Occurrence, synthesis, toxicity and detection methods for acrylamide determination in processed foods with special reference to biosensors: A review

Accepted Manuscript Occurrence, synthesis, toxicity and detection methods for acrylamide determination in processed foods with special reference to biosensors: A review Chandra S. Pundir, Neelam Yadav, Anil Kumar Chhillar PII:

S0924-2244(18)30021-9

DOI:

https://doi.org/10.1016/j.tifs.2019.01.003

Reference:

TIFS 2389

To appear in:

Trends in Food Science & Technology

Received Date: 10 January 2018 Revised Date:

24 November 2018

Accepted Date: 5 January 2019

Please cite this article as: Pundir, C.S, Yadav, N., Chhillar, A.K., Occurrence, synthesis, toxicity and detection methods for acrylamide determination in processed foods with special reference to biosensors: A review, Trends in Food Science & Technology, https://doi.org/10.1016/j.tifs.2019.01.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Abstract Background: Acrylamide (2-propanamide), an unsaturated amide, occurs in thermally processed (baked/fried) foods such as potato chips, biscuits, coffee, fried nuts and cereals. Acrylamide is

(amino acid), are heated at a very high temperature.

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generated, when baked food items consisting reducing sugars and protein containing asparagine

Scope and approach: Since acrylamide is potentially neurotoxic and carcinogenic in nature, its accurate determination in processed foods is very important. Among the various methods

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available for detection of acrylamide concentration, biosensors are comparatively more simple, rapid, sensitive and specific. The acrylamide biosensors work optimally within 2-10s, between

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pH 4.5-7.4 and have a shelf life upto 100 days at 40C.

Key findings: The present review describes in detail the occurrence, generation, toxicity and determination of acrylamide with special emphasis on biosensing methods. The miniaturization of laboratory model of acrylamide biosensor could be transformed into portable model. Key words: Acrylamide, Thermally processed foods, Neurotoxin, Carcinogen, Acrylamide

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detection methods, Acrylamide biosensors

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Graphical Abstract Occurrence, synthesis, toxicity and detection methods for acrylamide determination in processed foods with special reference to biosensors: A review

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Chandra S Pundir1*, Neelam Yadav2 and Anil Kumar Chhillar2

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Electrochemical reactions involved in functioning of acrylamide biosensor based on HbNPs

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Occurrence, synthesis, toxicity and detection methods for acrylamide determination in

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processed foods with special reference to biosensors: A review

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Chandra S. Pundir1*, Neelam Yadav2 and A.K. Chillar2

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Department of Biochemistry, M.D. University, Rohtak- 124001, Haryana, India 2

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Center for Biotechnology, M.D. University, Rohtak- 124001, Haryana, India

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Short title: Acrylamide determination: a review

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Key words: Acrylamide, Thermally processed foods, Carcinogen, Acrylamide detection

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methods, Acrylamide biosensors

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*Corresponding Author, email address: [email protected]

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Occurrence, synthesis, toxicity and detection methods for acrylamide determination in

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processed foods with special reference to biosensors: A review

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Abstract

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Background: Acrylamide (2-propanamide), an unsaturated amide, occurs in thermally processed

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(baked/fried) foods such as potato chips, biscuits, coffee, fried nuts and cereals. Acrylamide is

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generated, when baked food items consisting reducing sugars and protein containing asparagine

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(amino acid), are heated at a very high temperature.

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Scope and approach: Since acrylamide is potentially neurotoxic and carcinogenic in nature, its

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accurate determination in processed foods is very important. Among the various methods

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available for detection of acrylamide concentration, biosensing methods are comparatively more

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simple, rapid, sensitive and specific. The acrylamide biosensors work optimally within 2-10s,

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between pH 4.5-7.4 and have a shelf life upto 100 days at 40C.

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Key findings: The present review describes in detail the occurrence, generation, toxicity and

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determination of acrylamide with special emphasis on biosensing methods. The miniaturization

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of laboratory model of acrylamide biosensor could be transformed into portable model.

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Key words: Acrylamide, Thermally processed foods, Carcinogen, Acrylamide detection

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methods, Acrylamide biosensors

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Introduction

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Acrylamide (AA, 2-propenamide, C3H5NO, (Mr=71.09) is an unsaturated amide, occurs in

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various thermally processed (baked/fried) foods. It is generated by baking / cooking of food

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items that are rich in reducing sugars yielding starch and proteins containing asparagines (amino

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acid), at high temperature under low moist condition (Claeys et al., 2005; Mottaram et al., 2002).

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Acrylamide, a small unsaturated amide, is absorbed by humans and animals after ingestion and

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distributed into several vital organs such as thymus, heart, brain, liver and kidney (Hu et al.,

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2014).

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Acrylamide acts as a neurotoxicant, reproductive toxicant and carcinogen in animals

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(Sufian, 2009). Informations from different areas such as soil science, ecology, plant science,

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food science, microbiology, pharmacology, toxicology and medicine, have attracted the attention

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of scientific communities towards production of acrylamide. Hence, it is necessary to develop 2

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such economic, sensitive, specific and rapid system, which can detect acrylamide content in

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thermally processed foods. Biosensing methods have been employed for the detection of

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acrylamide in processed food products. These are electrochemical devices, which are simple,

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facile, rapid, cost effective and highly reproducible (Grieshaber et al., 2008). Thus, the present

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review describes in detail the occurrence, generation, toxicity and chemical analysis of

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acrylamide with special emphasis on biosensing methods

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2. Assessment of acrylamide in thermally processed foods Naturally, raw foods are not toxic, but when these are processed at high temperature, it

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causes significant toxicity. The food products obtained from the plant sources posses high level

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of acrylamide, as these are naturally rich source of glucose, fructose and asparagines.

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the range, 15-3700 µg/kg(ppb),being highest in potato chips and crisps (170-3700 µg/kg),

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biscuits, crackers(30-3200 µg/kg),popcorn(1635-1900 µg/kg) cereals breakfast (30-1346 µg/kg)

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and crisps bread (800-1200 µg/kg), while lowest in chocolate powder(15-90 µg/kg), fish

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products(30-39 µg/kg),boiled potato(48 µg/kg) and meat & poultry products(30-64 µg/kg) (Friedman, 2003) (Hu et al.., 2017).

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2.1 Maillard reaction during processing of foods

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The mechanism of formation of acrylamide in thermally processed foods has been

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accompanied by two mechanisms: (i) Strecker pathway or N-glycoside pathway (Fig.2) and (ii)

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Acrolein pathway. According to Strecker pathway, when cooking food items such as breads,

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biscuits, potato crisps, fried nuts and cereals rich in carbohydrates (glucose/fructose) and proteins

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containing asparagine amino acid, are heated at high temperature i.e. >120°C, these are

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converted into an intermediate, Schiff base, which is converted into acrylamide through a

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reaction known as Maillard reaction, also called as browning process (Claeys et al., 2005;

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Mottram et al.,2002; Stadler et al.., 2002).

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Acrolein pathway involves the decarboxylation of organic acid and then its conversion

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into acrylamide (Medeiros et al., 2012; Yaylayan and Stadler, 2005). Acrylamide is also known

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as triacylglycerols, which causes thermal degradation of products during food processing at

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elevated temperature (Oracz et al., 2011).

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Fig.2 Mechanism of formation of acrylamide in processed foods (Anese et al., 2009) The chemical

reaction between amino acid asparagine and glucose showing the

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formation of acrylamide in processed food is shown in Fig.2.

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3. Toxicological aspect of acrylamide

International Agency for Research on Cancer has documented the acrylamide for its various

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types of toxicity viz. neurotoxicity, carcinogenicity and genotoxicity (Erkekoglu and Baydar,

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2010; Hogervorst et al., 2010). Genotoxicity and carcinogenicity of acrylamide is due to its

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conversion into glycidamide metabolite, which is highly mutagenic as compared to acrylamide.

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Hence, this glycidamide persuades point mutations in the enzymes, which are involved in

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various physiological reactions (Fazendeiro, 2013). Moreover, acrylamide works as a Michael

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acceptor to form adducts with –SH, -OH

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damage (Watzek et al., 2012; Erkekoglu and Baydar, 2010).

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and –NH2 groups in DNA, which leads to DNA

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Acrylamide has been accounted as germinal cell mutagen for inducing dominant lethal

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mutations in spermatids of mice and rats due to chromosome aberrations. When both male and

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female rats provided with acrylamide with a concentration 50-200 ppm in solution form before

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mating followed by gestation and lactation period consequently, there was distraction in mating, 5

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intrusion in sperm ejaculation, decreased sperm count, reduced fertility, loss in body weight gain

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and taking dietary food, reduced pup body weight at birth and weight gain during lactation. The

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molecular basis of reproductive toxicity was the alkylation of -SH groups in the sperm nucleus

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and tail, which damages the testis DNA and reduction in glutathione (Friedman, 2003). Duru and

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co-workers studied the reproductive toxicity by exposing to acrylamide to mice oocytes, which

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was converted into glycidamide that caused severe toxicity to the oocyte of mouse (Duru et al.,

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2017).

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Moreover, acrylamide has been documented for causing neurotoxicity by inhibiting the

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transmission of neurotransmitters and axon transport based on kinesins proteins (Friedman,

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2003). It has been found that intraperitoneal introduction of acrylamide having concentration 100

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mg/kg has elevated the concentration of neurofilament proteins in the brain of rats due to

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alteration of gene expression of brain synthesizing proteins. Formation of acrylamide-sulfhydryl

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linkages has also damaged the regeneration activity of nerves and axons. Hence, mutilation of

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axonal nerve transport causes weakening of skeletal and paralysis of hind limb and walk

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impairment in animals has been observed. The biochemical basis for acrylamide neurotoxicity

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was due to the alteration of amino acids and proteins found in neurons which suppress the amino

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acid incorporation into proteins of nervous system (Friedman, 2003). The persons working in

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food industries get exposed continuously to acrylamide, as a result they suffer from the damage

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of both peripheral and central nervous system, due to increasing and persistent neurotoxic effects

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of acrylamide (Huang et al., 2011; Pennisi et al., 2013). The tolerable daily intake (TDI) of

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acrylamide is 40 µg/kg per day for neurotoxicity and 2.6 µg/kg per day for cancer.

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In addition to these toxicities, intake of acrylamide either in the form of dietry foods or

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from the environment is responsible for causing the cardiac developmental toxicity (CDT).

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Huang et al., (2018) have studied the CDT in zebrafish embryos. The results obtained from this

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study reveal that introduction of acrylamide after fertilization shrinked the heart and its abnormal

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morphological development. During atrioventricular valve development ,acrylamide suppress the

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expression of genes like my17, vmhc, myh6, bmp4, tbx2b and notch1b (Huang et al., 2018).

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Considering its toxicity and carcinogenic nature, the measurement of acrylamide level in foods

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has become a very important issue for the food safety (Tardiff et al.,2010).

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4. Detection methods for acrylamide determination in processed foods

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The consumption of processed food items containing acrylamide is responsible for various

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types of toxicity as stated above; therefore, it is necessary to detect acrylamide concentration in

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thermally processed foods. Various types of detection methods are available for the detection of

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acrylamide as described below:

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4.1 Acrylamide detection by LC-MS/MS

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Principle

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Acrylamide has been detected by LC-MS/MS, which requires pretreatment of sample before

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analysis. Firstly food sample is homogenized in water followed by addition of an internal

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standard (D3-AA, 13C1-AA, N,Ndimethylacrylamide, propionamide, and methacrylamide) and

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then fat extraction. Addition of internal standard helps in recovery, improves the accuracy and

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precision of the sample. Fat in the sample is extracted/ removed by adding hexane and

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cyclohexane (Wang et al., 2008) and Carrez reagents and other solvents such as potassium

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ferricyanide [II], zinc sulfate, acetone, ethanol, or methanol for deproteinization and

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removal/precipitation of protein in case of protein rich samples (Bagdonaite et al., 2008),

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followed by subsequent addition of ethyl acetate to get liquid-liquid extraction. The above

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preparation is purified by evaporation and solid phase extraction (SPE) cartridges. Recently,

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Lambert et al. (2018) have determined the levels of acrylamide in foods included in the ‘first

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French total diet study on infants and toddlers’ by LC-MS/MS technique.

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Merits

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This method is versatile, sensitive, selective, efficient method for acrylamide detection.

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Demerits

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This method is costly, requires time consuming sample pretreatment and costly equipment.

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4.2 Investigation of acrylamide by chromatic/color indicating methods Principle The principle of browning process involves the change in the color of the product when they

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are heated at high temperature and this change in color was measured earlier an L*a*b

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International marker for measurement where symbol L for luminescence/light emitting

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component, a* measure change in green color to red color and b* for change in color from blue

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to yellow color. a* acting as a marker for the detection of acrylamide concentration and it is

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directly proportional to the acrylamide concentration in the food sample. Better accuracy has

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been obtained by vision by computer for those parts of processed food which are uneven, based

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on classification, various algorithms have been used for analyzing their images and comparison

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between the brown ration and acrylamide concentration (Mogol & G€okmen, 2013). Chromatic

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method was used for the detection of acrylamide in potato chips based on nucleophile-initiated

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thiol-ene Micheal addition (Hu et al., 2016). A schematic representation for detection of

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acrylamide by chromatic method has been shown in Supporting Fig. 1.

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Supporting Fig.1 Schematic demonstration of the mechanism of the fluorescent sensing

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method for the detection of acrylamide based on CdSe/ZnS quantum dots. (Source: Hu et

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al., 2016)

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This method is simple and does not require costly instruments. It works by simply taking the

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images of processed food items and does not require sample pretreatment.

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Demerits

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Still this method has some drawbacks because of susceptibility of various parameters

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including light intensity, configuration and uniformity of food samples, focal length and

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aperture.

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4.3 Detection of acrylamide by fluorescence method Assessment of acrylamide in thermally processed foods has been made possible by using

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fluorescent methods and quantum dots (QDs), which exhibit unique photophysical properties

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(Hu et al., 2014). The acrylamide content in potato chips has been investigated for quantifying

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the acrylamide concentration by fluorescent sensing process based on acrylamide

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polymerization-induced increased distance between quantum dots (Hu et al., 2014).

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Principle

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Use of modified QDs by N-acryloxysuccinimide (NAS) under UV-radiation decreases the

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distance between C=C bonds of QDs, which in turn decreases the intensity of fluorescence (Liu

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et al., 2011, Noh et al., 2010 and Transakul et al., 2010). When these QDs were used for the

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detection of acrylamide, it increases the distance between C=C bonds of the QDs, which in turn

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increases the fluorescence intensity. Mechanism of detection of acrylamide based on CdSe/ZnS

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quantum dots has been illustrated in Supporting Fig. 2(a) and 2(b).

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Supporting Fig. 2(a) Diagrammatic depiction of the Michael addition reaction between

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GSH and acrylamide with catalysis of TCEP, as well as the mechanism of nucleophile

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initiated thiol–ene Michael addition reaction (Source: Hu et al., 2016).

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Supporting Fig. 2(b) Diagrammatic illustration of the detection mechanism based on the

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different dispersion of AuNPs in the presence or absence of acrylamide (Source: Hu et al.,

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2016)

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Merits

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The fluorescent method has offered many advantages such as easy to operate, visible signal

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and non requirement of large scale instrument (Qinqin et al., 2015).

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Demerits

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This method has poor sensitivity and selectivity when compared with the standard methods and electrochemical biosensing methods.

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4.4 Detection of acrylamide by enzyme-linked immunosorbent assay (ELISA)

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It is an immunological method which involves first recognition of suitable antigen followed by

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binding of specific high affinity antibody.

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Principle In this method a suitable analyte i.e. acrylamide is immobilized on the surface of Au electrode

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(recognition element) and specific antibodies with high affinity are produced against

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immobilized acrylamide which is confirmed by signal coming out from transducer in the form of

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either by emitting light or colored products which are labeled with specific enzyme. The

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intensity of coming out signal is directly produced to the analyte concentration on the

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immobilized acrylamide on the electrode. Supporting Fig. 3 shows the schematic depiction of the

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preparation of complete antigen, antibody and competitive indirect ELISA for acrylamide

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analysis (Qinqin et al., 2015).

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Supporting Fig.3 Schematic depiction of preparation of complete antigen, antibody and

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competitive indirect ELISA for the analysis of acrylamide (Source: Qinqin et al., 2015). 12

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Merits The use of ELISA for the quantification of acrylamide has certain promising advantages such

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as specificity, selectivity, simplicity, fast detection of acrylamide in food samples and

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independent of costly instruments.

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Demerits

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The major drawbacks of these biosensors are less availability of specific antibodies and low

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titre antibodies production in serum. This is due to the reason that acrylamide has small and low

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molecular weight. Therefore, epitopes present on the surface of acrylamide are not well exposed

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and confers the non-immunogenicity of acrylamide. 4.5. Investigation of acrylamide by supramolecules

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Acrylamide content in processed foods have also been detected by using recognition element

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composed of complex of two or more than two molecules (Kleefisch et al., 2004) having tiny

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configuration with unique integrity called as supramolecules, which help in binding of

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acrylamide to their specific site (Steed and Atwood, 2009).

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Principle

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Detection of acrylamide in food samples based on the concept of piezoelectric biosensor where

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piezoelectric crystals of opposite charges are prepared and these crystals possess active site for

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binding of acrylamide. Binding of acrylamide at the active site of piezoelectric crystals causes

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vibration at particular frequencies, which result in alteration of resonance frequencies which is

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measured by electronically. Now a days, another technique such as molecular imprinting

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technique (MIT) has been used for the detection of acrylamide, which involve the formation of

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“chemical antibody” by involving supramolecular chemistry. These chemical antibodies have

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been used in liquid chromatography, solid phase extraction and in sensation of food samples

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(Chen and Li, 2011). Nano-scale dummy-surface molecularly imprinted polymers (DSMIPs)

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have been used for the quantification of acrylamide in processed foods by immobilizing them on

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a magnetic graphene oxide (GO–Fe3O4) (Ning et al., 2017) Supporting Fig. 4.

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Supporting Fig. 4 Schematic representation of processes for preparation of AM-DSMIPs-

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GO-Fe3O4 (Source: Ning et al., 2017)

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Merits

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Biosensors based on supramolecules are specific and sensitive

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Demerits

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The major limitations of these methods are cumbersome preparation of supramolecules and

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requirement of skilled person and costly instrument. Therefore, this method has not been used in

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food science for the detection of acrylamide for routine.

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4.6 Biosensors for detection of acrylamide

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Although, chromatographic methods are highly sensitive, selective, stable and

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reproducible, still these methods have some drawbacks such as time consuming sample

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preparation, requirement of expensive equipment, trained persons to operate the instruments and

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high cost of analysis. Further, these techniques do not provide results of acrylamide analysis in

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the stipulated time, hence detection of acrylamide in foods is not possible online. On the other

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hand, ELISA also requires other robust confirmation methods for the better analytical results 14

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(Qinqin et al., 2015). Secondly, procurement of antibodies for acrylamide with high stability and

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affinity is also a big problem. Therefore, these limitations have been overcome by biosensing

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methods as these methods are simple, sensitive, specific and fast response measurement.

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Principle Biosensors are quantitative or semi quantitative analytical device which incorporates a

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living recognition entity viz. enzyme, antibodies, phages, aptamers or single stranded DNA with

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suitable physicochemical, optical, thermometric, piezoelectric and magnetic transducers (Elif et

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al., 2015)

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Supporting Fig. 5 Principle and working of a biosensor (Source: Bernal et al., 2014)

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Advanced biosensing techniques are focused on the preparation of devices/tools which

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are efficient in detection of analyte used for foods analysis (Palchett et al., 2008). Majority of

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biosensors employ entire microbial cells for determination of particular chemical constituents

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and quantification of their toxicity. Ignatov et al. (1997) have quantified acrylamide using

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biosensor composed with oxygen Clark electrode as a processor. They measured the respiratory

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activity of bacterial Brevibacterium sp. cells which was obstructed by the existence of

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acrylamide and acrylic acid in waste waters. Extent of oxygen consumption gives the analytical

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signal of amperometric sensor (Ignatov et al., 1997). The occurrence of analyte (acrylamide) in

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sensor’s cell reduced the consumption of oxygen on the outer surface of the sensor which is

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directly proportional to the decrease in intensity of current in Clark electrode. While analytical

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sample lacking acrylamide the respiratory activity of cells was taken as the measure of acrylic

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acid amount (Souzaw et al., 2001). This method offers high sensitivity (10 mg L−1),

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comparatively short duration of assays, simple sample preparation and advanced procedures for

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acrylamide measurement (Souzaw et al., 2001; Ignatov et al., 1996). Developments in biological

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methods have constructed novel sensors which are sensitive, specific and quantify trace amounts

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of acrylamide in samples comprising of various complex matrices (Silva et al., 2008).

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Researchers have designed a biosensor by using transgenic nematode known as

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Caenorhabditis elegant having fusion gene gst-4::gfp was constructed from the promoter gene

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gst-4 and reporter gene gfp. Occurrence of acrylamide induced the transcription of the reporter

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gene which in turn activates the synthesis of quantifiable green fluorescent protein (GFP)

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(Hasegawa et al., 2007). Moreover, development in biosensors construction has been

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accomplished by using acrylamide-binding tetralactam (a macrocyclic compound of Hunter–

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Vogtle type) as a functional constituent of a biosensor.

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Gravimetric sensor made up of a quartz crystal coated with tetralactam film and used for

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detection of acrylamide in a gaseous phase (Kleefish et al., 2004). This sensor quantify precise

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amount of acrylamide at the gas–solid interface (Erikson et al., 2005; Kleefish et al., 2004). As

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the vapors of the sample interacted with coated crystal film, acrylamide was accumulated on the

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tetralactam. This accumulation increases the mass of sensor which in turn altered the frequency

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of resonance vibrations of quartz crystal (Erikson et al., 2005). Quartz crystal microbalance

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technique (QCM) was used for measuring minor frequency alterations (Erikson et al., 2005).

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In piezoelectric sensors the macrocyclic compound of Hunter–Vogtle type was suitable host

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which help in detection of traces amount of acrylamide. This macrocyclic compound showed

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significant high affinity to acrylamide than to analogous compounds such as acrylic acid and

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propionamide. Thus, piezoelectric sensors having tetralactam exhibit unique selectivity and

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sensitivity of acrylamide measurements (10µg kg−1) and are not sensitive to alterations in the

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relative humidity (Krajewska et al., 2008; Erikson et al. 2005). In spite of these merits these

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biosensors are not used for acrylamide determination in all kind of foods.

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4.6.1 Electrochemical acrylamide biosensor

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Principle

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Determination of acrylamide in processed foods has been made possible by electrochemical

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acrylamide biosensor. In these electrochemical biosensors, either current is generated, due to

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oxidation-reduction in milliampere (mA) or voltage in V or impedance in electron transfer

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resistance (RCT) are measured, which is directly proportional to analyte concentration. The electrochemical/amperometric biosensors for the detection of acrylamide in processed

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foods have been developed by conjugating catalytic core of Hb with the acrylamide. During

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redox reactions, there is formation of an adduct by conversion of Hb-Fe(III) to Hb-Fe(II). The

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formation of Hb-Fe(II)- acrylamide adduct increases the distance from the electrode

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consequently, decline in current peaks (Friedman, 2003, Lineback et al., 2012). Fig. 3 shows the

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electrochemical reaction between Hb and acrylamide.

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Fig.3 Electrochemical reactions involved in functioning of acrylamide biosensor based

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on HbNPs (Source: Yadav et al., 2018).

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Acrylamide has been detected by constructing electrochemical biosensor. The first attempt

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was made for the detection of acrylamide in polluted water by involving the microbial

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biochemical reactions such as respiration and other enzymatic reactions. The report for

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determination of acrylamide was given by Ignatov (1997) in the form of specific respiratory

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activity, which after introduction of acrylamide, measure how much oxygen was consumed by

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the bacterial cells (Brevibacterium sp.) and endogenous cells. The decreased consumption of

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oxygen by the bacterial cells and reduction in current due to reduced metabolic activities of

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exposed bacterial cells, acted act as a signal for the detection of acrylamide.

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Merits These biosensors are stable, highly sensitive, selective, fast responsive with wider linear

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range (Stobiecka et al., 2007). Working of electrochemical biosensor based on Hb depends on

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the effective immobilization of Hb and increased movement of electrons from the immobilized

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electrode (Sun et al., 2013).

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Demerits

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Hindrance in removal of electrons from the surface of electrode due to presence of shielded

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polypeptide covering around the redox centre (Sun et al., 2013). A large amount of immobilized

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Hb provides more adducting sites for acrylamide to augment the signal intensity. Thus, to

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improve the immobilization and electron transfer of Hb, novel nanoparticles were used (Qinqin

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et al., 2015).

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4.6.1. (a) Principle of electrolyte acrylamide biosensor

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Electrolytes such as LiCl and cobalt (II) ions have also played a significant role in

16

determination of acrylamide (Niaz et al., 2008 and Zargar, 2009). These cobalt (II) ions reduced

17

the movement of electron flow which lower the current peaks.

18

Merits

20 21 22

These electrolytes based biosensors are highly sensitive and rapid. Demerits

The food sample consisting interfering compounds hinders the activity of biosensor.

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19

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15

4.6.1. (b) Role of nanoparticles in detection of acrylamide Nanoparticles are the particles with the diameter in the range of 10-9m to 10-7m. They may be

24

organic (SWNT, cMWCNT) or metallic (e.g. Au, Ag, Pt etc.). The use of these nanoparticles

25

have many advantages such as increase in the surface to volume ratio therefore more and more

26

binding of acrylamide with the immobilized Hb, which leads to increase in the movement of

27

electron, between presence of diverse functional groups (-COOH & -CO) on the active site.

28

4.6.1. (c) Enzymatic/protein nanoparticles

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The proteins such as Hb, myoglobin and cytochrome C possess four heme groups in their

30

redox center (Ye, 1988, Wu et al., 2015, Liu et al., 2012 and Sayyad et al., 2012) and thus

31

employed in construction of various biosensors (Reed, 1987 and Sezgintürk and Dinçkaya, 18

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2009). However, Hb is more appropriate in construction of acrylamide biosensor, due to its

2

commercial accessibility at low cost and relatively more stability and configuration [N-(2-

3

carbamoyl-ethyl)-L-valine] similar to one of the glycidamide [N-(2-carbamoyl-2-hydroxyethyl)-

4

RS-valine), which facilitate the formation of Hb-acrylamide adduct (Friedman, 2003). Hence,

5

the biosensors constructed with native Hb are more specific and cost effective (Xu et al., 2011).

6

4.6.2 (c) Protein based acrylamide biosensor

7

Earlier, a number of Hb based biosensors involving

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Hb/sol-gel film modified carbon paste

electrode (Wang et al., 2004), Hb/gold nanoparticles(AuNPs)/carboxylated multiwalled carbon

9

nanotubes (cMWCNT)/glassy carbon(GC) electrode (Chen et al., 2007), Poly(maleic anhydride-

10

alt-butyl vinyl ether (AM41)-polyethylene glycol(PEG)/Hb/citric acid NPs (Dessy et al., 2011),

11

Hb/ silver nanoparticles)(AgNPs) modified borron dopped diamond electrode (BDDE) (Jiang et

12

al.,

13

electrode) (Wu et al., 2015) have been fabricated for diverse applications.

2015) and

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dimethyldioctadecyl-ammonium bromide(DDAB)/Hb/glassy carbon(GC)

A number of voltametric and amperometric biosensors have been constructed for the

15

detection of acrylamide. The voltametric acrylamide biosensor employed Hb/DDAB/CP

16

electrode (Stobiecka et al., 2007), Hb/SWCNT/GC electrode (Krajewska et al., 2008), while

17

amperometric acrylamide biosensors were based on Hb/AuNPs (Garabagju et al., 2011),

18

Hb/cMWCNT/CuNPs/polyaniline(PANI)/pencil graphite(PG) electrode (Batra et al., 2013),

19

Hb/cMWCNT-Fe3O4NP/ chitosan(CHIT)/Au electrode (Batra et al., 2012). However, these

20

methods have some drawbacks such as complicated preparation of working electrode and its

21

poor analytical performance. Moreover, direct immobilization of native Hb onto Au surface

22

leads to slow rate of electron transfer from the surface of Hb to Au electrode, due to presence

23

of heme groups (redox center) inside the intensified globular structure of Hb (not exposed)

24

(Stobiecka et al., 2007). These problems can be overcome by use of HbNPs in place of native

25

Hb molecules, as

26

high optical, electrical, electronic, thermal properties, chemical and catalytic (ability to facilitate

27

electron transfer) properties, increased surface area, rapid detection of traces amount of

28

acrylamide with high sensitivity (Pundir, 2015).

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protein nanoparticles(100-200nm in size) have unique advantages such as

29

4.6.1. (d) Merits of using nanoparticles

30

Thus, nanoparticles increase the catalytic activity and electrical conductivity that rapidly detect

31

acrylamide from the food samples (Dreyer et al., 2010 and Sun et al., 2013). Efficiency of 19

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1

detection of trace amount of acrylamide was enhanced by modifying electrode with Hb-gold

2

nanoparticles (AuNPs) (Garabagiu and Mihaileshcu, 2011). Acrylamide had also been detected from the water extract of potato crisps by single walled

4

carbon nano tubes (SWCNT) modified glass electrode followed by immobilization of Hb that

5

showed good linear range and low limit of detection (LOD), compared to standard methods

6

(Krajewska et al., 2008).

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Earlier, it was assumed that the functional electrode could not be used repeatedly, because of

8

irreversible interaction between acrylamide and Hb (Garabagiu and Mihailescu, 2011). This

9

problem was solved using conducting polymers and layer-by-layer immobilization of

10

nanoparticles (Batra et al., 2013). Batra and co-workers prepared modified pencil graphite (PG)

11

electrode by electrodepositing polyaniline (PANI), and then mixture of multi walled carbon

12

nanotubes (MWCNTs) and copper nanoparticles (CuNPs) followed by immobilization of Hb.

13

This modified PG electrode showed good reproducibility, sensitivity, electrical conductivity as

14

well as consistency. The above mentioned, process could be employed for detection of

15

acrylamide in diverse foods just with single nanomaterial. Hence, future research could be

16

focused on construction of nanoparticles based microscopic electrochemical biosensor which is

17

portable and has vast application in food science for the detection of trace amounts of acrylamide

18

in processed foods (Qinqin et al., 2015). Table 1 summarizes the various detection methods for

19

acrylamide in foods.

20

Table 1:- A comparison of analytical parameters of various methods for detection of

21

acrylamide in processed food

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Name of Processed Food Products

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Methods Used For Acrylamide Detection LC-MS/MS (HPLC, UPLC)

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7

Potato chips, Coffee, Cereal-based foods, Tea, Infant foods

Limit of Detection (LOD) (µg/kg) 1-6

Sensitivity (µA/nM/cm2)

Reference

-

Senyuua, 2006, Liu et al., 2008, Zhang et al., 2005, Yamazaki et al., 2012

HPLC-UV

Products of rice, potato corn and wheat, Bread samples, Baked and deep-fried

1.5-3.0

-

Hua et al., 2017, Alpozen et al., 2015

MSPD-HPLC

Coffee, beans potato

1.5-3.6

-

Zhao et al., 2015

20

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3

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36.9 -72.5

Stobiecka et al., 2007,Batra et al., 2013

0.56

-

Demirhan et al., 2017

UV-Capillary electrophoresis

Potato, Egg plant, Chick peas, Soft wheat flour, Sorghum Durra flour

320-560

-

Omar et al., 2017

ELISA

Mashed potatoes, French fries and cracker, Potato chips

15-35

-

Fu et al., 2011, Sun et al., 2013 Hu et al., 2014

15

-

Liu et al., 2014

28.6 nmol L−1

-

Hu et al., 2016

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Potato chips, biscuits, baby foods, coffee cream, bread

French fries, fried puffs, fried chicken roll, bread, biscuits Potato chips

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Colorimetric method 2

De Vleeschouwer et al., 2007, Hariri et al., 2015

Mn-Doped ZnS Quantum Dots

Fluorescence

1

-

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Electrochemical Biosensors

5.0- 75

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GC-MS (GC based)

chips, French fries, Potato, flour, twisted cruller, potato chips and toast samples Chinese foods, Coffee, Cereal-based foods, Infant powdered formula, coffee and chocolate powders, corn snacks, bakery, products and tuber-, meat- and vegetablebased foods, Potato chips, Corn chips Potato crisps

4.6.1.1 (a) Amperometric acrylamide biosensors Amperometric biosensors work by exchanging the electrons from recognition element and

4

electrode followed by generation of current that can be monitored (Zang et al., 2000).

5

Principle

21

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Working of acrylamide biosensors depends on the interaction of hemoglobin (Hb) with

2

acrylamide and consequently, formation of Hb–acrylamide adduct. Hb, is a heterogenous protein

3

comprised with four prosthetic groups of heme–Fe (III). This modified electrode exhibits

4

reversible reduction–oxidation reactions of Hb–Fe3+ (III)/Hb–Fe2+. Hb-acrylamide adducts is

5

formed, because of reaction between the alpha-NH2 group of N-terminal valine of Hb and

6

acrylamide results in formation of Hb acrylamide adducts. Reaction between Hb and acrylamide

7

is aided by decline in current peaks of Hb–Fe3+ reduction, which could alter the electrochemistry

8

of Hb. Consequently, Hb–acrylamide adduct accumulation at the electrode surface get

9

augmented and this decrease the current peak of cyclic voltammogram (CV) (Fig.3). Thus,

10

declines in current act as an analytical signal, which is used as a basis of construction of highly

11

precise and sensitive amperometric determination of acrylamide. Hence, Hb acts as useful

12

analytical marker for detection of acrylamide. These amperometric acrylamide biosensors can be

13

classified based on types of electrodes.

14 15 16

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These biosensors are highly selective, specific, sensitive and rapid. Depending upon type of electrode used electrochemical acrylamide biosensors are of following

18

types:

19

4.6.2.1. (a). Carbon electrode-based amperometric acrylamide biosensors

20

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These biosensors were further classified on the basis of whether nanomaterials were used or not.

22

4.6.2.1. a. (i) Acrylamide biosensors without nanomaterials

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21

A carbon paste electrode was functionalized with Hb and exhibited 1.2 × 10−10 mol/L LOD

24

for acrylamide detection. It was appropriate for the direct quantification of acrylamide in

25

aqueous extract of potato chips (Stobiecka et al., 2007).

26 27 28

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Principle

29

To construct such biosensors, firstly, graphene oxide (GO) was deposited on the surface of

30

glassy carbon electrode (GCE), followed by immobilization of DNA onto GO/GCE through

31

electro adsorption. Because of more surface area of GO, DNA was immobilized efficiently on 22

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the GC electrode surface. Beside this, electrode has exclusive nanostructure and rapid electron

2

transfer ability due to GO, which has increased the direct electron transfer of DNA considerably.

3

Consequently, the presence of acrylamide was electrochemically signaled by the formation of

4

two strong oxidation peaks on GO/GCE due to immobilization DNA on GO/GCE.

5

Merits

6

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1

This biosensor had low limit of detection, good reproducibility and cost effective (Li et al., 2014).

8

Demerits

9

These biosensors are less stable, more susceptible to leaching or oxidative environment and

10

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exhibit toxic qualities, which disrupt the functioning of cell.

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4.6.2.1. a. (ii) Acrylamide biosensors based on nanomaterials

12

An amperometric biosensor was prepared by was prepared by coating the single-walled

14

carbon nanotubes (SWCNTs) and Hb onto glassy carbon electrode (GCE) for determination of

15

acrylamide in water. The biosensor also detected the occurrence of acrylamide in several foods

16

like potato crisps, French fries or bread and has a very low LOD (1.0 × 10−9 mol/L) (Krajewaska

17

et al., 2008).

18

Principle

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13

19

A highly sensitive electrochemical biosensor was constructed in our laboratory for the

20

detection of acrylamide, by immobilizing Hb covalently onto carboxylated multiwalled carbon

21

nanotube/copper

22

electrodeposited onto pencil graphite electrode (PGE). The biosensor works ideal conditions

23

having working potential 20 mV/s, pH 5.5 (0.1 M sodium acetate buffer) and temperature 35 °C.

24

Hb/cMWCNT/CuNP/PANI was exceptionally sensitive (72.5 µA/nM/cm2), rapid (response time

25

<2 s), better linear range (5 nM–75 mmol/L) with very low limit of detection (0.2 nmol/L).

26

Analytical recovery of biosensor at 20 nmol/L and 40nmol/L was found to be 95.40% and

27

97.56% respectively. Coefficients of variation were 2.35% and 4.50% for Within- and between-

28

batch Within- and between-batch respectively. The enzyme electrode was stable for 100 days,

29

when stored at 4 °C (Batra et al., 2013).

nanocomposite

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30 31

(cMWCNT/CuNP/PANI)

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nanoparticle/polyaniline

Merits 23

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1 2

The biosensor was highly sensitive, specific, rapid, low limit of detection and unique linear range.

3 4

4.6.2.1. a. (iii) Gold electrode-based amperometric acrylamide biosensor An improved amperometric acrylamide biosensor was developed in which Hb was attached

6

covalently onto nanocomposite of cMWCNT and iron oxide nanoparticles (Fe3O4NPs)

7

electrodeposited onto Au electrode through chitosan (CHIT) film. This improved biosensor

8

principally involved the interaction between acrylamide and Hb, which decline the redox

9

reactions of Hb; i.e. current produced during its reversible conversion [Fe2+ /Fe3+ ]. The

10

biosensor has optimized at pH 5.0, temperature 30 °C, response time 8 s and wide working range

11

3–90 nmol/L, with 0.02 nmol/L detection limit and sensitivity of 36.9 µA/ nmol/L /cm2. The

12

biosensor was examined and used for quantification acrylamide in potato crisps (Batra et al.,

13

2012).

14

Hb/cMWCNT/Fe3O4/CHIT/AuE has been illustrated in Supporting Fig. 7.

representation

of

amperometric

acrylamide

biosensor

based

on

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Schematic

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5

16

Supporting Fig. 7 Schematic representation of amperometric acrylamide biosensor based

17

on Hb/cMWCNT/Fe3O4/CHIT/AuE (Source: Batra et al., 2012)

18 19

4.6.2.1. a. (iv) HbNPs based improved amperometric acrylamide biosensor

20

An improved amperometric acrylamide biosensor was constructed in our laboratory by

21

immobilizing HbNPs directly onto Au electrode through thiolate bonding/covalent coupling

22

(Yadav et al., 2018). HbNPs were prepared by desolvation method and characterized them by

23

Transmission electron microscopy (TEM), Fourier transformation infra red (FTIR), X-ray 24

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diffraction (XRD) and Atomic force microscopy (AFM). The working electrode (AuE) was

2

characterized by scanning electron microscopy (SEM) and electrochemical impedance spectra

3

(EIS). The response of HbNPs/AuE was obtained by recording the decrease in current by cyclic

4

voltammetry, which occured due to the formation of Hb-acrylamide adduct during

5

electrochemical reaction on the surface of functional Au electrode (Fig. 4).

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7 8

Fig. 4 Reaction for adduct formation between HbNPs and acrylamide (Yadav et al., 2018) The working potential range of HbNPs/AuE was -0.750 to +0.500V and observed maximum

10

current at 0.26V. The HbNPs/AuE biosensor was optimized in terms of pH and substrate

11

concentration. The optimum pH of HbNPs/AuE was (5.0) and showed inverse linear relationship

12

between 0.1-100nmol/L. The analytical performance of HbNPs/AuE was better than earlier

13

reported biosensor for the detection of acrylamide various thermally processed foods with 0.1nM

14

limit of detection (LOD), within-and between batches, co-efficient of variations were 3.85% and

15

4.67% respectively. The analytical recovery of biosensor was found to be 10mM was 99% and

16

98% at 5mmol/L and 10 mmol/L

17

HbNPS/AuE and response measurement has been depicted in Fig. 5.

19

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acrylamide respectively. The scheme for construction

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9

25

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1 2 3

Fig. 5 Schematic illustration of electrochemical reaction of immobilized HbNPs involved in

4

fabrication of HbNPs/AuE (Yadav et al., 2018).

4.6.2.1.a. (v) Composite biosensor (Hb-DDAB/PtAuPd NPs/Ch-IL/MWCNTs-IL/GCE) for

6

the detection of acrylamide

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An ultrasensitive amperometric biosensor was constructed for the detection of acrylamide in

8

various thermally processed foods. The biosensor was designed by immobilizing Hb-

9

dimethyldioctadecylammonium bromide (Hb-DDAB), platinum-gold-palladium three metallic alloy

nanoparticles

(PtAuPd

11

bis(trifluoromethylsulfonyl)imide (Ch-IL) through multiwalled carbon nanotubes-IL (MWCNTs-

12

IL) onto glassy carbon electrode (GCE) (Supporting Fig. 8). The biosensor was based on

13

formation of an adduct by the reaction of acrylamide with α-NH2 group of N-terminal valine of

14

Hb which decline the peak current of Hb-Fe3+ reduction. Under ideal conditions, the biosensor

15

detected acrylamide by square wave voltammetry (SWV) in two linear concentration ranges of

16

0.03–39.0 nmol/L and 39.0– 150.0 nmol/L with 0.01 nmol/L limit of detection. The biosensor

17

was capable for precise detection of acrylamide even at elevated quantity of ordinary interfering

18

compounds that confirmed that the biosensor was exceptionally selective. Moreover, the results

19

obtained from recent studies revealed that the above proposed biosensor was stable, sensitive,

20

reproducible and rapid (response time less than 8 s). Hence, the biosensor has been effectively

NPs),

chitosan-1-ethyl-3-

methylimidazolium

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26

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1

applied for the detection of acrylamide in potato chips and results obtained were analogous to

2

results of gas chromatography-mass spectrometry (GC-MS) as standard method (Varmira et al.,

3

2018).

4

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6

Supporting Fig. 8 Schematic representation of construction of composite biosensor Hb-

8

DDAB/PtAuPd NPs/Ch-IL/MWCNTs-IL/GCE for acrylamide detection (Source: Varmira

9

et al., 2018).

10

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7

4.6.2.1. a. (vi) DNA based biosensor for acrylamide detection

11

The biosensor was fabricated by immobilizing single-stranded DNA (ssDNA) functionalized

12

with –SH group onto Au electrode (GE) covalently. The ssDNA/GE exhibited solitary effective

13

DPV oxidation peak, which was used as the electrochemical analytical signal for detecting

14

acrylamide. The interaction between acrylamide and ssDNA was confirmed by UV–vis

27

ACCEPTED MANUSCRIPT

absorption spectrometry and DPV (Differential pulse voltammetry). Acrylamide and ssDNA

2

formed an adduct and the capturing ratio of acrylamide with ssDNA was one acrylamide per

3

guanine (G) base of ssDNA. The electrochemical oxidation of acrylamide-ssDNA adduct onto

4

GE was an adsorption-controlled stable/irreversible reaction that is mediated by transfer of two-

5

electrons and two-protons. Under working conditions, ssDNA/GE was exhibited unique DPV

6

response, wide linear range 0.4–200 µmol/L, 8.1 nmol/L (3σ/slope) LOD. This biosensor was

7

used for the detection of acrylamide in tap water and potato crisps. Hence, the electro-chemical

8

biosensor was suitable and effective, offered significant potential for fabrication of

9

electrochemical biosensors for diverse toxic components ( Huang et al., 2016). The schematic

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representation for the construction of ssDNA/GE has been depicted in Supporting Fig. 9

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Supporting Fig. 9 Schematic representation of the construction of ssDNA/GE and its

13

application for detection of acrylamide (Source: Huang et al., 2016)

14

Merits 28

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1

Detection of acrylamide by ssDNA/GE is simple, stable, reproducible and sensitive.

2 3

4.6.3. Potentiometric acrylamide biosensors A potentiometric acrylamide biosensor was fabricated, that worked by direct biochemical

5

interaction between acrylamide and entire bacterial cells Supporting Fig. 10. These intact

6

bacterial cells of Pseudomonas aeruginosa were immobilized on biological recognition element,

7

which possessed amidase enzyme and catalyzed the breakdown of acrylamide releasing

8

ammonium ions (NH4+) and corresponding organic acid. The NH4+ ions were detected by an

9

ammonium ion selective electrode. Therefore, cells of P. aeruginosa immobilized on various

10

types of membranes in the presence of glutaraldehyde and an ammonium ion selective electrode

11

were used for construction of acrylamide biosensor. Bacterial acrylamide biosensor has showed a

12

linear response in the range of 0.1–4.0 × 10−3 M, LOD 4.48 × 10−5 M, sensitivity 58.99

13

mV/mM of acrylamide, minimum response time 55 s and can be stored for 54 days. The

14

selectivity of biosensor for related amides had also been investigated, which revealed that it

15

cross-reacted with acetamide and formamide, but no reaction with phenylacetamide, p-

16

nitrophenylacetamide, and acetanilide. The biosensor was employed effectively, for

17

quantification of acrylamide in real industrial effluents. The analytic experiments were carried

18

out, which revealed an average substrate recovery of 93.3%. The biosensor was cheap, since

19

whole cells of P. aeruginosa, could be used as source of amidase activity (Silva et al., 2011).

20

Polymeric membranes such as polyethersulfone, nylon, polyethersulfone and polycarbonate were

21

used for the disk preparation P. aeruginosa which were then followed by attachment to the

22

surface of the selective electrode.

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1 2

Supporting Fig.10 Fabrication of potentiometric acrylamide biosensor by immobilizing

3

intact bacterial cells of Pseudomonas aeruginosa (Source: Silva et al., 2011)

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Though, a considerable loss of cells occurred, for every preparation biosensor was used,

6

primarily at the initialization of the assay, ammonium electrode was used for the immobilization

7

of membranes, and after the assay these membranes were removed for storage purposes. This

8

confirmation suggested a premature decrease in the biosensor’s stability. As an alternative of

9

using single membrane disks, “sandwich” designed with two membrane disks were considered.

10

In this method cells were remain persisted between the membranes, never contacting the

11

electrode’s surface, avoiding their premature loss. Thus, the functioning of the biosensor could

12

be maintained for longer periods of time. The investigative performance of the biosensor was

13

then studied which showed characteristic response at 120 mV (after 6 min reaction time), a

14

Nernstian slope of 48 mV/decade, LOD 6.31 × 10−4 mol/L and a half-life time of 27 days (Silva

15

et al., 2011).

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Another marker-free cell-based electrochemical sensor was fabricated to scrutinize the lethal

17

consequences of acrylamide on the pheochromocytoma cells, which was simple as well as

18

sensitive. The surface of the electrode was altered by immobilizing gold nanoparticles (AuNPs)

19

and electrochemically reduced with GO. This modified AuNPs/GO electrode was then confirmed

20

by CV, EIS, and DPV. Reduced GO provide evidence to enhance electron-transfer rate between

21

the cell and surface of electrode, whereas AuNPs retained cell bioactivity. The biosensor showed

22

unique relationship to the logarithmic value of cell numbers ranging from 1.6 × 104 to 1.6 × 107

23

cells/mL, with RSD value of 1.68%. The value of DPV at cell adsorption concentration of 1.6 ×

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30

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107 cells/mL decreased with the concentration of acrylamide in the range, 0.1–5 mmol/L with

2

the detection limit as 0.04 mmol/L. SEM-based morphological and 3-(4,5-dimethylthiazol-2-yl)-

3

2,5-diphenyltetrazolium bromide analysis revealed the results of the electrochemical study. This

4

sensor acted as a valuable tool for determining the toxicity of cells and help in the development

5

of a label-free, easy, fast, and instantaneous detection method (Sun et al., 2013).

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A brief literature of existing electrochemical acrylamide biosensors has been described in

7

Table 2. The acrylamide biosensors work ideally within 2-10s, between pH 4.5-7.4 and have a

8

shelf life upto 100 days at 40C.

9

Merits

11 12 13

These biosensors were stable, measure fast response and long storage time. Demerits

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But the major drawback was the requirement of living organism, making it difficult to handle, delayed response time as well as keeping bacteria safe from the environment pollution.

14 15

Table 2:- Comparison of various electrochemical biosensors for detection of acrylamide Linear Respons Interfering Storage range in e time (s) compound stability (µmol/L) at 4°C in days

7.4

0.39

.70-70

ND

ND

9

0.0002

0.05-0.07

<2

ND

100

ND

ND

ND

ND

ND

ND

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Noncovalently/ covalently interaction

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MIP/Au NPsMWCNTsCS/GCE, NIP/Au NPs-MWCNTCS/GCE, MIP/MWCNTsCS/GCE and NIP/MWCNTsCS/GCE cMWCNT/CuNP/ PANI

Detection limit (µmol/L)

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Support for Methods of Optimum immobilization immobilization pH

Covalent 5.5 immobilizations

Hb/DDAB ND carbon paste electrode

4.8

1.2x10-10 M

-11

1.3x10 -3 5.6-10 -11

Hb/SWCNT

ND

1.ox10 -

5.0 31

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-10

Glassy carbon electrode

Covalent Hb/cMWCNTFe3O4NP/CHIT/A immobilization u Gold electrode

6.31x10-4

ND

5.0

0.2x10-6

3x10-6 90x10-6

ND

DNA/GO/GCE

ND

DDAB/Hb/GCE

ND

ssDNA/GE

ND

ND

1

0.1

to 8

4x10-5

ND

Ti/2-27

ND

ND

0.1-100

<2

Acrylic acid and propionic acid

1x10-4 5x10-3

to ND

ND

ND

to ND

ND

ND

ND

5x10-8 1x10-3

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND= Not detected, MIP = Molecular imprinting, CS= Chitosan, MWCNT= Multiwalled carbon

3

nano tubes, GR= Graphene, MWCNT= Multi walled carbon nanotubes, CILE= Carbon ionic

4

liquid

5

Dimethyldioctadecyle ammonium bromide, GE= Gold electrode, HbNPs =Hemoglobin

6

nanoparticles

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2

electrode,

GC=

120

ND

EP

Nafion/Hb-GRMWCNT/CILE

5.0

3.5 min.

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Covalent immobilization

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HbNPs/AuE

ND

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ion ND

1.0x10

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-9

1.2x10

Glassy

carbon,

GCE=

32

Glassy

carbon

electrode,

DDAB=

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5. Latest reports on acrylamide biosensors In our laboratory we have developed an amperometric acrylamide biosensor based on HbNPs. This HbNPs/AuE biosensor was employed for the detection of acrylamide in different brands of various thermally processed food items (Yadav et al., 2018).

The results obtained

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from this study revealed that the HbNPs/AuE acrylamide biosensor was highly sensitive, better reproducibility, improved analytical performance and minimum response time.

We have also fabricated paper based electrochemical biosensor based on HbNPs for acrylamide determination (Yadav et al., 2018). This paper based acrylamide biosensor has

SC

exhibited several advantages such as economic, sensitive, specific, low transient time and good

6. Conclusion and future perspective

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analytical performance.

It is found in the baked foods items and responsible for causing various types of potential carcinogenicity, neurotoxicity, reproductively toxicity and cardiac toxicity. Various conventional investigative methods have been used for the detection of acrylamide however, due to their limitations, biosensing methods have been considered better as they are simple sensitive,

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selective, rapid, accurate and cost effective. Hence, in our opinion, there is a need to develop such devices which should be very quick in detecting any kind of harmful analyte in all kinds of consumable food items. Moreover, people should be aware for the restricted or no use of thermally processed food items. Consequently, people are more suffering from various types of

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fatal diseases. Hence, there is a need to put more efforts in research and technology which provide a better option for resolving all these issues and challenges . The future research should

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be focused on the miniaturization of the laboratory model of acrylamide biosensor into a portable model for its use in the field.. 7. Acknowledgement: One of the authors (Neelam) is thankful to M.D University, Rohtak for awarding university research scholarship (URS) during the tenure of her Ph.D. work. 8. Author information

*Corresponding Author: Prof. C.S. Pundir; E-mail: [email protected] Phone: +919416492413 9. References

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1

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‘Table Captions’ Table 1:- A comparison of analytical parameters of methods for detection of acrylamide in processed foods

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Table 2:- Comparison of various electrochemical biosensors for detection of acrylamide

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Table 1:- A comparison of analytical parameters of various methods for detection of acrylamide in processed foods.

HPLCUV

Recovery (% age)

RSD (%)

Reference

81.6-99.0

0.4-4.5

Zhang et al., 2007

2-95

<5

1~200

1

3

Coffee

2~100

5

16

Cereal-based foods

1~2,000

6

Tea

1~20

1

Infant foods

0.1~200

1

Potato chips

10~1,000

18

90.6-98.5

1.8

5

74-79

1.6-8.3

3

87-96

<6.5

5

-

81.9-95.7

5.3-13.4

3µg/kg

10µg/kg

91-95.3

-

Bortolomea zzi et al., 2012 Senyuua 2006 Liu et al., 2008 Zhang et al., 2005 Yamazaki et al., 2012 Hua et al, 2017

0-750µg/kg 1.5µg/k g

5.0µg/kg

99.3

-

Alpozen al, 2015

8.0 mg/kg

25 mg/kg

89.0– 103%.

2.78.9%.

Coffee, beans potato 18-6968 chips and French fries

3.6

12.1

92-95

-

Potato, flour, twisted 0.005cruller, potato chips 50mg/L and toast samples

1.5µg/k g

-

85.3-94,6

2.256.75

10

84-97

2-10

2

36

91-99

<4

25µg/kg

75µg/kg

82.9-104.2

7.2-9.8

Products of rice, 10-3649 potato corn and wheat µg/kg

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Baked and deep-fried

GC-MS (GC based)

LOQ (µg/kg)

Potato chips

Bread samples

MSPDHPLC

LOD (µg/kg)

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Food Linear Range

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LCMS/MS (HPLC, UPLC)

of

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Standard methods

Name Sample

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Method

Chinese foods

Coffee Cereal-based foods

0~1,500

5

5~50,000

Infant powdered 150-5000 formula, coffee and chocolate powders, corn snacks, bakery 2

et

Wang et al, 2013 Oroian et al, 2015 Zhao et al, 2015

Soares et al., 2010 De Vleeschouw er et al., 2007 Pacettia et al, 2015)

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products and tuber-, meat- and vegetablebased foods. chips,

Corn -

0.5µg/L

5.0 µg/L

70-80

-

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SC

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Potato chips

3

Hariri et al, 2015

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Potato crisps

9.2x10-4 ~3.4x103

8.5x10-3

-

-

-

Stobiecka et al., 2007

Potato crisps

5.3x106

1.4x10-2

-

95.4097.56

-

Batra et al., 2013

MnDoped ZnS Quantu m Dots

Potato chips, biscuits, 2-20µg/ml baby foods, coffee cream, bread

0.56 µg/ml

0.56-1,85 µg/ml

98.3-101.5

0.230.47

Demirhan et al, 2017

UVCapillar y electrop horesis

Potato, Egg plant, 2.5Chick peas, Soft wheat 40mg/L flour, Sorghum Durra flour

0.320.56mg/ L

1.061.85mg/L

85-108

0.725.68

Omar et al, 2017

Pringles crisps

51.763,311.5

65.7

Mashed potatoes

50-1,280

-

-

Preston et al., 2008

50

350

92.6-95.5

-

-

-

90-110.5

-

Fu et al., 2011 Sun et al., 2013

35-350,000

35

-

-

-

French fries, fried 50-20,000 puffs, fried chicken roll, bread, biscuits Potato chips 0.1 µmol L−1 to 80 µmol L−1

15

-

66.0-110.6

-

28.6 nmol L−1,

-

-

-

French cracker

fries

and -

Comput er vision

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Colorim etric method

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Potato chips Fluoresc ence

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ELISA

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Electroc hemical Biosens ors

Potato chips

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Rapid methods

Cookies

Correlation coefficient

Hu et al., 2016

Prediction accuracy

0.989

98% at a threshold of 1000µg/kg

0.946

100% at a threshold of 150µg/kg

4

Hu et al., 2014 Liu et al., 2014

Gokmen et al., 2006 Gokmen et al., 2008

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Linear range (µM)

0.39

.70-70

Covalent 5.5 immobilizations

Hb/DDAB ND carbon paste electrode

4.8

Respons Interfering Storage Applications Reference in e time (s) compound stability at 4°C in days

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7.4

0.0002

0.05-0.07

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Noncovalently/ covalently interaction

1.2x10-10 M

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MIP/Au NPsMWCNTsCS/GCE, NIP/Au NPs-MWCNTCS/GCE, MIP/MWCNTsCS/GCE and NIP/MWCNTsCS/GCE cMWCNT/CuNP/ PANI

Detection limit (µM)

SC

Support for Methods of Optimum immobilization immobilization pH

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Table 2:- Comparison of various electrochemical biosensors for detection of acrylamide

-11

1.3x10 -3 5.6-10

ND

9

Detection of Xia et acrylamide 2015 in potato chips

al.,

<2

ND

100

ND

ND

ND

ND

Stobiecka al., 2007

ND

ND

ND

ND

Krajewska et al., 2008

3.5 min.

ND

Ti/2-27

ND

Silva et al.,

Detection of Batra et al., acrylamide 2012 in potato chips

et

-11

Hb/SWCNT ND Glassy carbon electrode

5.0

1.2x10

1.ox10 -9 1.0x10

Ammonium

ND

6.31x10-4

ND

ion ND

ND

-10

5

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selective

2011 3x10-6 90x10-6

HbNPs/AuE

Covalent immobilization

5.0

0.1

0.1-100

Nafion/Hb-GRMWCNT/CILE

ND

ND

4x10-5

1x10-4 5x10-3

DNA/GO/GCE

ND

ND

ND

5x10-8 1x10-3

DDAB/Hb/GCE

ND

ND

ssDNA/GE

ND

ND

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EP

to 8

ND

ND

Batra et 2013.

Acrylic acid and propionic acid

120

Detection of acrylamide in various types of biscuits, cakes, chips, bread, fried cereals, fried nuts and kurkure

Yadav et al., 2018

to ND

ND

ND

ND

Sun et 2013

to ND

ND

ND

ND

Li et al., 2014

Wu et al., 2015 Huang et al., 2016

<2

ND

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0.2x10-6

SC

5.0

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ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

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ND= Not detected, MIP = Molecular imprinting, CS= Chitosan, MWCNT= Multiwalled carbon nano tubes, GR= Graphene, MWCNT= Multi walled carbon nanotubes, CILE= Carbon ionic liquid electrode, GC= Glassy carbon, GCE= Glassy carbon electrode, DDAB= Dimethyldioctadecyle ammonium bromide, GE= Gold electrode, HbNPs =Hemoglobin nanoparticles 6

al,

al.,

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Highlights
Acrylamide (2-propanamide), an unsaturated amide, occurs in thermally processed (baked/fried) foods

containing asparagine are heated at a very high temperature.

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Acrylamide is generated when baked food items consisting reducing sugars and protein
Acrylamide is potentially neurotoxic and carcinogenic in nature.


Among the various methods available for detection of acrylamide, biosensing methods are comparatively more simple, rapid, sensitive and specific.

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Present review describes in detail the occurrence, generation, toxicity and determination

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methods of acrylamide with special emphasis on biosensors.

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'Figure Captions' Fig.1 Mechanisms of formation of acrylamide in processed foods

acrylamide in processed food (Friedman, 2003).

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Fig.2 Chemical reaction between amino acid asparagine and glucose showing the formation of

Fig.3 Various methods for acrylamide detection in thermally processed foods.

Fig.4 Schematic demonstration of the mechanism of the fluorescent sensing method for the

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detection of acrylamide based on CdSe/ZnS quantum dots (Qinqin et al, 2015)

Fig.5 (a) Diagrammatic depiction of the Michael addition reaction between GH and acrylamide

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with catalysis of TCEP, as well as the mechanism of nucleophile initiated thiol–ene Michael addition reaction (Hu et al., 2016).

Fig.5 (b) Diagrammatic depiction of the detection mechanism based on the different dispersion of AuNPs in the presence or absence of acrylamide (Hu et al, 2016) Fig.6 Schematic depiction of preparation of complete antigen, antibody and competitive indirect

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ELISA for acrylamide detection (Qinqin et al, 2015)

Fig. 7 Schematic representation of processes for preparation of AM-DSMIPs-GO-Fe3O4 (Hu et al, 2014)

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Fig.8 Principle and working of a biosensor

Fig.9 Electrochemical reactions involved in functioning of acrylamide biosensor based

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on HbNPs (Friedman, 2003).

Fig. 10 Depiction of first, second and third generation of amperometric biosensor Fig.11Schematic

representation

of

amperometric

acrylamide

biosensor

based

Hb/cMWCNT/Fe3O4/CHIT/AuE (Batra et al, 2012).

Fig.12 Reaction for adduct formation between HbNPs and acrylamide (Yadav et al, 2017)

on

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Fig.13 Schematic illustration of chemical reaction involved in the fabrication of HbNPs

onto

Au electrode (Yadav et al, 2017) Fig.14

Schematic

representation

of

construction

of

composite

biosensor-Hb-

DDAB/PtAuPdNPs/Ch-IL/MWCNTs-IL/GCE for acrylamide biosensor

acrylamide

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Fig.15 Schematic representation of construction of ssDNA/GE and application for detection of

Fig.16 Fabrication of potentiometric acrylamide biosensor by immobilizing intact bacterial cells

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of Pseudomonas aeruginosa (Source: Silva et al., 2011)

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