Food Control 56 (2015) 135e146

Contents lists available at ScienceDirect

Food Control
journal homepage: www.elsevier.com/locate/foodcont

Review

Rapid methods for detecting acrylamide in thermally processed foods:

A review
Qinqin Hu a, Xiahong Xu b, Yingchun Fu a, Yanbin Li a, c, *
a
College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China
b
Key Lab for Pesticide Residue Detection, Ministry of Agriculture, Institute of Quality and Standards for Agricultural Products, Zhejiang Academy of
Agricultural Sciences, Hangzhou 310021, China
c
Department of Biological & Agricultural Engineering, University of Arkansas, Fayetteville, AR 72701, USA

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

Article history: Acrylamide (AA), a neurotoxin and potential carcinogen, has been found in various thermally processed
Received 22 October 2014 foods such as potato chips, biscuits, and coffee. LCeMS/MS and GCeMS as standard detection methods
Received in revised form show high sensitivity, selectivity, stability, and repeatability. However, these methods require expensive
15 March 2015
instruments, skilled technicians in laboratories, and high testing costs, and cannot meet the needs for
Accepted 16 March 2015
Available online 1 April 2015
real-time and on-line detection of AA in foods. Therefore, rapid detection methods with merits of
simplicity and portability such as computer vision, ELISA, electrochemical biosensing, and fluorescent
biosensing have obtained an increasing amount of attention. Reported research on rapid methods has
Keywords:
Acrylamide
shown similar sensitivity and selectivity, but requires less time and cost in comparison with standard
Detection methods through the use of nanomaterials and biomolecules with high affinity to AA. These improve-
Rapid methods ments show great promise for high-throughput, real-time, and on-line detection of AA. This paper
Computer vision provides a comprehensive overview of rapid detection methods for AA in foods with comparison be-
ELISA tween rapid and standard methods. Meanwhile, suggestions for further research on rapid methods for
Electrochemical biosensing detecting AA are also discussed based on technical challenges and industry needs.
© 2015 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
1.1. Toxicity of AA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
1.2. Existence and formation of AA in foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
1.3. Standard and conventional methods for the detection of AA in foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
2. Rapid detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
2.1. Color indicating methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
2.2. Enzyme-linked immunosorbent assay (ELISA) methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
2.3. Supramolecular recognition based methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
2.4. Electrochemical biosensing methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
2.5. Fluorescent sensing methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
3. Sample pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
3.1. Sample pretreatment protocols used in standard detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
3.2. Sample pretreatment protocols used in rapid detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
4. Comparison and prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
4.1. Comparison of rapid detection methods with standard methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
4.2. Comparison of different rapid detection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

* Corresponding author. C523 Nongshenghuan Building, College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China. Tel.: þ86 571
88982536; fax: þ86 571 88982530.
E-mail address: yanbinli@zju.edu.cn (Y. Li).

http://dx.doi.org/10.1016/j.foodcont.2015.03.021
0956-7135/© 2015 Elsevier Ltd. All rights reserved.
136 Q. Hu et al. / Food Control 56 (2015) 135e146

4.3. Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

1. Introduction et al., 2009). Workers who experience occupational exposure to

AA suffer from the damage of both peripheral and central nervous
Acrylamide (AA, 2-propenamide, C3H5NO (71.09 g mol1, CAS No. systems, since the neurotoxic effects of AA are cumulative and
79-06-1)) is a colorless and odorless crystalline produced by the chronic (Huang et al., 2011; Pennisi et al., 2013). The no-observed-
hydration of acrylonitrile. It dissolves in water, alcohol, and other adverse-effect level for morphological changes in the nerve sys-
polar solvents, but tends to be hydrolyzed into acrylic acid in an acidic tems of rats was 200 mg kg1 b.w. per day. Tardiff, Gargas, Kirman,
and alkali environment. Polyacrylamide can be easily produced at Leigh Carson, and Sweeney (2010) reported that the tolerable daily
melting point or under ultraviolet light, which is widely used in in- intake (TDI) of AA was 40 mg kg1 per day for neurotoxicity, and
dustrial and academic fields (Friedman, 2003). Increasing research on 2.6 mg kg1 per day for cancer.
AA in foods has been investigated in the past thirty years, involving
toxicity, formation, mitigation, and detection, as shown in Fig. 1. 1.2. Existence and formation of AA in foods

1.1. Toxicity of AA In April 2002, Swedish National Food Agency and researchers
from Stockholm University reported a large amount of AA was
In 1994, International Agency for Research on Cancer (IARC) found in some thermally processed foods. Since then, AA has
classified AA as “Group 2A” (probably carcinogenic to humans) received broad concerns from FAO/WHO, JECFA, FDA, other national
(Lyon, 1994), due to its neurotoxicity, carcinogenicity, and geno- food agencies, and other research institutions.
toxicity (Erkekoglu & Baydar, 2010; Hogervorst et al., 2010). Sub- AA exists in different food products at different levels. A report
sequently, AA was classified as a Category 2 carcinogen and on monitoring AA levels in food from 2007 to 2010 by the European
Category 2 mutagen by European Commission (EC, 2002) as well as Food Safety Authority (EFSA) showed that mean AA levels range
a substance of “very high concern” by European Chemical Agency in from 31 mg kg1 (processed cereal based foods for infants and
2010 (ECHA, 2010). young children) to 1350 mg kg1 (coffee substitutes) (EFSA, 2011).
The 72nd meeting of the Joint FAO/WHO Expert Committee on The trend analysis did not show obvious changes in AA levels
Food Additives (JECFA) have systematically summarized and during the past four years. Fried potatoes (~272e570 mg kg1),
updated the studies of AA throughout the world since their 64th bakery products (~75e1044 mg kg1), and coffee and its substitutes
meeting (JECFA, 2005, 2011). The resulted document indicated that (~229e890 mg kg1) were still the top three categories of foods
multi-organ tumors were discovered in experimental animals after containing AA, which were also the major contributors to adult AA
the exposure to AA, although no significant association between AA exposure. The levels for mean and high dietary exposure to AA were
dietary intake and an increase in multiple cancers has been 1 and 4 mg kg1 b.w. per day (JECFA, 2011), respectively, as repre-
established from epidemiological research (Friedman, Dulak, & sented at the 64th JECFA meeting (JECFA, 2005). Based on long-
Stedham, 1995; Johnson et al., 1986). The genotoxicity of AA can term monitoring, the indicative values of AA in 10 food categories
be presented in two ways. First, AA can be converted to its were proposed in 2011 and updated in 2013, which may be a step to
metabolite glycidamide, which is 3 times higher in mutability set regulatory limits in the next several years (EU, 2011, 2013).
compared to AA and can induce point mutations in various systems AA has been found in numerous carbohydrate-rich foods after
(Fazendeiro, 2013). Secondly, AA can act as a Michael acceptor to being fried, baked, or roasted at a temperature higher than 120  C.
form adducts with thiol, hydroxyl, and amino groups in DNA, which The well-accepted mechanism of AA formation in foods is the
leads to DNA damage (Doroshyenko et al., 2009; Erkekoglu & asparagine-reducing sugars (usually fructose and glucose) pathway
Baydar, 2010; Hogervorst et al., 2010; Watzek et al., 2012; Zeiger accompanied by the Millard reaction, wherein an intermediate
Schiff base is first formed, followed by AA formation via the
Strecker pathway or N-glycoside pathway (Claeys, De
Vleeschouwer, & Hendrickx, 2005; Mottram, Wedzicha, &
Dodson, 2002; Stadler et al., 2002). Another pathway that has
been reported is the acrolein pathway after organic acid decar-
boxylation (Medeiros Vinci, Mestdagh, & De Meulenaer, 2012;
Yaylayan & Stadler, 2005). The research indicated raw materials
(cultivars, the contents of free amino acid and reducing sugars) and
processing parameters (heating duration, heating temperature, and
water activity) influence the level of AA in processed foods (De
Vleeschouwer, Plancken, Van Loey, & Hendrickx, 2007, 2010;
Halford et al., 2012; Muttucumaru et al., 2008; Wicklund et al.,
2006).

1.3. Standard and conventional methods for the detection of AA in

foods

Liquid Chromatography (LC) and high performance liquid

Fig. 1. Recent researches on AA in thermally processed foods. chromatography (HPLC) coupled with mass spectrometry (MS) are
 Q. Hu et al. / Food Control 56 (2015) 135e146
the most preferred methods for separation and quantification of AA
in foods due to their sensitivity, selectivity, and versatility. Profi-
ciency tests (n ¼ 10) organized by the Food Analysis Performance
Assessment Scheme (FAPAS) revealed that LCeMS/MS is a reliable
method and has broad applications (Tekkeli, Onal,€ €
& Onal, 2012).
Chen, Yuan, Liu, Zhao, and Hu (2008) located AA in 349 food
products obtained from the Chinese market using the LCeMS/MS
method with excellent sensitivity and a wide detection range.
Ultra-performance liquid chromatography (UPLC) allows better
separation of AA in food matrices within a shorter retention time
and higher sensitivity (Zhang, Jiao, Cai, Zhang, & Ren, 2007). A
UPLC-ESP (electrospray)-MS/MS method was reported for the
detection of AA in foods, which supplied a rapid quantitative pro-
cedure for AA within a run time of only 3 min, along with good
repeatability (RSD  4.5%) within a day (n ¼ 5) and day-to-day
(n ¼ 10) precision tests (Zhang et al., 2007). Recently, Zhang, Ren,
Jiao, Li, and Zhang (2011) expanded UPLC-MS/MS to successfully
validate and apply to simultaneous determination of AA and its
precursors and intermediates during Maillard reactions and kinetic
elucidation.
Both gas chromatography (GC) based methods with and without
derivatization were developed, showing satisfactory agreement
with LCeMS on detecting AA in various foods. Due to its high po-
larity and low volatility, AA can derivatize with potassium bromate
and potassium bromide in order to improve the volatility and
selectivity. A derivatized GC coupled with mass spectrometry
(GCeMS) method was proposed for the detection of trace levels
(<50 mg kg1) of AA in cereal-based foods due to its high sensitivity
(LOD ¼ 2 mg kg1) and great recoveries (93e104%) (Pittet, Pe risset,
& Oberson, 2004). Some research omitted the derivatization step
and measured AA directly after extraction and purification. Lee,
Chang, and Dou (2007) detected AA in aqueous matrices using
direct immersion solid-phase microextraction (SPME) coupled with
gas chromatographyepositive chemical ionization tandem mass
spectrometry (GCePCI-MSeMS), which showed ultrahigh sensi-
tivity (0.1 mg kg1).
Capillary electrophoresis (CE) is a powerful alternative for
analyzing organic compounds based on charge-to-mass differences
with high separation efficiency. Two in-line preconcentration
capillary zone electrophoresis (CZE) methods (field amplified
sample injection (FASI) and stacking with sample matrix removal
(LVSS)) have been evaluated for the analysis of AA in foodstuffs after
being derivatized with 2-mercaptobenzoic acid, both of which
showed similar sensitivity and precisions compared with
chromatography-based methods (Bermudo, Nún ~ ez, Moyano,
Puignou, & Galceran, 2007; Bermudo, Nún ~ ez, Puignou, &
Galceran, 2006). A laser-induced fluorescence detection method
mediated by CE with CdTe quantum dots (QDs) as amplifier was
studied for the detection of AA in potato crisps with good recoveries
(90e95%) and RSD (<5.7%) (Chen, Zhao & Fung, 2011).
The 72nd meeting of JECFA (2011) evaluated the majority of
validated and fit-for-purpose methods, including LCeMS/MS with
isotope dilution and GCeMS or GCeMS/MS after derivatization,
which are available for the analysis of AA in most relevant foods.
Most organizations and governments also have set or recom-
mended these methods as standard methods for the detection of
AA in thermally processed foods, as summarized in Table 1.
Applications of standard or conventional methods in detecting
AA have been reviewed elaborately for the past decade by many
researchers (Keramat, LeBail, Prost, & Soltanizadeh, 2011; Lineback,
Coughlin, & Stadler, 2012; Oracz, Nebesny, & Zy _ zelewicz,
_ 2011;
Tekkeli et al., 2012; Wenzl, De La Calle, & Anklam, 2003; Zhang,
Ren, & Zhang, 2009; Zhang, Zhang, & Zhang, 2005). However,
only some reviews (Oracz et al., 2011; Zhang, Ren, et al., 2009)
involved rapid detection methods, which lack systematical and
137
comprehensive analysis. Therefore, the objective of this review is to
provide a scientific overview of rapid methods for the detection of
AA in thermally processed foods and to give a comprehensive
comparison with standard methods. Meanwhile, some suggestions
for future research on rapid methods are also discussed based on
current challenges.
2. Rapid detection methods
Unlike LCeMS/MS and GCeMS, rapid detection methods are
mainly based on biochemical properties of AA, biomaterials with
high and specific interaction with AA, or changes of physiochemical
properties of thermally processed foods related to AA. The standard
and rapid methods for detection of AA in foods are summarized in
Table 2.
2.1. Color indicating methods
The formation of AA is accompanied with color changes called
the browning process in the Maillard reaction of reducing sugars
with asparagine (Mottram et al., 2002; Stadler et al., 2002). Some
studies have demonstrated the correlation between browning
levels and AA concentrations in different food products, such as
potato chips (Majcher & Jelen  , 2007; Pedreschi et al., 2007), coffee
(Go€ kmen & Şenyuva, 2006), and French fries (Pedreschi, Moyano,
Kaack, & Granby, 2005). Early research on measuring food color
adopted the L*a*b* system, an international standard for color
measurement, where L* is the luminance or lightness component,
a* (from green to red) and b* (from blue to yellow) are the two
chromatic components (Yam & Papadakis, 2004). Go € kmen and
Şenyuva (2006) reported a relationship between redness param-
eter (a*) changes and AA levels in green coffee, wheat flour, and
potato chips during heating. Therefore, the redness parameter (a*)
can be used as an indicator to evaluate AA levels in thermally
processed products.
However, for the uneven surfaces of food samples, solely using
the redness parameter cannot get accurate data to reveal the con-
tent of AA. Therefore, computer vision is proposed to solve this
problem based on classification, measurement with various algo-
rithms through image analysis, and a correlation between the
brown ratio and AA concentration during the Maillard reaction
(Go€ kmen & Mogol, 2010; Go € kmen, Şenyuva, Dülek, & Cetin, 2007;
Go€ kmen et al., 2006; Mogol & Go € kmen, 2013). Fig. 2 shows the
constituent elements of computer vision for monitoring the con-
tent of AA in potato chips. Specifically, Go€kmen et al. (2006) clas-
sified fried potato images into three segments: bright yellow (in
web version) (Region 1), yellowish brown (in web version) (Region
2), and darker brown (in web version) (Region 3) according to
image pixels with different mean values of red, blue, and green
components, corresponding to three sets of pixels (set1, set 2, and
set 3). A calibration curve and a linear regression equation were
established to evaluate the AA levels in potato chips according to
the correlation between AA concentration and the ratio of the
number of pixels in Set 2 to the total number of pixels of the entire
image (the normalized area of Set 2, NA2). This method based on
the ratio of browning as an indicator was also verified for detection
of AA in cookies regardless of the recipe formulation and baking
conditions (Go € kmen, Açar, Arribas-Lorenzo, & Morales, 2008).
In summary, detection of AA based on browning is easy to
operate and independent from complex instruments. Through
taking pictures of food samples and data analysis during process-
ing, the content of AA can be monitored without any pretreatment.
Besides, by optimizing the number of cameras and the interval of
detection, this method can achieve the goal of on-line, high-
throughput, and real-time monitoring. Different threshold values
138 Q. Hu et al. / Food Control 56 (2015) 135e146

Table 1
Standard detection methods issued or adopted by organizations and countries.

Countries/Organizations Standard methods References

FDA LCeMS/MS with13C3-labeled AM as internal standard and OASIS HLB 6 mL SPE cartridge for purification (FDA, 2003)
Joint FAO/WHO GCeMS with a LOD of 5e10 mg kg1; LCeMS/MS with a LOD of 20e50 mg kg1 (FAO/WHO, 2002)
EC GCeMS and LCeESIeMS/MS in potato-based products, cereal based products and coffee (EC 2010a, 2010b)
Sweden LCeMS/MS (NFA, 2002)
UK GCeMS (UK, 2010)
Australia & New Zealand GCeMS/MS (FSANZ, 2014)
China GCeMS with a LOD of 7 mg kg1 (China, 2005)

Note: FDA, Food and Drug Administration; FAO, Food and Agriculture Organization; WHO, World Health Organization; EC, European Commission; NFA, National Food Agency;
UK, the United Kingdom; FSANZ, Food Standards in Australia and New Zealand.

can be set according to different processed foods. The browning
based method is susceptible to many factors, such as intensity of
light, shape and flatness of food samples, focal length, aperture, and
other parameters of taking photos. Therefore, compared with other
novel methods, this method may be more suitable to on-line and
real-time preliminary screening of AA during food processing.
2.2. Enzyme-linked immunosorbent assay (ELISA) methods
ELISA is a rapid method based on the recognition of anti-
geneantibody binding with high specificity and affinity, as well as
the signal readout through optically detecting colored products
catalyzed by enzyme labels. Because of the high specificity/affinity
of antigeneantibody recognition and the high efficiency of enzy-
matic catalysis, ELISA has many advantages such as good sensitivity,
selectivity, high-throughput, and its ability of coupling with other
technologies, for example, biotin-avidin amplification and chem-
iluminescence. Therefore, ELISA has attracted increasing attention
in detecting AA in foods. In this new field, developing of appro-
priate antigens to obtain high-affinity antibodies and signal
amplification are two key points. Fig. 3 shows the schematic rep-
resentation for the preparation of complete antigen, antibody, and
ELISA for AA detection.
AA lacks strong epitope groups and shows no immunogenicity
due to its low molecular weight. Therefore, the complete antigen
should be synthesized first via conjugating AA with some immune-
stimulating carrier proteins. Then, the antibody of AA can be pro-
duced via immunoreactions stimulated by the complex antigen.
Specifically, the linkage between AA and the carrier protein as well
as the choice of appropriate hapten are two key issues. So far, three
approaches have been reported as follows.
The first approach is using glutaraldehyde for conjugating. The
two aldehyde groups at the terminals act as a bridge to connect
amino groups of the protein and AA through generating a Schiff
base (Chan, Husseinsyah, & Sam, 2013). Zhang, Gao, et al. (2009)
and Fu et al. (2011) utilized this method to couple AA with bovine

Table 2
Applications of standard methods and rapid methods for detecting AA in thermally processed foods.

Method Sample Linear range LOD LOQ Recovery RSD Reference

(mg kg1) (mg kg1)

Standard LCeMS/MS Potato chips 1 ~ 200 1 3 81.6e99.0% 0.4e4.5% (Zhang et al., 2007)
Methods (HPLC, UPLC) Coffee 2 ~ 100 5 16 92e95% <5% (Bortolomeazzi et al., 2012)
Cereal-based foods 1 ~ 2,000 6 18 90.6e98.5% 1.8% (Şenyuva & Go €kmen, 2006)
(breakfast cereal, cookies) (mean)
Tea 1 ~ 20 1 5 74e79% 1.6e8.3% (Liu, Zhao, Yuan, Chen, & Hu, 2008)
Infant foods 0.1~200 1 3 87e96% <6.5% (Zhang et al., 2005)
GCeMS Potato chips 10 ~1,000 5 e 81.9e95.7% 5.3e13.4% (Yamazaki et al., 2012)
(GC based) Coffee 0 ~ 1,500 5 10 84e97% 2e10% (Soares, Alves, Casal, Oliveira, &
Fernandes, 2010)
Cereal-based foods 5 ~ 50,000 2 36 91e99% <4% (De Vleeschouwer et al., 2007)
(biscuits, cracker,
breakfast cereals)
French fries 30~10,000 1 25 >96% <2% (Notardonato, Avino, Centola,
Cinelli, & Russo, 2013)
Instant noodles 10 ~ 5,000 5.1 13 87.3% (mean) 1.6% (Yamazaki et al., 2012)
Rapid Electrochemical Potato crisps 9.2  104 ~ 8.5  103 e e e (Stobiecka et al., 2007)
Methods Biosensors 3.4  103
Potato crisps 0.35~ 5.3  106 1.4  102 e 95.40e97.56% e (Batra, Lata, Sharma et al., 2013)
No food detected 0.71~710 2.84 7.1 e e (Garabagiu & Mihailescu, 2011)
ELISA Pringles crisps 51.76~3,311.5 65.7 e e e (Preston et al., 2008)
Mashed potatoes 50 ~ 1,280 50 350 92.6e95.5% e (Fu et al., 2011)
Potato crisps, instant 26.3 ~ 221.1 18.6 60.6 74.4e98.1% e (Quan et al., 2011)
noodles, biscuits, and cakes
Potato fries, biscuits 10 ~ 100,000 6 e 90e110% 6.3e9.9% (Zhou et al., 2008)
Potato chips, cookies, 0.25 ~ 24.15 0.036 e 73.7e107.7% 3.6e19.2% (Wu et al., 2014)
and coffee
Fluorescence Potato chips 35 ~ 350,000 35 e e e (Hu et al., 2014)
French fries, fried puffs, 50 ~ 20,000 15 e 66.0e110.6% e (Liu et al., 2014)
fried chicken roll, bread,
biscuits
Correlation coefficient Prediction accuracy Reference
Computer vision Potato chips 0.989 98% at a threshold of 1000 mg/kg € kmen et al., 2006)
(Go
Cookies 0.946 100% at a threshold of 150 mg/kg € kmen et al., 2008)
(Go
 Q. Hu et al. / Food Control 56 (2015) 135e146 139

Fig. 2. Schematic representation of the computer vision for monitoring the content of AA in potato chips.

serum albumin (BSA) to synthesize an artificial antigen. This
method is simple and convenient, but may result in low efficiency
and even cause further loss of antigenic epitopes (Zhou, Zhang,
Wang, & Zhao, 2008). For example, Quan, Chen, Zhan, and Zhang
(2011) coupled AA with keyhole limpet hemocyanin (KLH) via
glutaraldehyde, but no antibody was obtained because of the low
antiserum titers during immunoreactions in vivo of rabbits.
The most common method uses active ester to combine AA and
proteins, such as 1-ethyl-3-(3-dimethylaminopropyl)-carbodii-
mide (EDC) and N-hydroxysuccinimide (NHS). Studies showed the
length of linker between the “AA-group” and the carrier protein is
the key issue in obtaining antibody with high titer (Preston, Fodey,
& Elliott, 2008; Wang et al., 2012). Preston et al. (2008) used AA and
two molecular mimics of AA (acrylamidoglycolic acid and acryloyl-
X short for 6-((acryloyl)amino) hexanoic acid) to conjugate the
carrier protein via Michael addition and the EDC/NHS cross-link
reaction. Only the acryloyl-X immunogen could produce anti-
bodies with high affinity to acryloyl-X rather than AA, which
indicated AA needed to be derived to a larger molecule before
detection. Inspired by the research above, the polyclonal antibody
of AA was produced by stimulating the derivate of AA and 3-
mercaptobenzoic acid (Ade-3-MBA). Similar research was carried
out by Wang et al. (2012) who synthesized three kinds of AA
artificial antigens using EDC/NHS. One approach was to couple the
carboxyl of acrylic acid and a linker arm (acryloyl aminobutyric
acid) to proteins, and the other was to use Ade-3-MBA as hapten
which possessed higher specificity. Recently, a polyclonal antibody
was produced against 4-mercaptophenylacetic acid (4-MPA)-

Fig. 3. Schematic representation of the preparation of complete antigen, antibody and competitive indirect ELISA for AA analysis.
140 Q. Hu et al. / Food Control 56 (2015) 135e146
derivatized AA and also showed high affinity to the derivative (Wu
et al., 2014). Therefore, by attaching AA or its mimics to proteins via
longer molecular terminals, more immunogenic orientations can
be projected from the protein surface, resulting in higher proba-
bility of successful antibody production. However, the approach
using EDC/NHS is more complicated than the other two ap-
proaches, and needs to introduce other chemical groups, increasing
the detection time due to the derivatization of AA with bridge
molecules before analysis.
The third method is using N-acryloxysuccinimide (NAS) as the
hapten (Quan et al., 2011; Zhou et al., 2008). NAS contains the
structures of AA and N-hydroxysuccinimide (NHS), which favors
NAS to react with the amino group and conjugate AA to the carrier
protein directly (Jameson & Wong, 2009). Zhou et al. (2008) used
this method to synthesize a complete antigen with a high anti-
bodyeantigen association constant (kaff ¼ 6.7  107 L mol1), which
is comparable with antibodies stimulated by haptens with molec-
ular weights in the range from 200 to 300 Da. This method shows
advantages of high coupling efficiency, no requests of other re-
agents or activations, and preservation of the intact structure of AA
in the conjugation.
Although antibodies of AA have shown satisfactory affinity, the
requirement of highly sensitive detection still needs signal ampli-
fication. To date, several signal amplification methods based on
biotin-(strept)avidin system and enhanced chemiluminescent
system are investigated. The biotin-(strept)avidin system is based
on the interaction between avidin or streptavidin and biotin with
an affinity constant of around 1015 L mol1 Zhou et al. (2008)
established a biotin-avidin amplifying ELISA (BA-ELISA) to detect
AA with a similar wide linear range and sensitivity compared with
standard methods, as shown in Table 2. Moreover, BA-ELISA
showed a good consistency with the HPLC method in detecting
AA in fried potatoes and biscuits. Using chemiluminescence in
ELISA, especially luminaleH2O2ehorseradish system, the sensi-
tivity was enhanced two to three orders than the that in conven-
tional ELISA. The introduction of chemicals (such as r-iodophenol)
can render the intensity of chemiluminescence to increase
dramatically (one to two orders of magnitude). Moreover, the light
intensity of enhanced chemiluminescent (ECL) can reach its
maximum in only 1e2 min, which provides the possibility of rapid
detection. A direct ECL-ELISA method was designed to detect AA
using luminol, r-iodophenol, and H2O2, which displayed faster
response but lower sensitivity than standard methods (Quan et al.,
2011).
Compared with common analytical methods, ELISA is a method
with similar sensitivity, lower cost, shorter detection time, more
simplicity, and does not requires expensive equipment or complex
sample preparation. Therefore, ELISA kits available for commercial
application are of high interests and possibility. For example, an
ELISA kit was investigated showing good recovery (from 91.8% to
96.0%) with a LOD and a linear range of 5 mg kg1 and
10e10,000 mg kg1, respectively (Franek, Rubio, Diblikova, & Rubio,
2014). However, ELISA still needs to be improved and the analytical
results should be confirmed by other robust methods. Meanwhile,
how to obtain antibodies of AA with high stability and affinity is still
the key issue in the future.
2.3. Supramolecular recognition based methods
A method that used supramolecules as the recognition elements
to detect AA was studied by Kleefisch, Kreutz, Bargon, Silva, and
Schalley (2004). A supramolecule composed of two or more mol-
ecules has excellent integrity and subtle microscopic structure
making it possible to provide specific sites for detection of AA
(Steed & Atwood, 2009). Kleefisch et al. (2004) designed a
supramolecule that consisted of tetralactam macrocycles with high
affinity to AA via a cavity binding site, followed by determining the
changes in resonant frequency of the oscillating quartz chip
modified by the supramolecule. Piezoelectric sensors based on
tetralactam displayed high sensitivity (LOD of 10 mg kg1) and
selectivity (negligible interference from co-existing compounds).
However, no food samples have been tested for the applications of
this method.
The highly selective and stable recognition between the “host”
supramolecule and “guest” molecule is attributed to intermolecular
interaction and macrocyclic effect. Recently, the molecular
imprinting technique (MIT) which is based on the principle of su-
pramolecular chemistry and regarded as “chemical antibody”, has
been applied in liquid chromatography, solid phase extraction, and
sensing in food analysis fields (Chen, Xu, & Li, 2011). Therefore,
application of MIT in food sample pretreatment and recognition
shows promise for the rapid detection of AA in foods.
2.4. Electrochemical biosensing methods
A biosensor is a device used to detect an analyte that combines a
biological component (bioreceptor) with a physicochemical de-
tector (transducer) (Turner, Karube, & Wilson, 1987). Electro-
chemical biosensors, with electric signal (such as current and
potential) output, have shown advantages such as rapidity,
simplicity, automation, and sensitivity, leading to broad applica-
tions in food safety.
Electrochemical biosensors have been proposed to detect AA in
foods or complex matrices only in the past few years. The first trial
for determining AA in wastewater was based on biocatalysis from
microbial metabolism, including respiration and enzyme reactions.
Ignatov, Rogatcheva, Kozulin, and Khorkina (1997) first quantified
AA by defining specific respiratory activity (SRA) as the difference
between the rate of oxygen consumption of Brevibacterium sp. and
endogenous cell respiration after introduction of AA. The reduction
of the concentration of oxygen and the current created from the
metabolism of AA could be used to detect AA. Silva, Gil, Karmali,
and Matos (2009), Silva, Matos, Karmali, and Rocha (2011)
designed an electrochemical biosensor based on the electron
transfer of a direct biochemical interaction between AA and whole
cells of Pseudomonas aeruginosa containing intracellular amidase,
which catalyzed the hydrolysis of AA producing ammonium ion
and acrylic acid. Using biochemical reactions from microbial
metabolism can lead to real-time detection of AA. Good stability, a
long working life, and containing various enzymes in cells are the
advantages of microbial biosensors. However, the dependence on a
living organism makes it have long time responses. Also, protecting
the bacteria against pollution from the environment is another
problem.
Another detection principle is based on the catalysis of the
reduction of AA with the assistance of electrolytes, such as LiCl and
cobalt(Ⅱ) ions (Niaz et al., 2008; Zargar, Sahraie, & Khoshnam,
2009). Specifically, AA was reduced with the catalysis of the Co
ion accompanied by electron transfer and finally changes in current
for signal readout (Zargar et al., 2009). This method has strengths of
high sensitivity (LOD of 3.52 mg kg1) and rapid responses. How-
ever, anti-interference should be improved to meet the need for
detecting AA in complex food samples.
In most cases, AA is proposed to combine with a redox-active Hb
(Hb-Fe(Ⅲ) to Hb-Fe(Ⅱ)) to form an adduct, which enlarges the
distance between the redox center of Hb and the electrode, sup-
pressing the redox current (Friedman, 2003; Lineback et al., 2012).
Efficient conjugation between AA, Hb, and the redox response of
electrochemistry endow this method with high sensitivity, high
selectivity, rapid response, and a wide linear range (Stobiecka,
 Q. Hu et al. / Food Control 56 (2015) 135e146 141

Radecka, & Radecki, 2007), as listed in Table 2. However, the

immobilization efficiency of Hb on the electrode, including the
loading and activity of Hb and the influence on the electron transfer
needed much improvement, as discussed later.
For the electrochemical biosensors using Hb, highly efficient
immobilization of Hb and the electron transfer on the surface of the
working electrode are two key issues. However, the redox center of
Hb is located inside of insulating polypeptide shell, which greatly
inhibits the electron transfer (Sun et al., 2013). Meanwhile, a
greater amount of immobilized Hb should provide more adducting
sites for AA to enhance the signal readout. Therefore, novel mate-
rials and methods to enhance the immobilization and electron
transfer of Hb are highly desired.
Nanomaterials, especially carbon nanomaterials and metal
nanoparticles (MNP, such as Au, Ag, Pt, etc.), have shown extensive
applications to electrode modification due to their unique electric
properties. High surface-to-volume ratio and abundant functional
groups (for example, carboxyl and carbonyl groups on carbon
nanotubes) enable more Hb to be immobilized on the electrode,
leading to higher signal response. Nanomaterials can be used as
mediators between Hb and the electrode to accelerate the electron
transfer due to their excellent electrical conductivity, leading to a Fig. 4. Schematic representation of chemical reaction involved in the fabrication of Hb/
cMWCNT/CuNP/PANI/PG electrode (Batra, Lata, Sharma, et al., 2013). Adapted with
rapid signal response (Dreyer, Park, Bielawski, & Ruoff, 2010; Lee,
permission from copyright 2012 Elsevier.
Kim, Chen, Hammond, & Shao-Horn, 2010; Liu et al., 2012; Mai
et al., 2011; Sun et al., 2013; Yang et al., 2010). Garabagiu and
Mihailescu (2011) used an electrode modified with hemoglo-
binegold nanoparticles (AuNPs) to determine the trace content of premise of rapid, on-line, and high-throughput detection of AA in
AA, which showed a comparable sensitivity (2.84 mg kg1) and thermally processed foods.
linear range (0.71e710 mg kg1). Krajewska, Radecki, and Radecka
(2008) used a glassy carbon electrode modified with single-
walled carbon nanotubes (SWCNTs)/Hb to detect AA in water ex- 2.5. Fluorescent sensing methods
tracts from potato crisp, which showed an ultrawide linear range
(7.1  104 to 7.1  102 mg kg1) and an ultralow LOD Recently, a novel fluorescent sensing method based on AA
(0.071 mg kg1) compared with standard methods, as shown in polymerization and the unique photophysical properties of quan-
Table 2. tum dots (QDs) was proposed to detect AA (Hu et al., 2014), as
Some early research indicated that the working electrodes shown in Fig. 5. In this study, QDs containing carbonecarbon
could hardly be used repeatedly because of the irreversible double bonds after modification of NAS polymerized under the UV
interaction between AA and Hb (Garabagiu & Mihailescu, 2011; irradiation, resulting in the decrease of the distance between QDs
Krajewska et al., 2008; Stobiecka et al., 2007). Fortunately, layer- and the fluorescence intensity (Liu et al., 2011; Noh et al., 2010;
by-layer assembly of nanomaterials and conductive polymers Tansakul et al., 2010). In the presence of AA, the distance of QDs
provided new ways to solve the problem (Batra, Lata, & Pundir, became larger due to the participation of AA in the polymerization
2013; Batra, Lata, Sharma, & Pundir, 2013). Batra, Lata, Sharma reaction, resulting in an increase in fluorescence intensity (Fig. 5).
and Pundir (2013) designed a novel work electrode by depos- Therefore, a correlation was established between the concentra-
iting the mixture of multi-walled carbon nanotubes (MWCNTs) tions of AA and changes of fluorescence intensities after UV irra-
and copper nanoparticles (CuNPs) on the surface of a polyaniline diation. The linear range and LOD were in the range of
(PANI) coated pencil graphic electrode, followed by the immobi- 35e350,000 mg kg1 and 35 mg kg1, respectively. Compared with
lization of Hb, as shown in Fig. 4. The MWCNTs and CuNPs worked standard methods and electrochemical biosensing methods, the
as excellent mediators for electron transfer and the composite film lower sensitivity of this method limits it to be used for detecting AA
provided a large surface for Hb immobilization. This new electrode in various food samples.
could be used for 120 times over 100 days with good reproduc- Another simple fluorescence method was studied for the
tively and consistency. determination of AA in foods (Liu et al., 2014). AA was degraded
The high selective combination between Hb and AA, through the Hofmann reaction to generate vinyl amine which could
outstanding sensitivity to the redox of electrochemistry, and little react with fluorescamine to produce pyrrolione, resulting in a
susceptibility to inference from food matrix endow this method strong fluorescence emission at 480 nm. The fluorescence intensity
with good application to detect AA in potato products. Making use increased with an increase in AA, with a good correlation coeffi-
of nanomaterials can not only increase the amount of Hb on the cient (r2 ¼ 0.99). This method showed similar sensitivity and
electrode and retain the bioactivity of Hb, but also improve the repeatability compared with standard methods. However, high
sensitivity via accelerating electron transfer and repeatability of the temperature reaction conditions limit the method for online
electrode. However, multiple food samples such as breakfast cereal detection of AA in foods.
and coffee and its substitute should be tested to verify the versa- Fluorescent sensing methods show merits of visible signals,
tility of electrochemical biosensors in the future. Then, this method ease of operation, and no requirement for large scale in-
can be utilized in the detection of AA in various foods just with one struments. However, compared with standard methods and
device due to its good correlation and wide linear range. Moreover, electrochemical biosensing methods, low sensitivity and selec-
small, portable, highly integrated, and well organized array elec- tivity based on chemical reactions should be improved in further
trodes can be designed by using nanomaterials, which is the research.
142 Q. Hu et al. / Food Control 56 (2015) 135e146
Fig. 5. Schematic representation of the mechanism of the fluorescent sensing method for AA detection based on CdSe/ZnS quantum dots (Hu et al., 2014). Adapted with permission
from copyright 2013 Elsevier.
3. Sample pretreatment
Sample pretreatment is antecedent in the detection of AA,
coming before instrumental analysis in both standard and rapid
methods because of the necessity of the extraction of AA from
processed foods. Due to the high diversity of food samples, various
sample pretreatment procedures have been investigated but no
universal pretreatment has been developed for all thermally pro-
cessed foods (EU, 2013).
3.1. Sample pretreatment protocols used in standard detection
methods
For standard detection methods, such as LCeMS/MS and
GCeMS, the general procedures of pretreatment include homoge-
nization, spiking with an internal standard, extraction, defatting,
deproteinization, and purification. Fig. 6 shows the flow chart of the
general procedures of sample pretreatment for the detection of AA
using LCeMS/MS and GCeMS.
Addition of an internal standard in food samples can allow for
recoveries and keep track of the possible loss during the whole
sample preparation, which improves the accuracy, precision, and
repeatability of measurements (JECFA, 2005). Most published
research used 13C3-AA as internal standards for AA detection, which
has very similar properties to AA. However, D3-AA, 13C1-AA, N,N-
dimethylacrylamide, propionamide, and methacrylamide also have
been used as internal standards (Go€kmen, Morales, Ataç, Serpen, &
Arribas-Lorenzo, 2009; Johnson et al., 1986; Zhang et al., 2007).
Extraction is the critical procedure in food pretreatment for
detecting AA. According to the similarityeintermiscibility theory,
polar media is used to extract AA, including water, salt solutions
(formic acid) and organic solvents (acetone and acetonitrile) (Chen
et al., 2008; Chu & Metcalfe, 2007; Go € kmen et al. 2009; Klaffke
et al., 2005). Water can minimize the collection of hydrophobic
compounds in foods but other hydrophilic interferences still
remain that should be removed afterwards (Zhang et al., 2005). In
comparison with water, organic solvents have the advantages of
being able to be extracted even without centrifugation and con-
venience in evaporation. The combination of water (salt solutions)
and organic solvents facilitated the efficiency of extraction in some
research (Yamazaki, Isagawa, Kibune, & Urushiyama, 2012; Zhang
et al., 2007). For fat-rich food samples, solvents such as hexane
and cyclohexane were used to eliminate fat (Wang, Lee, Shuang, &
Choi, 2008). For protein-rich food samples, Carrez reagents ([I]
potassium ferricyanide and [II] zinc sulfate), acetone, ethanol, or
methanol was used to precipitate and remove proteins (Bagdonaite,
Derler, & Murkovic, 2008).
Solid phase extraction (SPE) has been used extensively in pur-
ifying the extracts of food samples due to its simplicity, stability,
automation, accuracy, precision, and repeatability of instrumental
analysis (JECFA, 2011). According to the cartridges used in SPE for
purifying AA from the extracts of food samples, two strategies have
been developed. One strategy absorbs AA from the complex extract
in the cartridges through hydrogen bonding, p-p interaction, and
cation exchange, followed by elution of AA using other polar sol-
vents. Some common cartridges are Oasis HLB, Oasis MCX,
IsoluteMulti-Mode, ENVI-Carb, and Isolute ENVþ (Zhang et al.,
2005). The second strategy is applied to retain the interferences
and collect the elution containing AA using cartridges of Oasis HLB
coupled with Bond Elut-Accucat and a homemade SPE column fil-
led with the mixture of C18, SCX, and SAX (Bortolomeazzi, Munari,
Anese, & Verardo, 2012). Among these cartridges, Oasis HLB and
Isolute Multimode are the most preferred. Recently, novel filling
materials such as carbon nanotubes, magnetic chitosan (Xu, Zhang,
Qiao, Xu, & Song, 2012), and molecule imprinted polymer (Xu, Qiao,
Ma, Zhang, & Xu, 2013) were used with effective purification.
3.2. Sample pretreatment protocols used in rapid detection methods
For rapid detection methods, food sample pretreatments for AA
analysis also use similar protocols to standard methods, especially
the procedures and solvents of the extraction. However, purifica-
tion via SPE cartridges is often replaced by other procedures or even
disregarded, as shown in Fig. 6. Moreover, for browning-based
 Q. Hu et al. / Food Control 56 (2015) 135e146
Fig. 6. The flow chart of general procedures of sample pretreatment for LCeMS/MS, GCeMS analysis, electrochemical biosensing and ELISA.
methods such as computer vision, no extraction and purification
procedures are required.
For immunoassays, Quan et al. (2011) used phosphate buffered
saline (PBS) or PBS containing Tween 20 (PBST) to extract AA and
then directly added the extract into microwells for detection,
wherein using the same solution both in extraction and detection
can eliminate matrix interference efficiently. While, for the com-
plete antigens formed by the AA derivative and the carrier protein,
the extract should be derived first before detection. Only when AA
was transformed into its derivative, the antibody could selectively
recognize the analyst in the extract (Preston et al., 2008). Because of
the ability for specific recognition of antibodies, simpler pretreat-
ment without purification, spiking with an internal standard,
defatting, and deproteinization can still satisfy the need of immu-
noassays for detecting AA in food matrices.
The pretreatment of electrochemical biosensing is more
complicated compared with immunoassays, but still simpler than
standard methods. Because the electrochemical signals are based
on redox reactions on the surface of working electrode, proteins
and fat hinder the electron transfer between the electrode and the
solution. Therefore, defatting by hexane and deproteinization by
Carrez reagents ([I] potassium ferricyanide and [II] zinc sulfate) are
always followed by the extraction of AA using polar solvents. In
some research, adjusting the PH and ionic strength of the extract by
adding acetic acid and NaCl solutions, respectively, are necessary
before the measurement (Krajewska et al., 2008; Stobiecka et al.,
2007). In addition, ultrasonic treatment and filtration can facili-
tate the efficiency of extract and purify the extract, respectively
(Zargar et al. 2009). Simpler pretreatment without purification by
SPE cartridges decreases the time and cost of the whole detection
process. However, more food samples should be detected to eval-
uate the versatility of simple pretreatment procedures.
Compared with standard methods, the procedures for sample
pretreatment of rapid analytical methods are timesaving, lower
cost, and used without SPE cartridges for purification, which
facilitate the rapid, high-throughput, and on-line detection of AA.
4. Comparison and prospects
4.1. Comparison of rapid detection methods with standard methods
Both standard and rapid methods for the detection of AA in
thermally processed foods are summarized in Table 2. The
143
comparison between rapid and standard methods in terms of
sensitivity, repeatability, versatility, cost, time, and portability
shows the strength and weakness of each method as follows. (1)
According to the EU Recommendations, analytical methods for the
detection of AA should achieve a LOQ of 30 mg kg1 for bread and
foods for infants and young children and 50 mg kg1 for potato
products, cereal products, coffee, and many other products (EC,
2007, 2010a, 2010b). Standard methods such as LC (HPLC/UPLC)-
MS/MS or GCeMS have a LOD ranging from 1 to 5 mg kg1 and a
LOQ ranging from 10 to 30 mg kg1 as shown in Table 2, which fulfill
the EU requirements regarding LOD and LOQ without any prob-
lems. For rapid detection methods, electrochemical biosensors
show an excellent low LOD value which is about two orders of
magnitude lower than those of standard methods. However, ELISA
and fluorescent analysis show around 20% and can even achieve a
much higher LOQ value than that required by EU. (2) The data for
recoveries (inter- and intra-) and relative standard deviations (RSD)
indicate standard methods are more stable, repeatable, and
reproducable than rapid methods. The RSD values of standard
methods are below 10% or even 5%. However, the absence of data on
inter- and intra-tests for rapid methods indicate that the repeat-
ability needs further evaluations. The RSD of ELISA is close to or
higher than 10%. (3) Standard methods have been applied to
detecting AA in most foods such as potato chips, cereal-based foods,
coffee, tea, and instant noodles while potato chips were usually
selected as the main food sample in rapid methods to evaluate their
practical application, which indicates that the versatility needs to
be confirmed or improved for rapid methods. (4) The purification of
SPE cartridges is necessary to guarantee the high selectivity of
standard methods, but it would increase the cost and complicity of
operations. In contrast, simpler pretreatment in rapid methods
based on the biochemical properties and characteristics of AA can
meet the needs of the low cost detection of AA. Taking pretreat-
ment, depreciation of equipment, labor fees, and material cost into
consideration, ELISA is an example of a rapid method that saves at
least 50% in costs when compared to standard methods. (5)
Regarding detection time, the pretreatment of food samples is the
rate determining step for standard methods. However, for rapid
detection methods such as electrochemical biosensors and com-
puter vision, simpler or even no pretreatment reduces detection
time by 40% or more. Although it takes about 6 h to detect AA in
foods using commercial ELISA kits, multi samples (maximum 24
samples) can be detected by one assay. (6) The requirements of
144 Q. Hu et al. / Food Control 56 (2015) 135e146
expensive instruments and skilled technicians limit standard
methods to be used only in a laboratory. In contrast, simple pro-
tocols and portable instruments in combination with nanotech-
nology enable rapid methods the possibility to achieve on-line and
real-time detection.
4.2. Comparison of different rapid detection methods
Different rapid detection methods have their strengths and
weaknesses which are compared as follows. Electrochemical bio-
sensing methods show the highest sensitivity (two orders of
magnitude higher than the recommendation of EU) and the widest
linear range for detection of AA in comparison with other types of
rapid detection methods, but the recovery and versatility should be
evaluated more with different food samples. ELISA methods pro-
vide the best recovery and versatility for detecting AA in various
food samples. However, multiple washing and incubation steps in
ELISA extend the detection time (4 times longer than electro-
chemical biosesnors per sample) and increase the measurement
error. The newest fluorescent sensing method shows some merits
of visible signals, ease of operation, low cost (80% lower than
ELISA), rapid response (about 20 min), and no requirement for
biomaterials. However, lower sensitivity compared with other
types of rapid detection methods makes it only suitable for foods
containing large amount of AA. So far, computer vison and the
browning based method are the only methods to encompass real-
time and on-line detection of AA contents during food processing,
which makes them more suitable for primary screening of food
products containing too high of a concentration of AA during pro-
cessing or on market.
4.3. Prospects
Rapid detection methods also face challenges and need further
improvements in the future. For computer vision, a portable and
integrated experimental box with the hardware including a cam-
era, a light source, and software such as image analysis and algo-
rithms is the main trend for on-line and real-time detection of AA in
foods.
For ELISA or immunoassays, obtaining a bio-recognition
element with high specificity, simplifying the pretreatment and
improving the sensitivity are the key issues for their applications to
on-line and real-time detection of AA. Designing a hapten with an
adequate molecular terminal and without the requirement of
derivatization during pretreatment should be one of approaches for
further research. Besides, screening other types of bioreceptors
such as aptamers or peptides with high affinity and specificity to AA
may provide new and promising ways to solve the recognition
problem of rapid methods. What is more, the combination of
immuno-technique with the high selectivity of magnetic separa-
tion technology may effectively reduce the cost and time of pre-
treatment. Also, nanomaterials will play a more important role in
the immobilization of antibodies, amplification of signals,
improvement of diversity, and integration of designed immune-
biosensors.
For electrochemical biosensors, efficient immobilization of Hb
or other bioreceptors on the working electrode and the improve-
ment of diversity and portability are two challenges when detect-
ing AA. Nanomaterials such as metal nanoparticles (Pt, Au, Ag, etc.),
semiconductor nanocrystals (ZnO, CdSe, CdSe/ZnS, etc.), carbon
nanomaterials (carbon nanotubes, grapheme oxide, carbon dots,
etc.), and their complexes with polymers are good choices to not
only improve the efficiency of immobilization, but also enhance the
sensitivity, stability, and diversity of detection. In addition, com-
bination of micro- or nano-electrodes and portable electrochemical
detectors may lead the way for methods to achieve on-line, high-
throughput, and real-time detection of AA in foods.
For fluorescent sensing methods, low sensitivity and selectivity
based on chemical reactions are two key points to improve in the
further research. The approach to combining the fluorescence of
nanomaterials with selective recognition elements may promote
the method to obtain on-line and real-time detection of AA in foods.
In summary, rapid detection methods including computer
vision, ELISA, electrochemical biosensors, and fluorescent methods
have the advantages of being low cost, simple, easy to handle, and
portable for detecting AA in thermally processed foods compared
with standard methods such as LCeMS/MS and GCeMS. These
rapid detection methods have the ability to satisfy the need of food
industries, regulatory agents, and customers. However, they still
need further improvement to make themselves more accurate,
sensitive, repeatable, reproducible, and/or portable to achieve on-
line and real-time detection of trace AA. Besides, simplified pre-
treatment is essential in the detection of AA using a rapid (except
for computer vision method) method.
Acknowledgments
The authors are grateful for the financial support in part by the
National Program on Key Basic Research Program of China Project
(No. 2012CB720806) and the National Key Technology Research
and Development Program of the Ministry of Science and Tech-
nology of China Project (No. #2013BAD19B02). The authors also
thank Zach Callaway at the University of Arkansas for reviewing
this manuscript with his valuable comments and suggestions.
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