Analytical Methods
Gold nanoparticle aggregation-based colorimetric assay for b-casein
detection in bovine milk samples
Y.S. Li, Y. Zhou
⇑
, X.Y. Meng, Y.Y. Zhang, F. Song, S.Y. Lu, H.L. Ren, P. Hu, Z.S. Liu, J.H. Zhang
Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis/College of Veterinary Medicine, Jilin University, Changchun 130062, PR China
article info
Article history:
Received 5 November 2013
Received in revised form 9 February 2014
Accepted 12 April 2014
Available online 24 April 2014
Keywords:
b-Casein
Detection
Milk
Gold nanoparticle
abstract
Traditional Kjeldahl method, used for quality evaluation of bovine milk, has intrinsic defects of time-con-
suming sample preparation and two analyses to determine the difference between non-protein nitrogen
content and total protein nitrogen content. Herein, based upon antibody functionalized gold nanoparti-
cles (AuNPs), we described a colorimetric method for b-casein (b-CN) detection in bovine milk samples.
The linear dynamic range and the LOD were 0.08–250
l
gmL
1
, and 0.03
l
gmL
1
respectively. In addi-
tion, the real content of b-CN in bovine milk was measured by using the developed assay. The results
are closely correlated with those from Kjeldahl method. The advantages of b-CN triggered AuNP aggrega-
tion-based colorimetric assay are simple signal generation, the high sensitivity and specificity as well as
no need of complicated sample preparation, which make it for on-site detection of b-CN in bovine milk
samples.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
There are four major caseins (CNs) in bovine milk, namely as1-,
as2-, b-, and
j
-CN. b-CN makes up 37% of the casein content and is
steady (Johansson et al., 2009). Therefore, the quantity of b-CN
could be used as an index to evaluate the quality of bovine milk.
Kjeldahl method is the officially recognized standard method for
quality evaluation of bovine milk (Kamizake, Gonçalves, Zaia, &
Dimas, 2003). However, it has two main problems: the relatively
long testing time and the necessity to carry out two analyses to
determine the difference between non-protein nitrogen content
and total protein nitrogen content. Other reported methods, such
as optical immunosensor based assay (Muller-Renaud, Dupont, &
Dulieu, 2004), electrospray-ionisation mass spectrometric detec-
tion (Gaucheron, Mollé, Léonil, & Maubois, 1995), and chromatog-
raphy method (Bramanti, Sortino, Onor, Beni, & Raspi, 2003;
Bramanti, Sortino, & Raspi, 2002) require complicated handling
procedures or technical expertise.
Mono-dispersed colloidal gold nanoparticles (AuNPs) solution
appears red, which has high extinction coefficients and different
colour (red to blue) in the visible region of the spectrum when
the AuNPs are well-spaced in comparison with when they are
aggregated (Vilela, González, & Escarpa, 2012). Therefore, the
chemical reactions between the analyte and AuNPs surroundings
can lead to a change of colour, which allows the colorimetric assay
of the target. Recently, various AuNPs aggregation-based strategies
have been widely employed in colorimetric analysis, such as thiol-
functionalized cyanuric acid derivative stabilized AuNPs for mela-
mine (Ai, Liu, & Lu, 2009), peptide-capped AuNPs for lysozyme and
cancer diagnosis (Huang, Zhang, Luo, & Zhao, 2012; Kang et al.,
2010), thioctic acid functionalized AuNPs for fumarate (Youk,
Kim, Chatterjee, & Ahn, 2008), N-benzyl-4-(pyridin-4-ylmethyl)
aniline ligand functionalized AuNPs for Cr(III) (Zhao, Jin, Yan, Liu,
& Zhu, 2012), 4
0
-(4-mercaptophenyl)-2,2
0
:6
0
,2
00
-ter-pyridine zinc(II)
complex functionalized AuNPs for PO
4
3+
(He, Zhao, Chen, Liu, & Zhu,
2013), sialic acid stabilized AuNPs for viral detection (Lee, Gaston,
Weiss, & Zhang, 2013), aptamer modified AuNPs for bisphenol A
(Mei et al., 2013), and ethylenediamine-capped AuNPs for trinitro-
toluene (Lin et al., 2012).
Herein, we utilized the mono- (McAb) and polyclonal (PcAb)
antibodies to functionalize AuNPs as colorimetric probe and devel-
oped an AuNPs aggregation-based colorimetric assay for the detec-
tion of b-CN in bovine milk samples. Fig. 1 illustrates the design
strategy. To synthesized probes, AuNPs (20 nm) are functionalized
with McAb and PcAb, respectively. In the presence of b-CN, the
probe 1 (AuNP-McAb) would firstly bind with b-CN. After addition
of probe 2 (PcAb-AuNP), the structure of AuNP-McAb–b-CN–PcAb-
AuNP would be formed. Consequently the wine red colour of the
suspension would be accordingly changed to a blue colour. The
degree of colour change is directly related to the amount of b-CN
which can be easily seen by naked eye and semiquantitative
http://dx.doi.org/10.1016/j.foodchem.2014.04.049
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
⇑
Corresponding author. Tel.: +86 0431 87835734; fax: +86 13634318992.
E-mail address: zhouyu69@sina.com (Y. Zhou).
Food Chemistry 162 (2014) 22–26
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Food Chemistry
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