images of FPNP-S. typhi Ab labeled different antigen concen-
mPC membrane labeled with fluorescent probe at high trations captured on mPC membrane a 106, b 104, and
magnification, b negative control, fields (1) CLSM (2) DIC c 102 cells/mL
images (B) Superimposed view of DIC and fluorescent CLSM

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

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acknowledge their generous financial support. The authors are

also thankful to CSIR-UGC and ICMR, India for their research
fellowships.

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