Sensors and Actuators B 233 (2016) 157–161

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Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

A simple method for detection of anionic detergents in milk using

unmodified gold nanoparticles
Pardeep Kumar a , Piyush Kumar b , Sanjeev Manhas c , Naveen Kumar Navani a,b,∗
a
Centre of Nanotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667 Uttarakhand, India
b
Chemical Biology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667 Uttarakhand, India
c
Department of Electronics and Communication Engineering, Indian Institute of Technology Roorkee, Roorkee, 247667 Uttarakhand, India

a r t i c l e i n f o a b s t r a c t

Article history: Economically Motivated Adulteration (EMA) incidences of milk and infant milk formula by hazardous
Received 6 December 2015 chemicals like urea, detergent and melamine have increased recently. Since milk and milk products are
Received in revised form 28 March 2016 consumed by infants, adults and geriatric population alike, such adulterations need to be tackled on
Accepted 13 April 2016
priority. In the present study, we report a novel approach to detect anionic detergents (ADs) in milk by
Available online 14 April 2016
exploiting the optical and spectral properties of gold nanoparticles (AuNPs). The developed method is
specific for AD detection and is free from interference of other adulterants. The limit of detection for
Keywords:
ADs like sodium dodecylbenzenesulfonate (SDBS) and commercial ADs in milk was 23 and 92 ␮g/ml,
Economically motivated adulteration
Gold nanoparticles
respectively.
Anionic detergent detection © 2016 Elsevier B.V. All rights reserved.

1. Introduction ifestations like acute renal failure, cardiac dysfunction, haemolysis,

ventricular tachycardia and coagulation dysfunctions [12,13]. This
Food fraud and economically motivated adulteration due to paper reports a method for the detection of ADs in milk in order to
addition of cheap constituents results in health risk for unsuspect- prevent fraudulent practices in the dairy industry.
ing consumers [1]. Milk, olive oil, honey, saffron, orange juice, coffee ADs mainly consist of linear alkylbenzenesulfonate (LABS) and
and apple juice are the most common targets for adulteration [2]. have been widely used as surfactants. Various methods have
Amongst all of the above, milk is a major target for EMA in the been proposed for the detection of ADs using spectrophotome-
food and dairy industry [3]. Milk is an indispensable nutritional try [14–18], liquid chromatography [19] and mass spectrometry
food for pregnant women, infants and geriatric population. Adulter- [20,21]. The extant methods for the detection of ADs are based on
ation of milk with synthetic milk made up of hazardous chemicals the principle of interaction of cationic dyes like methylene blue
is a matter of serious health concern and has been reported from and Azure A with ADs followed by extraction into organic solvents
several countries like India, China and USA [4,5]. Synthetic milk [14–18]. Although some of the methods are sensitive, yet use of
is prepared by adding approximately 1.2 g/l of detergent in water sophisticated instruments [21], tedious extractions with carcino-
[6–8]. The adulteration of synthetic milk is done at 5–10% rate in genic organic solvents like chloroform [11], requirement of pH
genuine milk [9]. ‘Synthetic milk’ is made up of low cost and easily adjustment and relatively long sample preparation time render
available ingredients like detergent, urea, melamine, sodium chlo- these methods complex and hazardous to humans and environ-
ride, sodium bicarbonate, vegetable oil and water, which resembles ment [22]. A quick, simple and reliable method to detect AD in milk
pure milk in color and consistency. Detergent is generally used to will hasten screening of milk at small scale dairies and collection
emulsify cheap non-dairy fats for concocting synthetic milk [10]. centres.
ADs are most commonly used for preparation of synthetic milk AuNPs have been widely used as colorimetric sensing platforms
[11]. Detergents interact with cell membrane proteins and form for detecting a wide range of biomolecules based on aggregation
micelles which damage the cell membrane. This leads to toxic man- and dispersion of the nanoparticles [23–25]. In this study a novel,
user-friendly, inexpensive and green approach has been used to
detect the EMA of AD in milk using unmodified AuNPs. This method
∗ Corresponding author at: Department of Biotechnology and Associate faculty overcomes the complex sample preparation steps and is free from
Centre of Nanotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667 the use of harmful organic solvents.
Uttarakhand, India.
E-mail addresses: [email protected], [email protected] (N.K. Navani).

http://dx.doi.org/10.1016/j.snb.2016.04.066
0925-4005/© 2016 Elsevier B.V. All rights reserved.
158 P. Kumar et al. / Sensors and Actuators B 233 (2016) 157–161

2. Methodology 2.6. Limit of detection (LOD)

2.1. Materials and reagents To estimate the detection limit of AuNPs based sensor, 50 ␮l of
processed milk samples spiked with increasing concentrations of
Chemicals such as Chloroauric acid (HAuCl4 ), Trisodium citrate detergent (0–600 ␮g/ml and 0–2000 ␮g/ml for SDBS and commer-
(Na3 C6 H5 O7 ), Sodium chloride (NaCl), Sodium hydroxide (NaOH), cial detergent) with 68 mM NaCl were added into AuNPs solution
Sodium dodecylsulfate (SDS), Sodium bicarbonate (NaHCO3 ), (2.75 nM). UV–vis spectra of each sample were recorded in the
Sodium sulphate (Na2 SO4 ), Aspartic acid (C4 H7 NO4 ), Hydrogen range of 350–750 nm. The limit of detection (LOD) was calcu-
peroxide (H2 O2 ), Lactose, Potassium chloride (KCl), Sodium dode- lated by using the equation LOD = LOB + 1.645(SDLCS ) [27]. The
cylbenzenesulfonate (SDBS), Cetyltrimethylammonium bromide limit of blank (LOB) was calculated by measuring blank sam-
(CTAB) and Triton X-100 were purchased from Sigma Aldrich, USA. ple in three replicates and computing the values in the equation
Commercially available detergent powder and shampoo were pur- LOB = Mean + 1.645(SDLCS ), where SDLCS is the standard deviation
chased from local market. All the solvents were of analytical reagent of low concentration sample.
grade obtained from Merck, Germany.

3. Results and discussion

2.2. Preparation of milk samples
3.1. Detection of anionic detergents (ADs) using unmodified
AuNPs
Milk samples (250 ␮l) were spiked with different concentrations
of AD (0–1000 ␮g/ml) followed by the addition of three volume ice-
Presence of more than 100,000 different molecular species in
cold methanol supplemented with NaCl (68 mM). Spiked samples
milk makes it a complex biological fluid for the detection of individ-
were vortexed for 5 min and centrifuged at 10,000 rpm for 10 min at
ual species [28]. For the analysis of adulterants in the milk, extant
4 ◦ C. Supernatant was collected and used for detection of detergent.
methods use hazardous chemicals viz chloroform, hydrochloric
acid, trichloroacetic acid [11,15,29,30]. We employed a simple and
quick extraction method to eliminate the interfering molecules
2.3. Synthesis and characterization of gold nanoparticles
from milk without using hazardous chemicals. AuNPs were pre-
pared by citrate reduction method and characterized by biophysical
The AuNPs were synthesized by chemical reduction of HAuCl4
techniques (Fig. S1). AuNPs were found to be monodispersed,
using trisodium citrate [26]. The detailed synthesis and character-
spherical in shape with size of ∼13 nm in diameter. The surface
ization are given in Supplementary material [S1.1].
charge of AuNPs was measured to be −44 ± 12 mV which con-
firmed the stability of AuNPs. Further, we used unmodified AuNPs
2.4. Detection of AD using unmodified AuNPs to design a simple colorimetric method to detect the EMA of AD
in milk. The working principle of AuNPs based assay is illustrated
Inhibition of aggregation of AuNPs was observed by color and in Fig. 1. The AuNPs in the solution are stabilized due to elec-
spectral changes for the presence or absence of AD in milk. For trostatic repulsion among negatively charged citrate ions on the
detection of AD, salt concentration was optimized by varying in surface, preventing them from aggregation (red color). Addition
the range of 10–100 mM against a fixed concentration of 2.75 nM of inducer (NaCl) to the AuNPs neutralizes the surface charge and
AuNPs and observing the visual color and spectral changes in AuNPs causes aggregation which is reflected in color change of the solution
solution [23]. We found 68 mM NaCl, 2.75 nM AuNPs (300 ␮l) and from red to purple. However, upon addition of AD spiked sample
50 ␮l of milk sample spiked with AD to be optimal for detection of with optimized NaCl concentration in AuNPs solution, inhibition
AD in assay volume of 350 ␮l. A milk sample without addition of of AuNPs aggregation was observed (Test), due to which the solu-
AD was used as a negative control. tion color remained red (Fig. 2a). The plausible explanation for
prevention of color change is that in the presence of anionic deter-
gent, the salt induced aggregation will be inhibited as negatively
2.5. Specificity and interference studies charged detergent neutralizes the sodium ions (Na+ ). AuNPs got
aggregated and changed its color in the absence of AD (Control
To ensure that the color change was only due to the presence of 2). In the absence of both inducer and AD, AuNPs remained red
AD, the specificity of the assay was investigated by using various (Control 1). The corresponding TEM images also corroborate the
ADs, (SDBS, labolene, SDS, and commercial ADs in the form of deter- inhibition of AuNPs aggregation in the presence of AD and NaCl
gent powder and shampoos) different anionic species (e.g. aspartic (Fig. 2b). To confirm the above observation, UV visible spectra of
acid and sodium sulphate) and representative from cationic (CTAB) AuNPs were measured under the above experimental conditions.
and non-ionic (Triton X-100) detergent category. We also inves- The corresponding shift in surface plasmon absorption spectra also
tigated the performance of AuNPs system in different types of revealed a clear red shift (aggregation) for only NaCl spiked sample
buffalo milk (full cream, toned and double toned) having differ- whereas no such shift was observed for AuNPs in the presence of
ent fat content and milk from different sources such as buffalo, AD (no aggregation) (Fig. 2c).
cow and goat having different fat and protein contents. Milk sam-
ples were spiked with 300 ␮g/ml of each detergent along with NaCl 3.2. Limit of detection (LOD)
(68 mM). 50 ␮l of each sample was added to AuNPs (2.75 nM) and
inhibition of aggregation (color stability) was observed. In addition, For the estimation of LOD, increasing concentrations of AD were
we examined the effect of interfering synthetic milk ingredients spiked in milk followed by addition of inducer (NaCl; 68 mM).
and excipients such as sodium hydroxide (1 mg/ml); sodium bicar- We investigated the sensing system for concentration dependent
bonate (1 mg/ml); urea (60 mM), sodium azide (1%; w/v) hydrogen inhibition of AuNPs aggregation for quantitative AD detection by
peroxide (100 ppm), potassium (1.74 mg/ml) and lactose (4.7% w/v) UV–vis spectroscopy. As shown in Fig. S2, with an increase in con-
in this system. Milk sample spiked with only NaCl (68 mM) was centration of added AD in label-free AuNPs system, the surface
taken as a negative control. plasmon peak blue shifted as compared to control 2. A good linear
 P. Kumar et al. / Sensors and Actuators B 233 (2016) 157–161 159

Fig. 1. Schematic representation of unmodified AuNPs based method for the detection of anionic detergent.

Fig. 2. Colorimetric detection of AD in processed milk sample: (a) Processed milk sample (PMS) + AuNPs (Control 1), PMS + 68 mM NaCl + AuNPs (Control 2) and PMS + 68 mM
NaCl and 300 ␮g/ml AD + AuNPs (Test), (b) corresponding TEM images and (c) UV–vis absorption spectra. (For interpretation of the references to colour in the text, the reader
is referred to the web version of this article.)

was obtained with a regression value (R2 ) of 0.993 (Fig. 3; inset).

The detection limit of the method was calculated to be 23 ␮g/ml. In
case of commercial anionic detergent, response signals increased
linearly in the range of 100–900 ␮g/ml with R2 value of 0.984
(Fig. 3; inset). The limit of detection (LOD) was estimated to be
92 ␮g/ml. A comparison of extant methods of AD detection with
the proposed method shows that the methods involving sophis-
ticated instruments like Gas chromatography-Mass spectrometer
(GC–MS) show higher sensitivity as compared to present method
(Table S1). However, the ease, simplicity, rapidity and use of non-
hazardous extraction method are the hallmark of the proposed AD
sensing method [11,31,32]. Further, the proposed method is col-
orimetric and therefore, will be useful in rapid in-field detection of
EMA of ADs in milk.

3.3. Specificity and interference studies

In order to confirm the specificity, we performed the assay

with anionic, cationic, non-ionic detergents and simple anionic
Fig. 3. Limit of detection of the developed AuNPs based colorimetric assay for molecules such as aspartic acid (C4 H7 NO4 ) and sodium sulphate
anionic detergents: SDBS () and commercial anionic detergent (CAD) (䊏). Plot of (Na2 SO4 ). Inhibition of AuNPs aggregation was observed only in
detergent concentration vs absorbance ratio (A525 /A600 ) for quantification of AD in case of ADs (Figs. 4 a I,II, S3a ). This observation confirmed that
milk. The data from the experiment was used to compute LOD (see text). Inset shows
the proposed method is specific for detection of AD. The obv\ious
the linearly fitted absorption spectra.
distinction between anionic and other category of detergents is
probably due to ability of ADs to neutralize the charge of sodium
correlation was observed while measuring the ratio of absorption ions whereas cationic and non-ionic detergents were not able to
spectra at A525 /A600 nm (Fig. 3). For SDBS, a linear relation between neutralize the charge of sodium ions, thus leading to aggregation.
A525 /A600 nm and increasing concentrations of AD (25–300 ␮g/ml) In addition, to further confirm the color change was specific to
160 P. Kumar et al. / Sensors and Actuators B 233 (2016) 157–161

Fig. 4. Specificity and interference studies of AuNPs sensor. (a)Specificity: (I) Color changes in AuNPs system in presence of (1) PMS + AuNPs (2) PMS + AuNPs + NaCl,
(3) PMS + AuNPs + NaCl + 300 ␮g/ml AD, (4) PMS + AuNPs + NaCl + 300 ␮g/ml cationic detergent (CTAB), (5) PMS + AuNPs + NaCl + 300 ␮g/ml non-ionic detergent (Triton X-
100), (6) PMS + AuNPs + NaCl + 300 ␮g/ml aspartic acid and (7) PMS + AuNPs + NaCl + 300 ␮g/ml sodium sulphate; (II) corresponding TEM images; (b) Interference: (I) Color
changes in AuNPs system in presence of (1) PMS + AuNPs, (2) PMS + AuNPs + NaCl, (3) PMS + AuNPs + NaCl + 300 ␮g/ml AD + 1 mg/ml NaOH, (4) PMS + AuNPs + NaCl + 300 ␮g/ml
AD + 1 mg/ml NaHCO3 , (5) PMS + AuNPs + NaCl + 300 ␮g/ml AD + 60 mM urea, (6) PMS + AuNPs + NaCl + 300 ␮g/ml AD + 1% sodium azide, (7) PMS + AuNPs + NaCl + 300 ␮g/ml
AD + 100 ppm H2 O2 , (8) PMS + AuNPs + NaCl + 300 ␮g/ml AD + 1.36 mg/ml potassium and (9) PMS + AuNPs + NaCl + 300 ␮g/ml AD + 4.7% lactose; (II) corresponding TEM images.

added ADs, we also tested milk from different species such as buf- cient than that of organic dyes [33]. This is also reflected in higher
falo (4.63% protein), cow (2.82% protein) and goat (3.48% protein) dynamic detection range (for SDBS, 23–300 ␮g/ml and for CAD,
for checking the effect of protein variation on the performance of 92–900 ␮g/ml) of our method as compared to earlier reported
the sensor. In addition, we have also tested efficacy of the method methods. The limit of detection of method is 23 ␮g/ml and 92 ␮g/ml
with milk having different fat contents [whole milk (6% fat), toned for SDBS and commercial ADs, respectively. Our method relies on
(3% fat) and double toned milk (1.5% fat)]. The AuNPs system is able prevention of salt induced aggregation of AuNPs in the presence of
to differentiate ADs adulterated milk with non-adulterated natural ADs, where ADs protect the AuNPs and cause a red shift in surface
milk samples (Fig. S4). plasmon resonance absorption. The proposed method has advan-
The method was tested for its performance in the presence of tage of being environment friendly and replaces use of carcinogenic
excipients, intrinsic molecules of natural milk and common com- chemicals like chloroform. The sensing method does not require
ponents of synthetic milk (hydrogen peroxide, lactose, potassium, specialized costly instrumentation and is thus easily adaptable at
sodium hydroxide, sodium bicarbonate, sodium azide and urea). small scale dairies and milk collection centres.
No color change (aggregation) was observed upon addition of dif-
ferent interfering agents with AD in the milk samples (Figs. 4 b I,II Acknowledgements
and S3b). Taken together, above results show that the developed
method is specific towards AD and is free from interference from This research was supported by National Agricultural Innova-
other components of synthetic milk and common adulterants. tion Project (NAIP) Grant no. C4-30032 of the Indian Council of
Agricultural Research (ICAR) funded by Government of India to
3.4. Validation of the method NKN. Pardeep Kumar and Piyush Kumar are thankful to Ministry
of Human Resource Development (MHRD), Government of India
The recovery assay was performed to test the validation of the for financial support.
proposed method for determination of AD in milk samples. For this,
milk samples were spiked with different concentrations (for SDBS, Appendix A. Supplementary data
75, 150 and 250 ␮g/ml; for commercial anionic detergent, 200, 400
and 600 ␮g/ml) of detergent and recovery of added detergent was Supplementary data associated with this article can be found, in
quantified using the standard curve for AD. The mean recoveries the online version, at http://dx.doi.org/10.1016/j.snb.2016.04.066.
of ADs in milk samples ranged from 97.5 ± 1.05% to 101.1 ± 1.85%
(Table S2), indicating the reliability and practicality of the method. References

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by gas chromatography/mass spectrometry, Anal. Chem. 62 (1990) ing as Associate Professor in Department of Electronics, IIT Roorkee. His research
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