RESEARCH PAPER

Fabrication of magnetic gold nanorod particlesfor immunomagnetic separation and SERS application

Ugur Tamer • Ismail H. Boyacı • Erhan Temur •

Adem Zengin • Ilker Dincer • Yalcın Elerman

Received: 26 July 2010 / Accepted: 27 December 2010 / Published online: 11 January 2011

� Springer Science+Business Media B.V. 2011

Abstract The preparation and application of rod-

shaped core–shell structured Fe3O4–Au nanoparticles

for immunomagnetic separation and sensing were

described for the first time with this study. To

synthesize magnetic gold nanorod particles, the seed-

mediated synthetic method was carried out and the

resulting nanoparticles were characterized with trans-

mission electron microscopy (TEM), ultraviolet visi-

ble spectroscopy (UV–Vis), energy-dispersive X-ray

(EDX), and X-ray diffraction (XRD). Magnetic prop-

erties of the nanoparticles were also examined.

Characterization of the magnetic gold nanorod parti-

cles has proven that the resulting nanoparticles were

composed of Fe3O4 core and the gold shell. The rod-

shaped gold-coated iron nanoparticles have an average

diameter of 16 ± 2 nm and an average length of about

50 ± 5 nm (corresponding aspect ratio of 3). The

saturation magnetization value for the magnetic gold

nanorod particles was found to be 37 emu/g at 300 K.

Rapid and room temperature reaction synthesis of

magnetic gold nanorod particles and subsequent sur-

face modification with E. coli antibodies provide

immunomagnetic separation and SERS application.

The analytical performance of the SERS-based

homogenous sandwich immunoassay system with

respect to linear range, detection limit, and response

time is also presented.

Keywords Magnetic gold nanorod � SERS �Immunomagnetic separation � E. coli

Introduction

Nanocomposites with different functionalities have

long been of scientific interest due to their controlled

structure and interface interactions (Yu et al. 2005;

Zeng et al. 2002; Zeng et al. 2004). Considering the

growing demands for detection and separation meth-

ods in genomics and proteomics analysis, new

materials and technologies that can take advantage

Electronic supplementary material The online version ofthis article (doi:10.1007/s11051-010-0213-y) containssupplementary material, which is available to authorized users.

U. Tamer (&) � E. Temur

Department of Analytical Chemistry,

Faculty of Pharmacy, Gazi University, Etiler,

06330 Ankara, Turkey

e-mail: utamer@gazi.edu.tr

I. H. BoyacıDepartment of Food Engineering, Hacettepe University

Beytepe, Ankara, Turkey

A. Zengin

Department of Chemistry, Faculty of Art and Science,

Gazi University, Besevler, Ankara, Turkey

I. Dincer � Y. Elerman

Department of Engineering Physics,

Faculty of Engineering, Ankara University, Besevler,

Ankara, Turkey

123

J Nanopart Res (2011) 13:3167–3176

DOI 10.1007/s11051-010-0213-y

of the nanocomposites would be powerful tools with

the potential applications (Bong et al. 2009). The

bifunctional nanocomposite containing the magnetic

core–gold shell hybrid structure has the potential

advantages of magnetic separation and the optical

properties (Gole et al. 2008; Lin et al. 2001; Cho

et al. 2005; Mikhaylova et al. 2004; Mandal et al.

2005; Lyon et al. 2004; Wang et al. 2005a, b).

Magnetic separation enables the specific capture of

the desired target from its matrix and the major

advantage of using nanoparticles is the high capture

efficiency due to high surface-to-volume ratio (Yang

et al. 2007). A permanent magnet can be used for

separation or purification. The surface modification

with gold may provide not only the stability to the

magnetic nanoparticles in solution but also helps in

binding the various chemical and biological agents

(Gupta and Gupta 2005). Functional molecules or

biological materials can be coated through well-

known chemistry of gold and these modified particles

can easily detect the desired analyte (Luo et al. 2008;

Li et al. 2009; Elghanian et al. 1997; Mirkin et al.

1996; Rosi and Mirkin 2005).

Since the introduction of the SERS, nanomaterials

are of significant interest as SERS substrates due to

their tunable optical properties. The design and

fabrication of a variety of nanomaterials with large

SERS enhancement factor have been reported (Ban-

holzer et al. 2008). For this aim, special substrates

have been prepared using gold and silver. SERS

substrates constructed from gold and silver nanopar-

ticles have been used to detect microorganism (Dutta

et al. 2009; Liu et al. 2008, b), protein (Fabris et al.

2008; Drachev et al. 2005) and nucleotides (Bell and

Sirimuthu 2006). Anisotropic metallic nanoparticles

such as gold or silver have shown promise as novel

SERS tags for immunoassay. The results suggest that

SERS tags can be used for multiplex and ultrasen-

sitive detection of biomolecules (Wang et al. 2008a,

b). Gold nanorod particle with controllable size and

numerous surface functionalities can be easily syn-

thesized (Schultz 2003) and used as SERS substrates

(Murphy et al. 2005; Jana et al. 2002; Caswell et al.

2003; Wang et al. 2008a, b). It is also demonstrated

that SERS enhancements were always greater for

aggregated gold nanorods than for aggregated spher-

ical gold nanoparticles (Nikoobakht and El-Sayed

2003a, b). The shape of the SERS substrates could be

important parameter to the SERS activity. Although

there are several methods to synthesize gold nanorod,

synthesis by wet chemistry has emerged as the

method of choice for obtaining nanorods due to the

simplicity and low cost (Tollan et al. 2009). On the

basis of this procedure, gold nanorods were prepared

by a seed-mediated growth approach use gold

nanospheres as the seeds and subsequent reduction

of metal salt with ascorbic acid in the presence of a

directing surfactant (CTAB). These modified shapes

of the gold nanorods directly influence their optical

properties (Gou and Murphy 2005). The study

described here was motivated by a recent report

which demonstrated that nanoparticle shape could

have a substantial impact on the intensity of the

SERS signal. The SERS responses of cube-like

CTAB-capped gold nanoparticles were found to be

200 times greater than spherical-shaped gold nano-

particles (Narayanan et al. 2008).

Up to date, the optical and magnetic properties of

the fluorescent silica-coated gold nanorods and

magnetic nanocrystals were reported (Heitsch et al.

2008). Murphy and coworkers reported Fe3O4-coated

gold nanorods (Gole et al. 2008). Similarly, Fe3O4

nanoparticles bearing gold nanorods were synthe-

sized and used as a magnetic resonance imaging and

fluorescence imaging to target cancer cells (Wang

et al. 2009). However, the production of monodi-

spersed nanoparticles achieving high magnetization

and providing well-known chemical surfaces with

various functional groups for coupling remains a

significant challenge.

Here, we report for the first time on the rod-shaped

core–shell nanostructures made from magnetic iron

oxide nanoparticles and gold, which demonstrates

remarkable optical and magnetic effects stemming

from collective interactions of gold nanorod and

Fe3O4. Subsequent surface modification for the

recognition of the target provides an effective means

for separation via application of the magnetic field.

To synthesize magnetic gold nanorod particles, the

seed-mediated synthetic method was carried out. The

magnetic separation of these nanoparticles was easily

accomplished and the resulting nanoparticles were

characterized with transmission electron microscopy

(TEM), ultraviolet visible spectroscopy (UV–Vis),

energy-dispersive X-ray (EDX), X-ray diffraction

(XRD), and magnetic properties of the nanoparticles

were also examined. Immunomagnetic separation

was performed at different E. coli concentration.

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As-synthesized gold nanorods particles were prepared

as an SERS tag. Then the SERS signals were obtained

and calibration curve was constructed to determine

the amount of E. coli in the samples. The analytical

performance of the SERS-based homogenous sand-

wich immunoassay system with respect to linear

range, detection limit, and response time is presented.

Experimental section

General

FeSO47H2O, sodium hydroxide, hydrogen peroxide,

sulfuric acid, and Tween 20 were purchased from

Merck (KgaA, Germany). Hydrogen tetrachloroaurate

(III) hydrate, FeCl3, N-ethyl-N0-(3-dimethylaminopro-

pyl)-carbodiimide (EDC), and N-hydroxysuccinimide

(NHS) were obtained from Fluka (Steinheim, Ger-

many). Biotin conjugated rabbit anti-E. coli polyclonal

antibodies were obtained from Abcam plc. (Cam-

bridge, UK). 11-mercaptoundecanoic acid (11-MUA),

3-mercaptopropionic acid (3-MPA), hexadecyltrime-

tyl-ammonium bromide (CTAB), L-ascorbic acid

(AA), 98% ethanolamine, 2-morpholinoethanesul-

fonic acid monohydrate (MES), trisodium citrate

dihydrate, sodium phosphate monobasic, sodium

phosphate dibasic, and absolute ethanol were obtained

from Sigma-Aldrich (Taufkirchen, Germany). Silver

nitrate (AgNO3) and sodiumborohydride (NaBH4)

were obtained from Merck (Darmstadt, Germany).

5,5-dithiobis (2-nitrobenzoic acid) (DTNB) was

obtained from Acros (NJ, USA). Immunopure avidin

was obtained from Pierce Biotechnology (IL, USA).

NaCl, Na2HPO4, and KH2PO4 were obtained from J.T.

Baker (Deventer, The Netherlands), used as phosphate

buffered saline (PBS). All reagents were used as

received.

The fabrication of the magnetic gold nanorod

The seed-mediated synthetic method was performed

in two steps to synthesize magnetic gold nanorod

particles. First, the spherical-shaped gold-coated iron

oxide nanoparticles were synthesized according to

our recent report (Tamer et al. 2010). Second, the

resulting spherical-shaped gold-coated Fe3O4 nano-

particles were used as seed and different amount of

seed particles were added to 4.75 mL growth solution

containing 0.1 M CTAB (capping agent), 0.01 M

HAuCl4, 0.01 M silver ions, and 0.1 M AA as a

reduction. This final mixture was stirred for a few

seconds and allowed to stay 3 h at room temperature

for rod-shaped gold-coated Fe3O4 nanoparticles

synthesis.

Gold nanorods were prepared by using a seed-

mediated growth technique with slight modification

(Nikoobakht and El-Sayed 2003a, b). Seed solution

was prepared by mixing 7.5 mL of 0.1 M CTAB and

250 lL of 0.01 M HAuCl4 solution. Once mixed,

600 lL of 0.01 M ice-cold NaBH4 was added rapidly

to the resulting solution, and allowed to stand for

5 min to form seed solution. To prepare rod-shaped

gold nanoparticles, 4.75 mL of 0.1 M CTAB, 1 mL

of 0.01 M HAuCl4, and 60 lL of 4 9 10-3 M

AgNO3 were mixed; the resulting color was dark

orange. When the dark orange color was observed,

250 lL of 0.1 M AA was added dropwise to the

resulting solution. The solution turned colorless after

adding 250 lL AA. Then, 5 lL seed solution was

added to the stock solution. The final mixture was

stirred for a few seconds and allowed to stand for 3 h

at room temperature to give the nanorods.

Immunomagnetic separation and SERS

measurements

Equal quantity (1 mg) of gold nanorods was used as

SERS tags by assembling DTNB on nanoparticles.

For constituting self-assembled monolayer, 50 mM

DTNB was used and, it was formed gold nanorod

particles in absolute ethanol overnight to track SERS

signal. After creating self-assembled monolayer

(SAM), modification was carried on from the

carboxylate group of DTNB to complete sandwich

immunoassay. Considering the density of gold as

19.3 g/mL and TEM images, the estimated number of

gold nanorods was 4 9 1012 particles/mL.

Usability of developed core–shell nanoparticles

was investigated in immunomagnetic separation of

E. coli using developed core–shell nanoparticles. To

compose assay, protocol includes two parts as

microorganism capture probe and SERS labels by

assembling DTNB on gold nanorod particles. In first

step, to fabricate the capture probe, magnetic gold

nanorods were modified with the mixture of

150 mM 11-MUA, 150 mM 3-MPA and 50 mM

1-decanethiol in absolute ethanol for overnight to

J Nanopart Res (2011) 13:3167–3176 3169

123

form SAM monolayer. Nanoparticles were collected

using a magnet and washed with 0.05 M MES (pH

6.5) buffer. For surface activation over carboxylate

groups, nanoparticles were stirred for 30 min in NHS/

EDC solution. After the surface activation, magnetic

gold nanorods were interacted with 0.5 mg/mL

avidin in MES buffer for 30 min. For avoiding non-

specific interaction 1% (v/v) ethanolamine was used

to block active groups or any holes on surface of

magnetic gold nanorods. Then, 0.1 mg/mL biotin-

labeled antibody was interacted with magnetic gold

nanorods in PBS solution to form the biotin–avidin

complex on the surface of nanoparticles. After this

procedure, washing was performed with PBS buffer

to remove the unconjugated and loosely conjugated

biotin-labeled antibodies. In all washing steps, nano-

particles in washing buffer were placed in an

ultrasound bath containing ultra pure water for 10 s.

The Ab-coated magnetic gold nanorods were mixed

with 250 lL of E. coli (102–107 cfu/mL) and incu-

bated for 30 min at 37 �C on a vortex mixer. The

nanoparticles were then separated magnetically and

washed with PBST solution. In the second step,

50 mM DTNB was formed as SAM on gold nanorods

in absolute ethanol overnight. In the second step,

SAM was formed on gold nanorods using 50 mM

DTNB in absolute ethanol overnight. Completing of

the IMS with optimized magnetic nanoparticle–

antibody conjugates, the same biotinylated antibody

binding procedure was performed for DTNB labeled

gold nanorods. To form sandwich complex in solu-

tion, DTNB labeled gold nanorods and magnetic gold

nanorod particles that are surrounded with E. coli

were combined and incubated for 30 min at room

temperature. Then sandwich complex was separated

magnetically from the solution. When assay was

completed, washing procedure was begun with usage

of PBST for nanoparticles surfaces. After three times

washing with PBST, nanoparticles were washed with

three times PBS buffer and they were placed into the

ultrasound bath containing ultra pure water for 10 s.

The complexes were resuspended in 10 lL PBS,

1.0 lL of suspension was placed on the thin layer

chromatography (TLC) paper, and the signals of

DTNB were observed with SERS. The Raman spectra

corresponding to each concentration of E. coli were

recorded. The Raman intensity versus concentration

calibration curve for E. coli (102–107 cfu/mL) is

obtained by calculating the average reading of the

response of duplicate assays under the same condi-

tions. The number of cells (cfu/mL) in each sample

solution was estimated by plating on LB agar,

incubating at 37 �C for 18 h and counting the number

of colonies. The relation between the E. coli

concentrations and the peak intensity at 1325 cm-1

was used for prediction of linearity and the coeffi-

cient of determination (R2). The LOD and LOQ

values were calculated from the calibration curves.

Preparation of buffer solutions and bacteria

samples

Phosphate buffered saline (PBS) (67 mM, pH 7.4)

was prepared by using Na2HPO4 and KH2PO4, NaCl,

and pH adjusted with HCl or NaOH. PBST solution

was prepared by mixing 67 mM, PBS, pH 7.4 with

0.05% Tween 20 [v/v]. PBST solution is the mixture

of 0.1 M PBS buffer at pH 7.4 and 0.05% Tween.

MES buffer (0.05 M, pH 6.5) was prepared by

adjusting the pH with NaOH. EDC/NHS solution

(0.2 M EDC and 0.05 M NHS) was prepared with

0.05 M MES buffer at pH 6.5. Avidin solution

(0.5 M) was prepared using 0.05 M MES buffer.

All solutions were prepared with Milli-Q quality

water (18 MX cm) to reach the desired concentra-

tions. Aqua regia solution (1:3 nitric acid/hydrochlo-

ric acids) was used to clean the quartz cuvettes and

the glassware that is used to synthesize the

nanoparticles.

E. coli K12 was obtained from Refik Saydam

National Type Culture Collections, Ankara, Turkey.

The culture for assay was grown on Tryptic Soy

Broth (TSB) (Merck KgaA, Germany) for 18 h at

37 �C. Bacteria samples were prepared by diluting

the culture (1:10) with 0.1 M PBS at pH 7.5 and

100 lL diluted solution of the culture was plated.

After incubation at 37 �C for 18 h colonies on plates

were counted to determine the number of colony-

forming units per milliliter (cfu/mL).

Instrumentation

Optical absorption spectroscopy measurements were

performed in a Spectronics, Genesis model single

beam spectrophotometer using 1 cm path length

quartz cuvettes. Spectra were collected within a

range 300–800 nm. TEM measurements were per-

formed with the Tecnai G2 F30 instrument. TEM

3170 J Nanopart Res (2011) 13:3167–3176

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samples were prepared by pipetting 10 lL of nano-

particle solution onto TEM grids and allowed to stand

for 10 min. X-ray diffraction measurements were

done using of Philips PW-1140 model diffractometer

with CuKa radiation at a wavelength of 1.5406 A.

DeltaNu Examiner Raman microscope (Deltanu Inc.,

Laramie, WY) with a 785-nm laser source, a

motorized microscope stage sample holder, and a

CCD detector was used to detect E. coli. Instrument

parameters were as follows: 920 objective, 30-lm

laser spot size, 100-mW laser power, and 60-s

acquisition time. Baseline correction was performed

for all measurements. The magnetic field and tem-

perature dependence of magnetization were measured

by using a physical properties measurement system

(PPMS) from quantum design.

Results and discussion

Several methods utilizing seed-mediated growth

based on a wet chemistry approach were developed

to synthesize gold nanorods, with controllable size

and aspect ratio (Yu et al. 2007). Concentrated CTAB

has a tendency to form elongated rodlike micellar

structures (Tornblom and Henriksson 1997). Jana and

coworkers (2002) prepared uniformly shaped gold

nanorods with high aspect ratio by the seeding growth

process in the reduction of HAuCl4 with borohydride

in the presence of CTAB as a miceller template. In

this study, we have employed a different strategy to

construct an Au–Fe3O4 hybrid particle which exhibits

the optical properties of Au metal while the magnetic

properties are very similar to that of Fe3O4. This

method involved a two-step seed-mediated growth

process. First, spherical gold-coated Fe3O4 particles

with an average diameter 12.5 ± 3 nm were synthe-

sized by our previously described procedure (Tamer

et al. 2010). Second, these nanoparticles are added to

a growth solution containing CTAB, HAuCl4, silver

ions, and ascorbic acid as a reduction. In order to find

the optimal synthesizing conditions of magnetic gold

nanorods, various synthesis parameters were investi-

gated. It was observed that shape of nanoparticle was

affected substantially by the amount of HAuCl4 and

seed used in synthesizing conditions. The magnetic

gold nanorod particles were prepared by adding

70 lL seed (spherical gold-coated Fe3O4 particles) to

a solution containing 4.75 mL of 0.1 M CTAB,

50 lL of 0.01 M AgNO3, 250 lL of 0.01 M HAuCl4,

and 50 lL of 0.1 M AA. This mixture was stirred for

10 s and left undisturbed for 3 h to form magnetic

gold nanorod particles. The surfactant solution used

is CTAB and the addition of gold chloride salt

solution to a mixture of water and CTAB leads to the

formation of a complex containing both CTA? and

gold (III) which appeared as an orange solid. The AA

solution was used to reduce Au?3 into Au? and

additional gold was reactively deposited onto the

gold seeds until they merged to form a shell around

the iron oxide particles. As the half wave potential of

ascorbic acid is 0.39 V versus NHE and that of

Au(III) ions is 0.8 V, the AA would reduce the

Au(III) ions on the spherical gold-coated iron nano-

particles. Based on this procedure, magnetic gold

nanorods were prepared by a seed-mediated growth

approach using magnetic gold nanospheres as the

seeds and subsequent reduction of metal salt with

ascorbic acid in the presence of a directing surfactant

(CTAB).

Figure 1 shows representative TEM images of rod-

shaped gold iron nanoparticles. The shape distribu-

tions were obtained by measuring 50 individual

particles from TEM images. Based on the TEM

images, the rod-shaped gold-coated iron nanoparti-

cles have an average diameter of 16 ± 2 nm and an

average length of about 50 ± 5 nm (corresponding

aspect ratio of 3). The percentage of spherical-shaped

Fig. 1 TEM image of magnetic gold nanorods

J Nanopart Res (2011) 13:3167–3176 3171

123

nanoparticles is about 15%. The rod-shaped nano-

particles were appeared as clustered on the TEM

images. This clustering occurred even when the

samples were diluted and sonicated before placement

on the TEM grids. This is a result of the magnetic

properties of nanoparticles to form the observed

clusters. Observed clusters are well separated and can

be distinguished as individual on TEM images. The

central portion of the TEM image is blurred as a

consequence of orientation. Similar behavior was

also observed (Yuan et al. 2006; Spycher et al. 1991).

In addition, the gold coating is composed of (III) planes

with a d-spacing of 0.236 nm as shown in Fig. S1.

Figure 2 shows the extinction spectra of the rod-

shaped gold-coated iron nanoparticle as well as the

spherical gold-coated iron nanoparticle synthesized

using the modified seed-mediated growth method.

The characteristics of the surface plasmon are

correlated to the shape, size, and aggregation of the

nanostructured surface. The spectra for the spherical

gold-coated iron nanoparticle and magnetic gold

nanorod particles are virtually distinguishable, first

plasmon band of magnetic gold nanorod particle

belongs to transverse plasmon band at 545 nm and

second plasmon band belongs to longitudinal plas-

mon band at 695 nm. On the other hand, spherical

gold-coated iron nanoparticles have a plasmon band

at 545 nm as shown in Fig. 2.

The magnetic gold nanorod particles were also

characterized using XRD method. The XRD pattern

of magnetic gold nanorod particles is shown in Fig.

S2. All the reflections correspond to gold shell. As

labeled in Fig. S1, the peaks at 38.148, 44.368, 64.588are assigned to the Au–Fe position of (111), (200),

(220), which are located in the positions of the

corresponding materials (JCPDS card No: 04-0784).

The crystalline structure of gold iron nanoparticles

has proven that the particles are face centered cubic

with the dominant crystal planes of 111. In addition,

the peaks at 5�, 17�, 20.5�, 21.5�, 24�, and 27� are

assigned to CTAB crystalline structure as indicated

the presence of CTAB layer on magnetic gold

nanorods. These core–shell structures were also

analyzed by energy-dispersive X-ray (EDX) spec-

troscopy as shown in Fig. S3. EDX result has proven

that the resulting nanoparticles are composed of iron

core and gold shell. The source of copper peak is due

to the copper grid substrates.

Magnetic nanoparticles may exhibit different

magnetic properties due to their synthetic conditions

(Gupta and Gupta 2005). Figure S4 shows the

temperature dependence of magnetization of mag-

netic gold nanorod particles. The roding temperature

of magnetic gold nanorod particles is about

TB = 145 K and higher than that of magnetic gold

nanosphere (TB = 40 K) and Fe3O4 nanosphere

(TB = 35 K) in this sample (Pal et al. 2009). Figure 3

shows the magnetic field and temperature dependence

of magnetization of magnetic gold nanorod particles,

magnetic gold nanosphere, and Fe3O4 nanosphere

samples. The values of the saturation magnetization

(MS) for these samples are 37 emu g-1, 30 emu g-1,

and 4.7 emu g-1, respectively, which are signifi-

cantly lower than that of the bulk Fe3O4 (Pal et al.

Fig. 2 UV–Vis absorption spectra for magnetic gold nanorod

and nanosphere particlesFig. 3 Magnetic field dependence of magnetization of mag-

netic particles

3172 J Nanopart Res (2011) 13:3167–3176

123

2009) (MS = 84 emu g-1). The MS value of gold-

coated Fe3O4 nanosphere is smaller than that of

Fe3O4 nanosphere because gold can affect the

magnetic ordering of the iron magnetic moments.

According to Fig. 3, the MS value of magnetic gold

nanorod is higher than that of the gold-coated Fe3O4

nanosphere and Fe3O4 nanosphere. Magnetic anisot-

ropy of magnetic gold nanorod is extremely higher

than nanosphere and this should be the reason that the

MS value of magnetic gold nanorod is higher than that

of the nanosphere. Shown in Fig. S5, magnetic gold

nanorod particles synthesized using our system

display strong magnetism.

The performance of the magnetic gold nanorod

particle is tested by using the immunomagnetic

separation of E. coli and SERS spectroscopy as a

benchmark system for E. coli detection. To enhance

Raman scattering, it is crucial to control both the

shape and arrangement of the nanoparticles. It is

reported that the nanorods with aspect ratios of

2.5–3.5 enhance Raman scattering substantially when

the photon energy of the laser used for Raman

spectroscopy is approximately 1.6 eV (Suziki et al.

2008). In this experiment, the gold-based surface

protein binding reactivity and the magnetic separa-

tion capability were examined. Avidin-modified

magnetic gold nanorods particles were coupled to

the antibody via covalent coupling, forming anti-

body-immobilized nanoparticles. The antibody-

immobilized magnetic nanorod particles were reacted

with E. coli as indicated in Fig. 4. The magnetic

separation of these nanoparticles was easily accom-

plished and magnetic gold nanorods were separated

only by a magnet.

The SERS spectra for E. coli assays conducted by

using SERS tag constructed from gold nanorod are

shown in Fig. 5, which displays a typical SERS

response of the immunoassay system after addition of

various concentrations of E. coli (3.5 9 102–

3.5 9 107 cfu/mL). The spectra contain features

which are attributable to SERS tag and are dominated

by bands representative of the DTNB-based adlayer

(e.g., the symmetric nitro stretching (Vs(NO2)) at

1325 cm-1). It is important to note that for aspect

ratio threee magnetic rods with the presence spherical

shapes in the SERS sample, the resulting SERS

spectra would be convolution of those for bacteria

samples in immunoassay system. The detection is

based on a characteristic feature of the Raman tag and

then quantified by its intensity. The calibration curve

was plotted with the changes of the peak intensities of

DTNB versus the different concentrations of E. coli

Fig. 4 Shematic

illustration of the surface

modification of magnetic

gold nanorod particles and

E. coli assay

J Nanopart Res (2011) 13:3167–3176 3173

123

as shown in Fig. S6. The SERS signal tracks with

E. coli concentration and become distinguishable

when 350 bacteria exist in a sample matrix. A good

linear correlation (R2 = 0.980) was obtained within

the range 3.5 9 102–3.5 9 107 cfu/mL E. coli con-

centration. The sensitivity of the developed immu-

noasssay was investigated and the LOD and LOQ of

the proposed method for E. coli were found to be

35 cfu/mL and 3.5 9 102 cfu/mL, respectively. By

using surface-enhanced Raman scattering (SERS)-

based immunoassay detection, even single bacterium

can be detected label-free microarray detection

(Knauer et al. 2010) or using gold particle-covered

glassy substrates (Premasiri et al. 2005). The detec-

tion and identification of bacterial samples by SERS

have been conducted by mixing suspensions of

bacteria with a suspension of colloidal silver particles

and its limit of detection (LOD) was reported to be

103 cfu/mL (Sengupta et al. 2006; Kahraman et al.

2007). The assembly of Ag nanocrystals into Ag

nanospheres used for pathogenic bacteria detection

could detect cells as few as 10 cfu/mL (Wang et al.

2010). We have developed a new magnetic approach

to immunoassay detection of E.coli using SERS.

However, the result (LOD 35 cfu/mL) reported in

this manuscript is not better than our previous

strategy (LOD 5 cfu/mL) (Temur et al. 2010). The

slide surface was used in their study; however, the

magnetic gold nanorod particles were used in this

study for the immunomagnetic separation. There is

no difference in terms of the SERS tag gold nanorod

used in both experiments. The reason for this high

detection limit could be due to the low capture

efficiency of magnetic gold nanorod particles.

Conclusions

The major advantage of using nanomaterials instead

of microbeads is the higher capture efficiency due to

the high surface-to-volume ratio. Other advantages of

using nanoparticles include faster reaction kinetics

and minimal sample preparation. The sensitivity of

the assay described here is not high enough to detect

low concentrations of bacteria; however, proper

optimization such as antibody concentration, anti-

body–nanoparticle incubation time may provide

opportunity to reach low detection limits of regarding

bacteria. SERS-labeled gold nanorods were inter-

acted with magnetic gold nanorod–antibody–E. coli

complex in solution and the assay time is greatly

reduced to less than 2 h. The correlation between the

concentration of bacteria and SERS signal was found

to be linear within the range 3.5 9 102–

3.5 9 107 cfu/mL (R2 = 0.980) with a detection

limit of 35 cfu/mL. The fabrication of high quality

hybrid core–shell nanoparticles endowed with sens-

ing ability using optical and magnetic properties

could show a novel SERS platform.

Acknowledgments The authors are grateful for the financial

support provided by The Scientific and Technological Research

Council of Turkey; Project Number: COST-MP091-108T794.

Fig. 5 Symmetric NO2

stretching bands of DTNB

at different E coliconcentrations obtained

with magnetic gold nanorod

a No E coli, b 101 cfu/mL,

c 102 cfu/mL, d 103 cfu/

mL, e 104 cfu/mL,

f 105 cfu/mL, g 106 cfu/mL,

h 107 cfu/mL

3174 J Nanopart Res (2011) 13:3167–3176

123

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