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Detection of Acrylamide by Biosensors
CHAPTER 26
Detection of Acrylamide by Biosensors Bhawna Batra, Chandra S. Pundir Department of Biochemistry, M.D. University, Rohtak, Haryana, India
INTRODUCTION Acrylamide is a well-known neurotoxin and potential carcinogen. It is a probable human carcinogen, which is formed during high-temperature cooking of many commonly consumed foods. It is derived from the well-known reaction between reducing sugars and proteins/amino acids (mainly asparagine). However, other routes are also possible.Various reports suggest that high temperatures (>120 °C) are needed for forming acrylamide in foods. High levels of this compound have been found in potato crisps, French fries, and several other common foods [1–3]. Other parameters such as oil type and amount, the glucose–asparagine ratio, and storage practices during potato frying influence acrylamide formation and its content in food. A very high amount of this toxic compound (4 mg/kg) has been found in potato chips. Among analytical methods used for determination of acrylamide levels, the expensive and time-consuming chromatographic techniques such as GC–MS [4], GC–MS–MS [5], HPLC–MS [5], and LC–MS–MS [6,7] predominate. These chromatographic methods require preparation of food samples, which involves extraction using water or methanol and the cleanup step typically consisting of a combination of several solid-phase extractions. GC–MS often needs an additionally laborious bromination step to form a more volatile acrylamide derivative and increase the selectivity of the determination.The growing demand for rapid and precise determination of acrylamide has stimulated to develop alternative methods for screening this neurotoxin cum carcinogen. A few techniques for detection of this toxic compound are available. The biosensors offer great promise for detection of biomolecules because of their simplicity, rapidity, and high sensitivity.
BASIC CONCEPT OF BIOSENSOR A biosensor can be defined as a compact analytical device incorporating a biological or biologically derived sensing element either integrated within or intimately associated with a physicochemical transducer. A biosensor consists of a bio-element and a sensorelement. The bio-element may be an enzyme, antibody, living cells, tissue, or so on, and the sensing element may be electric current, electric potential, or so on. Different combinations of bio-elements and sensor-elements constitute several types of biosensors to Acrylamide in Food http://dx.doi.org/10.1016/B978-0-12-802832-2.00026-7
Copyright © 2016 Elsevier Inc. All rights reserved.
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suit a vast pool of applications. The usual aim of a biosensor is to produce either discrete or continuous digital electronic signals, which are proportional to the concentration of single analyte or a related group of analytes. The key part of the biosensor is the transducer, which makes use of the physicochemical change accompanying the reaction. Several types of transducers have been used in the development of biosensors [8]. This chapter provides a comprehensive overview of recent advances in electrochemical acrylamide biosensors.
ACRYLAMIDE BIOSENSORS Only two types of acrylamide biosensors namely amperometric and potentiometric have been described in the literature.
Classification of Acrylamide Biosensors Amperometric Acrylamide Biosensors Amperometric biosensors monitor current generated, when electrons are exchanged either directly or indirectly between a biological system and an electrode [9]. Principle: The idea of acrylamide biosensors relay on the reaction of hemoglobin (Hb) with acrylamide, which leads to the creation of a Hb–acrylamide adduct, which could alter the electroactivity of Hb. As a consequence, in parallel with an increase in Hb–acrylamide adduct concentration at the electrode surface, the current peak of cyclic voltammogram (CV) decreases. Thus, a decrease in current can be treated as the analytical signal, and could be a base for very selective and sensitive amperometric acrylamide. Therefore, hemoglobin serves as useful biomarker of human exposure to acrylamide. Classification of Amperometric Acrylamide Biosensor Based on Types of Electrode
Carbon Electrode-Based Acrylamide Biosensors These can be classified further on the basis of whether nanomaterials were employed or not. Acrylamide Biosensors Without Nanomaterials Stobiecka et al. [10] constructed a carbonpaste electrode modified with Hb, which contains four prosthetic groups of heme– Fe(III). Such an electrode displays a reversible reduction–oxidation process of Hb–Fe(III)/ Hb–Fe(II). Acrylamide forms adduct with Hb as a result of the reaction with the alphaNH2 group of N-terminal valine of Hb. Interaction between Hb and acrylamide occurred through decreasing of the peak current of Hb–Fe(III) reduction. The electrodes presented a very low detection limit (1.2 × 10−10 M). The validation made in the matrix obtained by water extraction of potato chips showed that the electrodes were suitable for the direct determination of acrylamide in food samples. Li et al. [11] proposed an acrylamide biosensor based on the interaction between acrylamide and DNA, which was confirmed by UV–visible spectroscopy. According to
Detection of Acrylamide by Biosensors
such an interaction, a label-free DNA biosensor for electrochemical determination of acrylamide was proposed. To fabricate the sensor, graphene oxide (GO) was first coated on glassy carbon electrode (GCE) surface. Then, DNA was immobilized on GO/GCE by electroadsorption. Due to large surface area of GO, DNA was effectively immobilized on the electrode surface. Moreover, the unique nanostructure and excellent electron transfer ability of GO promoted the direct electron transfer of DNA significantly. Thus, DNA showed two strong oxidation peaks on GO/GCE, which was the electrochemical signal for acrylamide sensing.The influences of adsorption potential and adsorption time on the immobilized DNA and pH for acrylamide sensing were systematically investigated. Under optimum conditions, the response of DNA/GO/GCE was linear to the concentration of acrylamide from 5.0 × 10−8 to 1.0 × 10−3 mol/L. Moreover, the sensor showed good reproducibility and high stability. Acrylamide Biosensors Employing Nanomaterials Krajewaska et al. [12] presented the use of glassy carbon electrodes coated with single-walled carbon nanotubes (SWCNTs) and Hb for amperometric detection of acrylamide in water solutions. The presence of toxic acrylamide in a wide range of food products such as potato crisps, French fries, or bread was confirmed. The biosensor had a very low detection limit (1.0 × 10−9 M). The validation made in the matrix obtained by water extraction of potato crisps showed that the electrodes were suitable for direct determination of acrylamide in food samples. Batra et al. [13] described the construction of a highly sensitive electrochemical biosensor for the detection of acrylamide, based on covalent immobilization of Hb onto carboxylated multiwalled carbon nanotube/copper nanoparticle/polyaniline (cMWCNT/CuNP/PANI) nanocomposite electrodeposited onto pencil graphite electrode. The enzyme electrode was characterized by CV, scanning electron microscopy (SEM), X-ray diffraction, transmission electron microscopy, Fourier transform infrared (FTIR) spectroscopy, and electrochemical impedance spectroscopy (EIS). The biosensor showed an optimal response at pH 5.5 (0.1 M sodium acetate buffer) and 35 °C when operated at 20 mV/s. It exhibited low detection limit (0.2 nM) with high sensitivity (72.5 μA/nM/cm2), fast response time (<2 s), and wide linear range (5 nM–75 mM). Analytical recovery of added acrylamide was 95.40–97.56%. Within- and between-batch coefficients of variation were 2.35% and 4.50%, respectively. The enzyme electrode was used 120 times over a period of 100 days, when stored at 4 °C. Gold Electrode-Based Amperometric Acrylamide Biosensor Batra et al. [14] described the construction of an improved amperometric acrylamide biosensor based on covalent immobilization of Hb onto nanocomposite of cMWCNT and iron oxide nanoparticles (Fe3O4NPs) electrodeposited onto Au electrode through chitosan (CHIT) film. The Hb/cMWCNT/Fe3O4NP/CHIT/Au electrode was characterized
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by SEM, FTIR spectroscopy, EIS, and differential pulse voltammetry at different stages of its construction. The biosensor was based on the interaction between acrylamide and Hb, which led to decrease in the electroactivity of Hb; that is, current generated during its reversible conversion [Fe(II)/Fe(III)]. The biosensor showed optimum response within 8 s at pH 5.0 and 30 °C. The linear/working range for acrylamide was 3–90 nM, with a detection limit of 0.02 nM and sensitivity of 36.9 μA/ nM/cm2. The biosensor was evaluated and employed for determination of acrylamide in potato crisps. Potentiometric Acrylamide Biosensors Principle: The potentiometric acrylamide biosensor is based on a direct biochemical interaction between acrylamide and intact bacterial cells.The biological recognition element consisted of whole cells of Pseudomonas aeruginosa containing intracellular amidase activity, which catalyzes the hydrolysis of acrylamide-producing ammonium ion (NH4 + ) detected by an ammonium ion selective electrode. Silva et al. [15] developed a biosensor for toxic amides using whole cells of P. aeruginosa containing amidase activity, which catalyzes the hydrolysis of amides such as acrylamide-producing ammonia and the corresponding organic acid.Whole cells immobilized in several types of membrane in the presence of glutaraldehyde and an ammonium ionselective electrode were used for biosensor development. This biosensor exhibited a linear response in the range of 0.1–4.0 × 10−3 M of acrylamide, a detection limit of 4.48 × 10−5 M acrylamide, a response time of 55 s, a sensitivity of 58.99 mV/mM of acrylamide and a maximum t1/2 of 54 days.The selectivity of this biosensor toward other amides was investigated, which revealed that it cross-reacted with acetamide and formamide, but no activity was detected with phenylacetamide, p-nitrophenylacetamide, and acetanilide. It was successfully used for quantification of acrylamide in real industrial effluents, and recovery experiments were carried out, which revealed an average substrate recovery of 93.3%.The biosensor was cheap, since whole cells of P. aeruginosa could be used as source of amidase activity. The first electrochemical biosensor for acrylamide determination was based on a direct biochemical interaction between the analyte and intact bacterial cells, with intracellular enzymatic activity.The biological recognition element consisted of whole cells of P. aeruginosa containing intracellular amidase activity, which catalyzes the hydrolysis of acrylamide-producing ammonium ion (NH4 + ) and acrylic acid. The transduction process was accomplished by means of an ammonium ion selective electrode. Whole cells were first immobilized on single disks of polymeric membranes, such as polyethersulfone, nylon, and polycarbonate, which were, then, attached to the surface of the selective electrode. However, a significant loss of cells occurred, each time the biosensor was used, mainly at the beginning of the assay, when the membranes were attached to the ammonium electrode, and after the assay, when removed
Detection of Acrylamide by Biosensors
for storage purposes.This evidence determined a premature decrease in the biosensor’s stability. Instead of using single membrane disks, a “sandwich” design with two membrane disks was considered. This way the cells remain contained between the membranes, never contacting the electrode’s surface, preventing their premature loss. Consequently, the activity of the biosensor could be maintained for longer periods of time. The analytical performance of the biosensor was evaluated. The best results were obtained when polyethersulfone double membranes were used. A typical response of 120 mV (after 6 min reaction time), a Nernstian slope of 48 mV/decade, a limit of detection of 6.31 × 10−4 M and a half-life time of 27 days, were examples of some figures of merit observed for this biosensor [16]. A sensitive, simple, label-free cell-based electrochemical sensor was constructed to monitor the toxic effect of acrylamide on the pheochromocytoma cells. The surface of the electrode was modified with gold nanoparticles (AuNPs) and electrochemically reduced GO. The modified electrode was characterized by CV, EIS, and DPV. Reduced GO was proved to increase electron-transfer rate between the cell and the surface of electrode, while AuNPs retained cell bioactivity. The sensor exhibited good correlation to the logarithmic value of cell numbers ranging from 1.6 × 104 to 1.6 × 107 cells/mL, with RSD value of 1.68%. The value of DPV at cell adsorption concentration of 1.6 × 107 cells/mL decreased with the concentration of acrylamide in the range, 0.1–5 mM with the detection limit as 0.04 mM. SEM-based morphological and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis confirmed the results of the electrochemical study. This sensor was proved to be a useful tool for probing the toxicity of cells, and assisted in the development of a labelling-free, simple, rapid, and immediate detection method [17]. A novel fluorescent sensing method based on acrylamide polymerization-induced distance increase between quantum dots (QDs) was proposed for detecting acrylamide in potato chips. The functional QDs were prepared by their binding with N-acryloxysuccinimide (NAS), which was characterized by FTIR spectra. The carbon–carbon double bonds of NAS modified QDs polymerized with assistance of photo initiator under UV irradiation, leading to QDs getting closer along with fluorescence intensity decreasing. Acrylamide in the sample participated in the polymerization and induced an increase of fluorescence intensity. This method possessed a linear range from 3.5 × 10−5 to 3.5 g/L (r2 = 0.94) and a limit of detection of 3.5 × 10−5 g/L. Although the sensitivity and specificity could not be compared with standard LC–MS–MS analysis, this new method required much less time and cost, which was promising for online rapid detection of acrylamide in food processing [18].
Comparison of Various Acrylamide Biosensors Table 1 summarizes the comparison of analytical characteristics of various acrylamide biosensors.
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Table 1 A comparison of analytic characteristics of different acrylamide biosensors Properties
Type of electrode used Optimum pH Linearity Detection limit Response time Sensitivity Storage stability
Stobiecka et al. [10]
Krajewaska et al. [12]
Carbon paste electrode
Glassy carbon electrode
4.8
5.0
1.3 × 10−11 to 5.6 × 10−3 M 1.2 × 10−10 M
1.0 × 10−11 to 1.0 × 10−3 M 1.0 × 10−9 M
0.1 to 4.0 × 10−3 M 4.48 × 10−5 M
–
–
– –
Silva et al. [15]
Silva et al. [16]
Batra et al. [14]
Batra et al. [14]
Ammonium ion selective electrode
Ammonium ion selective electrode
Pencil graphite electrode
Gold electrode
5.5
5.0
6.31 × 10−4 M
5 × 10−6 to 75 × 10−6 M 0.2 × 10−6 M
3 × 10−6 to 90 × 10−6 M 0.02 × 10−6 M
55 s
3.5 min
2 s
8 s
58.99 mV/mM t1/2-54 days
t1/2-27 days
72.5 μA/nM/cm2 100 days
36.9 μA/nM/cm2 –
Sun et al. [17]
Li et al. [11]
Gold electrode
Glassy carbon electrode
1 × 10−4 to 5 × 10−3 M 4 × 10−5 M
5.0 × 10−8 to 1 × 10−3 M
Detection of Acrylamide by Biosensors
CONCLUSION AND FUTURE OUTLOOK The acrylamide biosensors worked optimally in following conditions: pH 7.5–7.75, concentration range 1–1950 μM, detection limit 2–111 μM, and storage stability 40–95 days. Recently, acrylamide bio-nanosensors were developed, but the futuristic goal of lowcost, high-throughput, multiplexed diagnostic laboratory on-a-chip devices is yet to be truly realized. A great deal of excitement has been generated by bio-nanosensors, due to their ability to detect a wide range of materials at incredibly small concentrations with high sensitivity. The growth in sensor technology to enter the commercial market has accelerated tremendously, due to nanotechnology.
KEY FACTS • A crylamide, a neurotoxin and potential carcinogen, is formed during high-temperature cooking of food. • GC–MS, GC–MS–MS, and HPLC–MS are prominent methods but expensive, timeconsuming, and cumbersome. • Biosensing methods are simple, rapid, sensitive, and specific. • Two types of acrylamide biosensors are amperometric and potentiometric biosensors. • Acrylamide biosensors work optimally at pH 7.50–0.7.75 and acrylamide concentration range of 1–1950 μM. • Detection limit and storage stability of acrylamide biosensor are in the range 2–111 μM and 40–95 days, respectively.
MINI DICTIONARY Amperometric biosensor It measures signal in current (in amp.) and is linearly dependent to the amount of analyte. Biosensor An analytical device that converts a biological response into a quantifiable and processable signal. It consists of a biological receptor, transducer, and processor. Carbon nanotubes (CNTs) These are long thin cylinders of carbon.These are large macromolecules that are unique for their size, shape, and remarkable physical properties. CNTs with single cylindrical tubes are called SWCNTs and multiple-walled tubes are named MWCNTs. Functionalized/carboxylated MWCNTs are known as cMWCNT. Chitosan A polysaccharide of glucosamine, obtained by partial deacetylation of chitin, is the major component of crustacean shell. It has free –NH2 groups and is used widely for immobilization of enzymes, due to its excellent, biocompatibility, film-forming ability, physiological inertness, and high mechanical strength. Copper nanoparticles (CuNPs) These are a kind of NPs that are formed by the reduction of cupric chloride in solution. Iron oxide/magnetic nanoparticles (Fe3O4 NPs) These are a kind of NPs that have large surface area and good biocompatibility.
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Nanomaterials The materials when reduced down to 1–100 nm in their dimensions show drastic changes with respect of their physical, chemical, magnetic, mechanical, and electrochemical properties. Different nanostructures include nanotubes, nanofibers, nanorods, nanoparticles (NPs), and thin films. NPs have attracted attention, due to their applications in construction of improved analytical devices. Polyanline (PANI) It is a polymer of aniline, which is formed by cyclic voltammetry of analine. Potentiometric biosensor It measures signal in potential (in volts) and has logarithmic relationship with the amount of product formed from analyte.
SUMMARY POINTS • A crylamide, neurotoxin and potential carcinogen, is formed during high-temperature (>120 °C) cooking of many commonly consumed foods. It is derived from reactions between reducing sugars and proteins/amino acids (mainly asparagine). • Prominent chromatographic methods for determination of acrylamide levels such as GC–MS, GC–MS–MS, HPLC–MS, and LC–MS–MS are expensive, time-consuming, and require sample preparation. • Biosensors offer great promise for the detection of acrylamide, because of their simplicity, rapidity, and high sensitivity. • Two types of acrylamide biosensors are reported with or without nanomaterials: amperometric and potentiometric biosensors. • The acrylamide biosensors worked optimally under following conditions: pH 7.5–7.75, concentration range, 1–1950 μM, detection limit 2–111 μM, and storage stability 40–95 days. • The miniaturization of these bio-nanosensors is expected to provide smart sensing devices.
REFERENCES [1] Tareke E, Rydberg P, Karlsson S, Eriksson M, Törnqvist M. Analysis of acrylamide, a carcinogen formed in heated foodstuffs. J Agric Food Chem 2002;50:4998–5006. [2] Gökmen V, Palazoğlu TK, Şenyuva HZ. Relation between the acrylamide formation and time– temperature history of surface and core regions of French fries. J Food Eng 2006;77:972–6. [3] Becalski A, Lau BP-Y, Lewis D, Seaman S. Acrylamide in foods: occurrence, sources and modeling. J Agric Food Chem 2003;51:802–8. [4] Motaghi MM, Ardebili MS, Honarvar M, Mehrabani M, Baghizadeh A. Determination of acrylamide in selected types of Iranian breads by SPME technique. J Food Biosci Technol 2012;2:57–64. [5] Ono H, Chuda Y, Ohnishi-Kameyama M,Yada H, Ishizaka M, Kobayashi H, et al. Analysis of acrylamide by LC-MS/MS and GC-MS in processed Japanese foods. Food Addit Contam 2003;20(3):215–20. [6] Rosen J, Hellanas KE. Analysis of acrylamide in cooked foods by liquid chromatography tandem mass spectrometry. Analyst 2002;127:880–2. [7] Cheng WC, Kao YM, Shih DYC, Chou SS,Yeh AI.Validation of an improved LC/MS/MS method for acrylamide analysis in foods. J Food Drug Anal 2009;17:190–7. [8] Turner APF, et al. In: Biolayer modelling and optimization for SPARROW biosensor. Chapter 1.UMI. Ann Arbor (MI, USA): ProQuest Information & Learning Company; 1987. p. 1–18. [9] Zang S, Wright G, Yang Y. Materials and techniques for electrochemical biosensor design and construction. Biosens Bioelectron 2000;15:273–82.
Detection of Acrylamide by Biosensors
[10] Stobiecka A, Radecka H, Radecki J. Novel voltametric biosensor for determining acrylamide in food samples. Biosens Bioelectron 2007;22:2165–70. [11] Li D, Xu Y, Zhang L, Tong H. A label free electrochemical biosensor for acrylamide based on immobilization on grapheme oxide–modified glassy carbon electrode. Int J Electrochem Sci 2014;9:7217–8. [12] Krajewaska A, Rodecki J, Rodecka H. A voltametric biosensor based on glassy carbon electrode with SWCNT/Hb for detection of acrylamide in water extracts from potato crisps. Sensors 2008;8:5832–44. [13] Batra B, Lata S, Sharma M, Pundir CS. An acrylamide biosensor based on immobilization of haemoglobin onto multiwalled carbon nanotubes/copper nanoparticles/polyaniline hybrid film.Anal Bichem 2012;433: 210–7. [14] Batra B, Lata S, Pundir CS. Construction of an improved acrylamide biosensor based on hemoglobin immobilized onto cMWCNT/Fe3O4NPs/CHIT composite film. Bioprocess Biosys Engg 2013;36:1591–9. [15] Silva N, Gil D, Karmali A, Matos M. Biosensor for acrylamide based on ion selective electrode using whole cells of Pseudomonas aeruginosa containing amidase activity. Biocat Biotransform 2008;27:143–51. [16] Silva NAF, Matos MJ, Karmali A, Rocha MM. An electrochemical biosensor for acrylamide determination: merits and limitations. Port Electrochim Acta 2011;29:361–73. [17] Sun X, Jin J, Jiang D, et al. Development of novel electrochemical sensor using pheochromocytoma cells and its assessment of acrylamide toxicity. Biosens Bioelectron 2013;44:122–6. [18] Hu Q, Xu X, Zhang Y, Wang J, Fu Y, Li Y. Detection of acrylamide in potato chips using fluorescent sensing method based on acrylamide polymerization induced distance increase in quantum dots. Biosens Bioelectron 2014;15:54–64.
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Detection of Acrylamide by Biosensors Bhawna Batra, Chandra S. Pundir Department of Biochemistry, M.D. University, Rohtak, Haryana, India
INTRODUCTION Acrylamide is a well-known neurotoxin and potential carcinogen. It is a probable human carcinogen, which is formed during high-temperature cooking of many commonly consumed foods. It is derived from the well-known reaction between reducing sugars and proteins/amino acids (mainly asparagine). However, other routes are also possible.Various reports suggest that high temperatures (>120 °C) are needed for forming acrylamide in foods. High levels of this compound have been found in potato crisps, French fries, and several other common foods [1–3]. Other parameters such as oil type and amount, the glucose–asparagine ratio, and storage practices during potato frying influence acrylamide formation and its content in food. A very high amount of this toxic compound (4 mg/kg) has been found in potato chips. Among analytical methods used for determination of acrylamide levels, the expensive and time-consuming chromatographic techniques such as GC–MS [4], GC–MS–MS [5], HPLC–MS [5], and LC–MS–MS [6,7] predominate. These chromatographic methods require preparation of food samples, which involves extraction using water or methanol and the cleanup step typically consisting of a combination of several solid-phase extractions. GC–MS often needs an additionally laborious bromination step to form a more volatile acrylamide derivative and increase the selectivity of the determination.The growing demand for rapid and precise determination of acrylamide has stimulated to develop alternative methods for screening this neurotoxin cum carcinogen. A few techniques for detection of this toxic compound are available. The biosensors offer great promise for detection of biomolecules because of their simplicity, rapidity, and high sensitivity.
BASIC CONCEPT OF BIOSENSOR A biosensor can be defined as a compact analytical device incorporating a biological or biologically derived sensing element either integrated within or intimately associated with a physicochemical transducer. A biosensor consists of a bio-element and a sensorelement. The bio-element may be an enzyme, antibody, living cells, tissue, or so on, and the sensing element may be electric current, electric potential, or so on. Different combinations of bio-elements and sensor-elements constitute several types of biosensors to Acrylamide in Food http://dx.doi.org/10.1016/B978-0-12-802832-2.00026-7
Copyright © 2016 Elsevier Inc. All rights reserved.
497
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suit a vast pool of applications. The usual aim of a biosensor is to produce either discrete or continuous digital electronic signals, which are proportional to the concentration of single analyte or a related group of analytes. The key part of the biosensor is the transducer, which makes use of the physicochemical change accompanying the reaction. Several types of transducers have been used in the development of biosensors [8]. This chapter provides a comprehensive overview of recent advances in electrochemical acrylamide biosensors.
ACRYLAMIDE BIOSENSORS Only two types of acrylamide biosensors namely amperometric and potentiometric have been described in the literature.
Classification of Acrylamide Biosensors Amperometric Acrylamide Biosensors Amperometric biosensors monitor current generated, when electrons are exchanged either directly or indirectly between a biological system and an electrode [9]. Principle: The idea of acrylamide biosensors relay on the reaction of hemoglobin (Hb) with acrylamide, which leads to the creation of a Hb–acrylamide adduct, which could alter the electroactivity of Hb. As a consequence, in parallel with an increase in Hb–acrylamide adduct concentration at the electrode surface, the current peak of cyclic voltammogram (CV) decreases. Thus, a decrease in current can be treated as the analytical signal, and could be a base for very selective and sensitive amperometric acrylamide. Therefore, hemoglobin serves as useful biomarker of human exposure to acrylamide. Classification of Amperometric Acrylamide Biosensor Based on Types of Electrode
Carbon Electrode-Based Acrylamide Biosensors These can be classified further on the basis of whether nanomaterials were employed or not. Acrylamide Biosensors Without Nanomaterials Stobiecka et al. [10] constructed a carbonpaste electrode modified with Hb, which contains four prosthetic groups of heme– Fe(III). Such an electrode displays a reversible reduction–oxidation process of Hb–Fe(III)/ Hb–Fe(II). Acrylamide forms adduct with Hb as a result of the reaction with the alphaNH2 group of N-terminal valine of Hb. Interaction between Hb and acrylamide occurred through decreasing of the peak current of Hb–Fe(III) reduction. The electrodes presented a very low detection limit (1.2 × 10−10 M). The validation made in the matrix obtained by water extraction of potato chips showed that the electrodes were suitable for the direct determination of acrylamide in food samples. Li et al. [11] proposed an acrylamide biosensor based on the interaction between acrylamide and DNA, which was confirmed by UV–visible spectroscopy. According to
Detection of Acrylamide by Biosensors
such an interaction, a label-free DNA biosensor for electrochemical determination of acrylamide was proposed. To fabricate the sensor, graphene oxide (GO) was first coated on glassy carbon electrode (GCE) surface. Then, DNA was immobilized on GO/GCE by electroadsorption. Due to large surface area of GO, DNA was effectively immobilized on the electrode surface. Moreover, the unique nanostructure and excellent electron transfer ability of GO promoted the direct electron transfer of DNA significantly. Thus, DNA showed two strong oxidation peaks on GO/GCE, which was the electrochemical signal for acrylamide sensing.The influences of adsorption potential and adsorption time on the immobilized DNA and pH for acrylamide sensing were systematically investigated. Under optimum conditions, the response of DNA/GO/GCE was linear to the concentration of acrylamide from 5.0 × 10−8 to 1.0 × 10−3 mol/L. Moreover, the sensor showed good reproducibility and high stability. Acrylamide Biosensors Employing Nanomaterials Krajewaska et al. [12] presented the use of glassy carbon electrodes coated with single-walled carbon nanotubes (SWCNTs) and Hb for amperometric detection of acrylamide in water solutions. The presence of toxic acrylamide in a wide range of food products such as potato crisps, French fries, or bread was confirmed. The biosensor had a very low detection limit (1.0 × 10−9 M). The validation made in the matrix obtained by water extraction of potato crisps showed that the electrodes were suitable for direct determination of acrylamide in food samples. Batra et al. [13] described the construction of a highly sensitive electrochemical biosensor for the detection of acrylamide, based on covalent immobilization of Hb onto carboxylated multiwalled carbon nanotube/copper nanoparticle/polyaniline (cMWCNT/CuNP/PANI) nanocomposite electrodeposited onto pencil graphite electrode. The enzyme electrode was characterized by CV, scanning electron microscopy (SEM), X-ray diffraction, transmission electron microscopy, Fourier transform infrared (FTIR) spectroscopy, and electrochemical impedance spectroscopy (EIS). The biosensor showed an optimal response at pH 5.5 (0.1 M sodium acetate buffer) and 35 °C when operated at 20 mV/s. It exhibited low detection limit (0.2 nM) with high sensitivity (72.5 μA/nM/cm2), fast response time (<2 s), and wide linear range (5 nM–75 mM). Analytical recovery of added acrylamide was 95.40–97.56%. Within- and between-batch coefficients of variation were 2.35% and 4.50%, respectively. The enzyme electrode was used 120 times over a period of 100 days, when stored at 4 °C. Gold Electrode-Based Amperometric Acrylamide Biosensor Batra et al. [14] described the construction of an improved amperometric acrylamide biosensor based on covalent immobilization of Hb onto nanocomposite of cMWCNT and iron oxide nanoparticles (Fe3O4NPs) electrodeposited onto Au electrode through chitosan (CHIT) film. The Hb/cMWCNT/Fe3O4NP/CHIT/Au electrode was characterized
499
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by SEM, FTIR spectroscopy, EIS, and differential pulse voltammetry at different stages of its construction. The biosensor was based on the interaction between acrylamide and Hb, which led to decrease in the electroactivity of Hb; that is, current generated during its reversible conversion [Fe(II)/Fe(III)]. The biosensor showed optimum response within 8 s at pH 5.0 and 30 °C. The linear/working range for acrylamide was 3–90 nM, with a detection limit of 0.02 nM and sensitivity of 36.9 μA/ nM/cm2. The biosensor was evaluated and employed for determination of acrylamide in potato crisps. Potentiometric Acrylamide Biosensors Principle: The potentiometric acrylamide biosensor is based on a direct biochemical interaction between acrylamide and intact bacterial cells.The biological recognition element consisted of whole cells of Pseudomonas aeruginosa containing intracellular amidase activity, which catalyzes the hydrolysis of acrylamide-producing ammonium ion (NH4 + ) detected by an ammonium ion selective electrode. Silva et al. [15] developed a biosensor for toxic amides using whole cells of P. aeruginosa containing amidase activity, which catalyzes the hydrolysis of amides such as acrylamide-producing ammonia and the corresponding organic acid.Whole cells immobilized in several types of membrane in the presence of glutaraldehyde and an ammonium ionselective electrode were used for biosensor development. This biosensor exhibited a linear response in the range of 0.1–4.0 × 10−3 M of acrylamide, a detection limit of 4.48 × 10−5 M acrylamide, a response time of 55 s, a sensitivity of 58.99 mV/mM of acrylamide and a maximum t1/2 of 54 days.The selectivity of this biosensor toward other amides was investigated, which revealed that it cross-reacted with acetamide and formamide, but no activity was detected with phenylacetamide, p-nitrophenylacetamide, and acetanilide. It was successfully used for quantification of acrylamide in real industrial effluents, and recovery experiments were carried out, which revealed an average substrate recovery of 93.3%.The biosensor was cheap, since whole cells of P. aeruginosa could be used as source of amidase activity. The first electrochemical biosensor for acrylamide determination was based on a direct biochemical interaction between the analyte and intact bacterial cells, with intracellular enzymatic activity.The biological recognition element consisted of whole cells of P. aeruginosa containing intracellular amidase activity, which catalyzes the hydrolysis of acrylamide-producing ammonium ion (NH4 + ) and acrylic acid. The transduction process was accomplished by means of an ammonium ion selective electrode. Whole cells were first immobilized on single disks of polymeric membranes, such as polyethersulfone, nylon, and polycarbonate, which were, then, attached to the surface of the selective electrode. However, a significant loss of cells occurred, each time the biosensor was used, mainly at the beginning of the assay, when the membranes were attached to the ammonium electrode, and after the assay, when removed
Detection of Acrylamide by Biosensors
for storage purposes.This evidence determined a premature decrease in the biosensor’s stability. Instead of using single membrane disks, a “sandwich” design with two membrane disks was considered. This way the cells remain contained between the membranes, never contacting the electrode’s surface, preventing their premature loss. Consequently, the activity of the biosensor could be maintained for longer periods of time. The analytical performance of the biosensor was evaluated. The best results were obtained when polyethersulfone double membranes were used. A typical response of 120 mV (after 6 min reaction time), a Nernstian slope of 48 mV/decade, a limit of detection of 6.31 × 10−4 M and a half-life time of 27 days, were examples of some figures of merit observed for this biosensor [16]. A sensitive, simple, label-free cell-based electrochemical sensor was constructed to monitor the toxic effect of acrylamide on the pheochromocytoma cells. The surface of the electrode was modified with gold nanoparticles (AuNPs) and electrochemically reduced GO. The modified electrode was characterized by CV, EIS, and DPV. Reduced GO was proved to increase electron-transfer rate between the cell and the surface of electrode, while AuNPs retained cell bioactivity. The sensor exhibited good correlation to the logarithmic value of cell numbers ranging from 1.6 × 104 to 1.6 × 107 cells/mL, with RSD value of 1.68%. The value of DPV at cell adsorption concentration of 1.6 × 107 cells/mL decreased with the concentration of acrylamide in the range, 0.1–5 mM with the detection limit as 0.04 mM. SEM-based morphological and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis confirmed the results of the electrochemical study. This sensor was proved to be a useful tool for probing the toxicity of cells, and assisted in the development of a labelling-free, simple, rapid, and immediate detection method [17]. A novel fluorescent sensing method based on acrylamide polymerization-induced distance increase between quantum dots (QDs) was proposed for detecting acrylamide in potato chips. The functional QDs were prepared by their binding with N-acryloxysuccinimide (NAS), which was characterized by FTIR spectra. The carbon–carbon double bonds of NAS modified QDs polymerized with assistance of photo initiator under UV irradiation, leading to QDs getting closer along with fluorescence intensity decreasing. Acrylamide in the sample participated in the polymerization and induced an increase of fluorescence intensity. This method possessed a linear range from 3.5 × 10−5 to 3.5 g/L (r2 = 0.94) and a limit of detection of 3.5 × 10−5 g/L. Although the sensitivity and specificity could not be compared with standard LC–MS–MS analysis, this new method required much less time and cost, which was promising for online rapid detection of acrylamide in food processing [18].
Comparison of Various Acrylamide Biosensors Table 1 summarizes the comparison of analytical characteristics of various acrylamide biosensors.
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Table 1 A comparison of analytic characteristics of different acrylamide biosensors Properties
Type of electrode used Optimum pH Linearity Detection limit Response time Sensitivity Storage stability
Stobiecka et al. [10]
Krajewaska et al. [12]
Carbon paste electrode
Glassy carbon electrode
4.8
5.0
1.3 × 10−11 to 5.6 × 10−3 M 1.2 × 10−10 M
1.0 × 10−11 to 1.0 × 10−3 M 1.0 × 10−9 M
0.1 to 4.0 × 10−3 M 4.48 × 10−5 M
–
–
– –
Silva et al. [15]
Silva et al. [16]
Batra et al. [14]
Batra et al. [14]
Ammonium ion selective electrode
Ammonium ion selective electrode
Pencil graphite electrode
Gold electrode
5.5
5.0
6.31 × 10−4 M
5 × 10−6 to 75 × 10−6 M 0.2 × 10−6 M
3 × 10−6 to 90 × 10−6 M 0.02 × 10−6 M
55 s
3.5 min
2 s
8 s
58.99 mV/mM t1/2-54 days
t1/2-27 days
72.5 μA/nM/cm2 100 days
36.9 μA/nM/cm2 –
Sun et al. [17]
Li et al. [11]
Gold electrode
Glassy carbon electrode
1 × 10−4 to 5 × 10−3 M 4 × 10−5 M
5.0 × 10−8 to 1 × 10−3 M
Detection of Acrylamide by Biosensors
CONCLUSION AND FUTURE OUTLOOK The acrylamide biosensors worked optimally in following conditions: pH 7.5–7.75, concentration range 1–1950 μM, detection limit 2–111 μM, and storage stability 40–95 days. Recently, acrylamide bio-nanosensors were developed, but the futuristic goal of lowcost, high-throughput, multiplexed diagnostic laboratory on-a-chip devices is yet to be truly realized. A great deal of excitement has been generated by bio-nanosensors, due to their ability to detect a wide range of materials at incredibly small concentrations with high sensitivity. The growth in sensor technology to enter the commercial market has accelerated tremendously, due to nanotechnology.
KEY FACTS • A crylamide, a neurotoxin and potential carcinogen, is formed during high-temperature cooking of food. • GC–MS, GC–MS–MS, and HPLC–MS are prominent methods but expensive, timeconsuming, and cumbersome. • Biosensing methods are simple, rapid, sensitive, and specific. • Two types of acrylamide biosensors are amperometric and potentiometric biosensors. • Acrylamide biosensors work optimally at pH 7.50–0.7.75 and acrylamide concentration range of 1–1950 μM. • Detection limit and storage stability of acrylamide biosensor are in the range 2–111 μM and 40–95 days, respectively.
MINI DICTIONARY Amperometric biosensor It measures signal in current (in amp.) and is linearly dependent to the amount of analyte. Biosensor An analytical device that converts a biological response into a quantifiable and processable signal. It consists of a biological receptor, transducer, and processor. Carbon nanotubes (CNTs) These are long thin cylinders of carbon.These are large macromolecules that are unique for their size, shape, and remarkable physical properties. CNTs with single cylindrical tubes are called SWCNTs and multiple-walled tubes are named MWCNTs. Functionalized/carboxylated MWCNTs are known as cMWCNT. Chitosan A polysaccharide of glucosamine, obtained by partial deacetylation of chitin, is the major component of crustacean shell. It has free –NH2 groups and is used widely for immobilization of enzymes, due to its excellent, biocompatibility, film-forming ability, physiological inertness, and high mechanical strength. Copper nanoparticles (CuNPs) These are a kind of NPs that are formed by the reduction of cupric chloride in solution. Iron oxide/magnetic nanoparticles (Fe3O4 NPs) These are a kind of NPs that have large surface area and good biocompatibility.
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Acrylamide in Food
Nanomaterials The materials when reduced down to 1–100 nm in their dimensions show drastic changes with respect of their physical, chemical, magnetic, mechanical, and electrochemical properties. Different nanostructures include nanotubes, nanofibers, nanorods, nanoparticles (NPs), and thin films. NPs have attracted attention, due to their applications in construction of improved analytical devices. Polyanline (PANI) It is a polymer of aniline, which is formed by cyclic voltammetry of analine. Potentiometric biosensor It measures signal in potential (in volts) and has logarithmic relationship with the amount of product formed from analyte.
SUMMARY POINTS • A crylamide, neurotoxin and potential carcinogen, is formed during high-temperature (>120 °C) cooking of many commonly consumed foods. It is derived from reactions between reducing sugars and proteins/amino acids (mainly asparagine). • Prominent chromatographic methods for determination of acrylamide levels such as GC–MS, GC–MS–MS, HPLC–MS, and LC–MS–MS are expensive, time-consuming, and require sample preparation. • Biosensors offer great promise for the detection of acrylamide, because of their simplicity, rapidity, and high sensitivity. • Two types of acrylamide biosensors are reported with or without nanomaterials: amperometric and potentiometric biosensors. • The acrylamide biosensors worked optimally under following conditions: pH 7.5–7.75, concentration range, 1–1950 μM, detection limit 2–111 μM, and storage stability 40–95 days. • The miniaturization of these bio-nanosensors is expected to provide smart sensing devices.
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Detection of Acrylamide by Biosensors
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