ARTICLE IN PRESS

Journal of Quantitative Spectroscopy &

Radiative Transfer 89 (2004) 133142
www.elsevier.com/locate/jqsrt

Differential light-scattering spectroscopy: a new approach to

studying of colloidal gold nanosensors
N.G. Khlebtsov, V.A. Bogatyrev, A.G. Melnikov, L.A. Dykman,
B.N. Khlebtsov, Ya.M. Krasnov
Institute of Biochemistry and Physiology of Plants and Microorganisms, RAS, 13 Pr. Entuziastov, Saratov 410049,
Russian Federation

Received 17 May 2004

Abstract

Addition of complementary components to a bioconjugate probe results in aggregation of nanoparticles

that can be monitored by extinction spectra. This is the core idea of the well-known sol particle
immunoassay (SPIA) introduced by Leuvering et al. (J. Immunoassay 1 (1980) 77). Here, we describe a new
approach to study the biospecic interactions in systems of colloidal gold bioconjugates. The method is
based on measuring static light-scattering spectra (at 90
) within a wavelength range of 350800 nm. To this
end, we have developed a special attachment to the Specord M-40 spectrophotometer and a corresponding
measurement procedure called by us differential light-scattering spectroscopy (DLSS). The DLSS
technique has been compared with conventional spectrophotometry as applied to colloidal gold conjugates
with various biopolymers. Our theoretical simulations and experiments with gold particles of various sizes
showed a higher potential sensitivity of the method proposed as compared with conventional extinction
spectroscopy. It is expected that DLSS can be used to develop an analytical biospecic test for various
biopolymers.
r 2004 Elsevier Ltd. All rights reserved.

Keywords: Gold nanoparticles; Bioconjugates; Aggregation; Resonance light scattering; Plasmon resonance; T-matrix
method

Corresponding author. Tel.: +7-8452-970-403; fax: +7-8452-970-383.

E-mail address: khlebtsov@ibppm.sgu.ru (N.G. Khlebtsov).

0022-4073/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jqsrt.2004.05.017
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134 N.G. Khlebtsov et al. / Journal of Quantitative Spectroscopy & Radiative Transfer 89 (2004) 133142

1. Introduction

The intense red color of colloidal gold (CG) sols is attributed to the localized plasmon
resonance (LPR) of noble metal nanoparticles. The optics of LPR can be understood in terms of
collective oscillations of free metal electrons (classical point of view) or in terms of volume
plasmon excitations (quantum mechanical point of view) [1]. In addition to the adsorption of
light, the LPR excitation is accomplished by light scattering, as noted already by the founders of
the scientic study of CG-solutions, Faraday and Zsigmondy [2,3]. The relative contributions of
light scattering and absorption to the total extinction strongly depend on the CG particle size.
According to the usual Mie calculations and experimental measurements [4,5], the light scattering
gives an appreciable contribution to sol extinction only for 40-nm gold particles, whereas for
1015-nm particles (most popular in biomedical applications) the LPR absorption dominates.
Conjugation of gold particles with biopolymers leads to the formation of an adsorbed dielectric
shell that does not change the spectral properties of conjugates signicantly, as compared with
bare particles. Addition of complementary molecules to a bioconjugate probe results in two
possible modes of biospecic interaction between the adsorbed recognizing molecules and the
complementary (target) molecules.
In the rst mode, the interaction between target molecules and bioconjugate probe results in an
aggregation of nanoparticles that can be monitored by extinction spectra. It is the key idea of the
well-known sol particle immunoassay (SPIA) introduced by Leuvering et al. in 1980 [6]. The
physical origin of pronounced changes in sol color and in extinction spectra is the strong
electrodynamic interaction of gold particles caused by their approaching each other due to
biospecic or salt aggregation [79]. The same physical picture is applicable to CG particles with
chemically attached oligonucleotides that can interact with complementary nucleotide chains
[10,11] or to the gold nanoparticle aggregation induced by a non-cross-linking DNA hybridization
[12]. It should be emphasized that such types of interaction are possible when both
complementary components (conjugateconjugate or conjugatemolecule) are non monovalent;
i.e., when they possess several sites for cross-linked binding.
In the second mode, the binding of target molecules results in formation of a secondary
polymer shell without any aggregation. Such a scenario is typical for interaction between the
conjugates of CG to monoclonal antibodies (only one clone) and the corresponding target
antigens, or between the conjugates of CG to polyclonal antibodies and the low-molecular
antigens (haptens). In some cases, DNADNA hybridization on gold particles also does not lead
to particle aggregation [12]. The second interaction mode has been utilized for real-time
monitoring of biomolecular proteinprotein binding and high-throughput clinical screening
[13,14]. The optical signal arises from the dependence of the extinction maximum and its position
on the local dielectric properties near the particle surface, which are altered due to biospecic
interaction [15]. Recently, the second interaction mode has been studied in terms of a two-layer [5]
and multilayer [16] conjugate models.
Application of extinction spectra to the conjugate-based biomolecular interaction monitoring
has two essential drawbacks. First, at the initial stages of aggregation (the rst interaction mode)
the changes in extinction are often rather small, especially when the interparticle spacing within an
aggregate is greater than 13 nm [9,17,18]. Analogously, the changes in extinction spectra are also
small for the second interaction mode, which does not involve conjugate aggregation. In these
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N.G. Khlebtsov et al. / Journal of Quantitative Spectroscopy & Radiative Transfer 89 (2004) 133142 135

cases, light-scattering spectroscopy can be considered as a reasonable alternative to the usual

absorption spectroscopy. As far as we are aware, such an approach was proposed for the rst time
in our work [19], with subsequent development of the differential light-scattering spectroscopy
(DLSS) technique reported in papers [9,2022].
In line with our studies [9,1922], the resonance light-scattering (RLS) technique has found
several promising applications in recent years. For example, Roll et al. [23] used RLS spectra
excited by white LED radiation to detect CG aggregation induced by avidinbiotin interaction.
They showed that the scattering intensity ratio (e.g., I 530 =I 680 ) is a reliable indicator of the extent
of colloid aggregation. Quite recently, RLS spectroscopy was applied to detect a trace amount of
thiamazole that enhanced scattering intensity due to aggregation of gold colloid [24] and to detect
Au nanoparticles inside single brome mosaic virus (BMV) capsids [25]. Besides, RLS spectroscopy
was shown [26] to be useful in monitoring the preparation of gold nanoparticle-supported DNA
probes. We would like to stress that all the above-mentioned studies (and also Ref. [27]) actually
did not utilize the DLSS technique. Instead, the RLS spectra of works [24,26] were recorded as the
difference between the enhanced scattering intensity of aggregated sample and the scattering
intensity from a blank (non-aggregated) sol, whereas simple white light illumination was used in
[23].
In this work, we describe the theoretical background and experimental implementation of the
DLSS technique as a new, effective tool for control over the gold nanoparticle and conjugate
preparation as well as for optical monitoring of the biomolecular interaction between
nanoparticle biosensors with attached recognizing molecules and target molecular components.
Unlike in previous publications [21,22], we start here with a brief theoretical consideration based
on exact T-matrix codes designed for aggregated core/mantle monomers [9,28]. Then, we discuss
the basic principles of the DLSS technique and provide illustrative experimental examples.

2. Theoretical background

To explain our basic motivation, let us rst consider qualitatively a simplest case where two
isolated nanoparticles with diameters about 1530 nm are combined into a trivial cluster (a
bisphere). If the average interparticle spacing is around 13 nm [9,18], a weak electrodynamic
interaction of particles will not change the extinction spectrum after particle clustering. Because
the particle size and interparticle distance are much less than the incident light wavelength, the
particles scatter light coherently and the scattered elds should interfere constructively. This
means that the coupling of particles into a bisphere results in a twofold increase in the scattered
intensity. We can formulate this conjecture in terms of the well-known properties of dipolar
Rayleigh scattering: the extinction and scattering cross-sections are proportional to the particle
volume and to the square of volume, respectively. So, after particle coupling, their extinction will
not change whereas the scattering should be increased twofold.
The above qualitative speculations have been conrmed by the exact cluster T-matrix (CTM)
method [29] generalized to include two-layer spherical cluster particles [9,28]. In CTM, the
expansion coefcients amnp for the total scattered eld are given by equation [28]
amnp T pq
mnmn pmnq ; 1
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136 N.G. Khlebtsov et al. / Journal of Quantitative Spectroscopy & Radiative Transfer 89 (2004) 133142

where T pq
mnmn is the elements of the cluster T-matrix, and pmnq are the expansion coefcients of the
incident led. In Eq. (1) and hereafter we adopt the well-known tensorial convention currently
used in light-scattering works [28,29]. According to [30], the cluster T-matrix can be obtained
from the following linear system:
0 0 0
l0 pp lp q
T pq
mnmn Amn T mnm0 n0 ; 2

0lpq 0 0 0
T lpq  lmn A mnmn dll 0  1Almnm
mnmn a
lpp lp q
0 n0 T m0 n0 mn ; 3
0 0
where a lmn are the usual Mie coefcients of a homogeneous sphere, Almnm lpp
0 n0 are vector translation

coefcients for translating incident and scattered elds of individual particles between primary
0lpq
and particle-centered reference frames, and A mnmn are vector translation coefcients to translate
the incident plane wave from the primary into the reference system centered on a cluster particle
(for details, see Ref. [28]). In our modication of the T-matrix algorithm, the usual Mie
coefcients a lmn for the homogeneous gold particles in Eq. (3) were replaced with the
corresponding expansion coefcients of a two-layer CG conjugate. Calculations were carried
out for 15- and 30-nm gold particles covered with a 2.5-nm-thick polymer shell. The number of
cluster particles N varied from 1 to 50, the shell refractive index equaled 1.40, and the optical
constants of gold particles and the surrounding medium (water at 20
C) were calculated according
to Ref. [30]. To study the polymer shell effects, we carried out a special calculations with variation
of shell thickness from 0.25 to 5 nm. The results did not differ essentially from those presented in
this work.
Monodisperse (i.e. N const) cluster ensembles were simulated using the ballistic clusterluster
aggregation (BCCA) model described in Ref. [8] and were implemented on a 3D simple cubic
lattice. Extinction (absorption+scattering) A was computed for a constant gold concentration of
57 mg/ml and a 1-cm cuvette. The scattered intensity I 90 (at 90
) was calculated for the same
conditions and is given in arbitrary units. All optical parameters were averaged over random
cluster orientations by using an analytical T-matrix approach [28,29] and over 10 statistical
congurations by simply using 10-independent cluster simulations.
Fig. 1 shows the extinction (panels (a) and (c)) and scattering (b,d) spectra calculated for BCCA
clusters built from conjugates with a 15 nm (a,b) and 30 nm (c,d) gold core covered by a 2.5 nm
polymer shell. These results clearly demonstrate a noticeable increase in scattered intensity with an
increase in the average aggregate size (in terms of the cluster particle number N) at a constant
concentration of gold in suspension, whereas the extinction spectra remain very close to the
monomer spectrum. It should be emphasized that the interparticle spacing (5 nm) was rather
large, so an appreciable electrodynamic coupling between cluster particles was eliminated or at
least essentially diminished. That is way the extinction spectra do not differ substantially from the
monomer spectrum. On the other hand, our clusters were rather small, as compared with the
wavelength, and they were not compact. Therefore, the multiple intracluster scattering should give
a minor contribution to the optical properties of our clusters. So, the predicted increase in the
right angle scattering could be explained simply by constructive interference of elds scattered
from almost independent cluster particles.
Comparison of graphs for 15- and 30-nm aggregated conjugates allows one to note the
following peculiarities of the spectra. First, other things being equal, the aggregation of larger
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N.G. Khlebtsov et al. / Journal of Quantitative Spectroscopy & Radiative Transfer 89 (2004) 133142 137

1.4 0.12
15 nm 15 nm
1.2 0.10
- N=1 - N=1
1.0 -2 -2
- 10 0.08 - 10
0.8 - 20 - 20

I90
A

- 50 0.06 - 50
0.6
0.04
0.4

0.2 0.02

0.0 0.00
400 500 600 700 800 400 500 600 700 800
(a) Wavelength, nm (b) Wavelength, nm

1.6 0.35
30 nm 30 nm
1.4 0.30
1.2 - N=1 - N=1
-2 0.25 -2
1.0 - 10 - 10
- 20 0.20 - 20
I90
A

0.8 - 50 - 50
0.15
0.6
0.10
0.4
0.2 0.05

0.0 0.00
400 500 600 700 800 400 500 600 700 800
(c) Wavelength, nm (d) Wavelength, nm

Fig. 1. Extinction (a,c) and static light-scattering (b,d) spectra for aggregates with the number of conjugates N ranging from 1 to 50. T-
matrix calculations were carried out for random BCCA clusters built from CG conjugates with gold core diameters of 15 (a,b) and
30 nm (c,d); the polymer shell thickness and the refractive index equal 2.5 nm and 1.40, respectively. All results were averaged over
random cluster orientations and over 10 statistical congurations.

30-nm conjugates gives more pronounced alterations in extinction spectra, as compared with the
15-nm conjugate case. Second, by contrast with Fig. 3b, the spectra in Fig. 3d indicate rapid
saturation with an increase in the cluster particle number (compare Fig. 3d with 3c for N 10, 20,
and 50). It is evident that both observations are related with two effects: different cluster size and
different relative interparticle distances.

3. Principles of DLSS measurements

For DLSS measurements, we used an attachment to a Specord M-40 spectrophotometer (Fig.

2). A detailed description of the attachment and the corresponding procedure of spectral
measurements have been discussed in Refs. [21,22], so we limit ourselves here to a short summary.
During calibration of the device (100%-line calibration), the diffusive reectors 4 and 5 are
placed in both channels, and the calibration transmittances T 0 are stored in the device memory
I 1 lr1 l
T 0 l ; 4
I 2 lr2 l
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138 N.G. Khlebtsov et al. / Journal of Quantitative Spectroscopy & Radiative Transfer 89 (2004) 133142

2 I2 9b 4

7
10 8
3
6

1 I1 9a
5

Fig. 2. Schematic of DLSS measurements: 1 and 2sample and blank light beams, 3monochromator, 4 and 5mirrors, 6 and 7
diffusive reectors, 8photodetector (PM). For extinction measurements, the sample and blank cuvettes are placed in positions 9a and
9b respectively. For scattering measurements, the diffusive reector 6 is replaced by a sample cuvette 10, and the other sample cuvette is
placed in position 9b.

where I 1;2 l are the intensities of sample and blank monochromator beams, and r1;2 l are the
reection coefcients of diffusive reectors. For extinction measurements, the sample and blank
cuvettes are placed in positions 9a and b, respectively, and the measured transmittance is equal to
I 1 lr1 lT b l10Al
T A l 10Al ; 5
I 2 lr2 lT b lT 0 l
where T b is the blank transmittance, and A is the measured sol extinction. In the case of scattering
spectrum measurements, one of the two identical sample cuvettes is placed in position 9b and the
other (10) is replaced with reector 6 (Fig. 2). It can be shown [10] that the light-scattering ux S90
is given by the equation
S90 l bI 1 lT b l10Al I 90 l; 6
where b is a constant coefcient. Hence, the measured transmission T s equals
S90 l b
T s l Al
I 90 l: 7
I 2 lr2 lT b l10 T 0 l r1 l

For experiments described in this work, glass plates coated with barium sulfate were used as
scattering reectors. These plates possessed an almost neutral reection spectrum (r1 l const,
l 400700 nm), so that T s is directly proportional to the single-scattering quantity I 90 l.

4. Experimental verication of the DLSS technique

CG preparations were obtained by Frenss procedure following a published protocol [30]. The
average sizes of CG particles were controlled by spectrophotometric calibration [30]. In addition,
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we used dynamic light scattering to evaluate the average size of CG particles before and after
conjugation with biopolymers. Using a laboratory-made setup [16], we measured the
autocorrelation function of photocurrent uctuations g2 t
hitit ti and then determined
the average translation diffusion coefcient and the hydrodynamic diameter d by standard
procedures [32].
For comparative experiments, the conjugates of CG with Tween 20, Protein A (PrA), human
IgG, gelatin, trypsin, and BSA were prepared according to the protocols described previously (see
Refs. [16,31]). In this paper, only two experimental examples are given for illustrative purposes.
Other experimental applications of the DLSS technique can be found elsewhere [16,21,22].
Fig. 3 demonstrates different sensitivities of the extinction and scattering techniques to the
changes in the dielectric environment of gold nanoparticles. Curves with open circles represent
spectra for bare gold particles, whereas curves with dark circles correspond to the spectra
measured for conjugates of CG with trypsin (Tr). According to the dynamic light-scattering data
(Fig. 4), the average diameters of CG particles and CG conjugates were equal to 18.5 and 29 nm,
respectively. Thus, the thickness of the adsorbed trypsin shell is about 5 nm. The polymer
adsorption results in a small red shift of the LPR extinction (about 3 nm) and a small increase in
the extinction maximum (about 9%). By contrast, the corresponding change in the scattering
intensity maximum is about 75%. Possibly, some of these changes is due to the imperceptible
aggregation of gold nanoparticles after addition of K2CO3 and trypsin. Nevertheless, this example
clearly proves the advantages of the DLSS technique as a tool for controlling the conjugate
preparation.
Fig. 5 illustrates the time evolution of the extinction and scattering spectra caused by biospecic
aggregation. The conjugate of CG particles (15 nm in diameter) with PrA (Sigma, USA) (CG-15
+ PrA) and immunoglobulin G (IgG, Serva, Germany) was used as a model system. As the PrA
molecule has two or more sites of specic interaction with the IgG molecule [31], addition of IgG

1.6 2.0

Ext
Sca
1.2 1.5
I90

0.8 1.0
A

0.4 0.5
CG
CG+Tr

0.0 0.0
450 500 550 600 650
Wavelength, nm

Fig. 3. Extinction (solid curves) and scattering (dashed curves) spectra measured for 18-nm gold particles before (open circles) and
after (solid circles) conjugation with trypsin. Protocol of conjugation: 35 ml gold sol+1.5 ml 0.2 M K2 CO3 0:5 ml trypsin solution
(1 mg/ml).
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1.0

CG+Tr
d=29 nm
g2() shell~5nm

CG
d=18.5 nm

0.1
0.0 0.1 0.2 0.3
Time, ms

Fig. 4. Autocorrelation functions g2 t measured for 18-nm gold particles before (circles and crosses, two parallel runs) and after
(squares) conjugation with trypsin as described in legend to Fig. 4. The average particle hydrodynamic diameters equal 18.5 and 29 nm,
respectively.

1.5 20

2 1 1 Ext
2 Sca
0
15
3 3
1.0
I90

10
A

0.5
5

0
0.0 0
400 500 600 700 800
Wavelength, nm

Fig. 5. Evolution of extinction (solid curves) and differential scattering (dashed curves) spectra with time: t 0 ( 0, initial sol), 1 (1),
2(1), and 30 min (3). Experimental example for aggregation of conjugates CG-15+PrA after addition of IgG solution.

to a conjugate suspension results in cross-linked binding and biospecic aggregation. The data in
Fig. 5 approximately correspond to the equimolar reagent ratio with respect to binding sites (the
nal concentration of IgG added is 36 mg/ml; the PrA concentration of 5mg/ml approximately
corresponds to the gold number [31]). Comparing the observed variations in extinction and
light-scattering spectra we conclude that the DLSS technique is much more sensitive to biospecic
aggregation than common extinction measurements.
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5. Conclusion

In contrast to the known works on the optics of colloidal gold bioconjugates, in this paper we
reported data on the kinetics of the SLS spectra during nonspecic (data not shown) and
biospecic aggregation. In both cases, we observed an increase in the resonant scattering
maximum by a factor of 20 or even 40, already 12 min after mixing the reagents. For the same
samples, the corresponding changes in the extinction peak were much less signicant. This
observation allows us to suggest that the method described in this work is very promising as an
analytical or diagnostic test of biospecic interactions between colloidal gold conjugates and
target molecules.

Acknowledgements

This work was partially supported by RFBR Grant 04-04-48224, by Russian Education
Ministry Grants A03-2.11-608 and 2.11.03, by CRDF Grant REC-006, and by the President of
Russian Federation Grant NSH-25.2003.2.

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