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11 Colorimetric detection of acrylamide in potato chips based on
12
13 nucleophile-initiated thiol-ene Michael addition
14 Received 00th January 20xx, a a b a c a
Accepted 00th January 20xx Qinqin Hu, Yingchun Fu, Xiahong Xu,* Zhaohui Qiao, Ronghui Wang, Ying Zhang and Yanbin
15 Li
*a, c

16 DOI: 10.1039/x0xx00000x

Analyst Accepted Manuscript

17 Acrylamide (AA), a neurotoxin and potential carcinogen, has been found in various thermally processed foods such as
www.rsc.org/
18 potato chips, biscuits, and coffee. Simple, cost-effective, and sensitive methods for rapid detection of AA are needed to
19 ensure food safety. Herein, a novel colorimetric method was proposed for the visual detection of AA based on
20 nucleophile-initiated thiol-ene Michael addition reaction. Gold nanoparticles (AuNPs) were aggregated by glutathione
(GSH) because of ligand-replacement, accompanied with color change from red to purple. In the presence of AA, after the
21
thiol-ene Michael addition reaction between GSH and AA with the catalysis of nucleophile, the sulfydryl group of GSH was
22
consumed by AA, which hindered the followed ligand-replacement and the aggregation of AuNPs. Therefore, the
23
concentration of AA could be determined by the disperse state change of AuNPs. This new method showed high sensitivity
24
with a linear range from 0.1 μmol L-1 to 80 μmol L-1 and a detection limit of 28.6 nmol L-1, and especially revealed better
25
selectivity than the fluorescent sensing method reported previously. Moreover, this new method was used to detect AA in
26
potato chips with a satisfactory result in comparison with the standard methods based on chromatography, which
27 indicated that the colorimetric method can be expanded for rapid detection of AA in thermally processed foods.
28
29 fluorescent method to determine AA based on Hofmann
30 Introduction reaction to degrade AA and then react with fluorescamine
31 leading to the increase of fluorescence intensity. However,
Because of the neurotoxicity, potential carcinogenicity, and
32 high temperature reaction (> 90°C) limited its application.
genotoxicity, acrylamide (AA) has been classified as a
33 “probably carcinogenic to humans” (“2A group”) substance,1 a Recently, our group proposed a fluorescent chemical sensor
34 “very high concern” substance,2 a Category 2 carcinogen, and for the detection of AA based on polymerization-induced
35 Category 2 mutagen.3 Moreover, AA was verified for its high distance increase between quantum dots, initiating optical
15
36 content in common thermally processed foods in 2002,4,5 such sensor for analysis of AA. However, L-asparaginase should be
37 as potato chips, bread, biscuits, and coffee. Therefore, the added to guarantee the specificity when detecting AA in food
38 research on AA in diet, especially its detection, has captured samples. Thus far, simple, rapid, sensitive, and cost-effective
39 attentions for food safety and clinic diagnosis/therapy.6 methods for the analysis of AA in thermally processed foods
40 Standard methods based on chromatography for the detection are still of high demand but very challenging.
41 of AA in foods such as LC-MS/MS and GC-MS have high Colorimetric method based on aggregation or re-dispersion
42 sensitivity, specificity, and repeatability, but cannot satisfy the of gold nanoparticles (AuNPs) has been applied in the
43 need of simple, rapid and cost-effective detection due to the detection of DNA, proteins, small molecules, and metal ions
44 requirement of expensive instruments and skilled.7-10 due to its simplicity, low cost, high sensitivity, rapid response,
45 Therefore, new methods including electrochemical naked-eye visible signal readout, and no requirement of
16
biosensors,11 ELISA,12,13 and fluorescent detection methods14,15 expensive or complex instruments. Generally, the factors
46
have been studied to detect AA in foods. Liu et al.14 designed a that trigger distance change between AuNPs are the key points
47
to design novel colorimetric method. To date, there are two
48
main strategies, biological recognition such as hybridization of
49 17-19 li
DNA and immune-recognition, as well as physical
50 22-25
interactions such as electrostatic and steric effects.
51
However, the interaction depends on the properties that the
52 targets possess, such as biological activity or functional groups.
53 For those without special merits such as AA, it is difficult to
54 design a colorimetric method for facile detection.
55 Thiol-ene Michael addition reaction has been routinely
56 classified as one type of thiol click reactions because it carries
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attributes of click reaction such as achieving quantitative (M81201, Mediwax, USA) with Oasis HLB SPE cartridges (200
3
yields, requiring small amount of catalyst, and having rapid mg, 6 cc, Waters, Milford, MA, USA) was used for clean-up.
4
reaction rates. Recently, a strategy of using powerful and Nitrogen blowing instrument (NBI, HSC-24B, Tianjin Hengao
5 efficient nucleophilic alkyl catalysts (usually primary/second Technology Development Co. Ltd, Tianjin, China) was applied
6 amine or phosphine) has emerged as a model for extremely to concentrate the extract.
7 efficient thiol-ene Michael addition reactions, which has been Preparation of AuNPs
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successfully used for organic synthesis and polymer AuNPs aqueous colloid, with an average particle size of 12.83
9 functionalization. Nucleophilic attack at the electron-deficient nm (determined from the TEM images) and a concentration of
10 ene generates the intermediate, strong (zwitterionic) enolate -8 -1
1.80 × 10 mol L was synthesized according to the Frens’
11 base that is responsible for deprotonating thiol, generating the 29,30 -1
method. Briefly, one hundred milliliters of 1 mmol L
12 thiolate anion. Once the thiolate is formed the anionic chain -1
HAuCl4 solution was reduced by 10 mL of 38.8 mmol L
13 process begins with extremely rapid formation of the thiol-ene 31
trisodium citrate solution. The resulting AuNPs colloid was
14 product.
25-28
Considering that thiols are commonly used stored at 4 °C in the dark before use.
15 reagents to stabilize or aggregate AuNPs, application of thiol- General procedures for detection of AA
16 ene reaction between thiol and AA may explore a novel way to

Analyst Accepted Manuscript

-1
17 realize colorimetric detection of AA. Two hundred microliters of 80 μmol L GSH (1 eq) dissolved in
-1
18 Herein, we present a simple, cost-effective, rapid, and disodium hydrogen phosphate buffer (DHP buffer, 2 mmol L ,
pH = 9.00) was reacted with 200 μL of AA solutions at different
19 sensitive colorimetric method to detect AA based on thiol-ene
concentrations with assistance of TCEP for 2.5 h at the room
20 Michael addition reaction and the optical character of AuNPs.
Glutathione (GSH) contributes the thiol group to react with AA temperature. The ratio of GSH to TCEP was 1 to 0.05 (eq). Then
21
as well as aggregate AuNPs at the acidic environment. The 10 μL of the reaction solution was added into 90 μL of AuNPs
22
established colorimetric biosensor was also employed to dispersed in citric acid-disodium hydrogen phosphate buffer
23 -1
detect AA in potato chips with a satisfactory result. To the best (CD buffer, 2 mmol L , pH = 3.37). After 1 min, the changes in
24 absorption spectra were monitored with a microplate reader
25 of our knowledge, this is the first time to determine AA
intuitively using AuNPs-based colorimetric method with high at absorbance model in the wavelength range from 400 to 750
26 nm. The ratio of AuNPs absorbance at 520 nm and 650 nm
27 sensitivity and selectivity as well as mild reaction conditions.
(A520/650) was used as the signal response to the concentration
28 of AA.
29 Experimental To determine the selectivity, 80 μmol L
-1
different
30 compounds similar to AA in chemical structure, including L-
31 Materials and apparatus
asparagine, methylacrylamide, succinic acid, 6-aminocaproic
32 AA, GSH, Chloroauric acid, hydrate (HAuCl4), L-asparagine, acid, acrylic acid, methylacrylic acid, propionic acid, N-butyric
33 acrylic acid, and methylacrylamide were purchased from -1
acid, and acetic acid reacted with 80 μmol L GSH with the
34 Sangon Biotech Co. Ltd. (Shanghai, China). Succinic acid, 6- catalysis of TCEP (0.05 eq), followed by adding the mixture into
35 aminocaproic acid, N-butyric acid, propionic acid, acetic acid, AuNPs solutions. The changes in absorption spectra were
36 formic acid, citric acid monohydrate, trisodium citrate measured under the same condition as the measurement of
37 dehydrate, disodium hydrogen phosphate dodecahydrate, AA. All of these tests were conducted in triplicate.
38 hydrochloric acid, and nitric acid were from Sinopharm Detection of AA in potato chips
39 Chemical Reagent Co., Ltd. (Shanghai, China). Tris (2-
For sample homogenization, potato chips which were
40 carboxyethyl) phosphine hydrochloride (TCEP) were obtained
purchased from a local supermarket were minced with a
41 from Sigma-Aldrich (St. Louis, MO, USA). All aqueous solutions
blender. A portion of 1.0 ± 0.0100 g was weighed in a 50 mL
42 were prepared using freshly deionized water (18.2 MΩ.cm
centrifugal tube. AA was extracted by adding 10 mL of 0.1%
43 specific resistivity) obtained with Milli-Q water purification
formic acid and mixed well by a vortex for 1 min. In order to
44 system (Millipore, Billerica, MA, USA).
clarify the solution, the mixture was centrifuged at 4°C, 10,000
The UV-vis absorption spectra were carried out on Synergy
45 rpm for 10 min. A thin white fat layer at the top was discarded,
H1 Hybrid Multi-Mode Microplate reader (BioTek, Vinooski,
46 and the supernatant was transferred to another centrifugal
VT, USA) and UV-vis Spectrophotometer (8453, Agilent
47 tube. The steps above were repeated to extract the residues
Technologies, Santa Clara, USA). The pH values were
48 again. Two supernatants were merged after the second
monitored by a sens ION+ PH31 meter (HACH, Loveland, CO,
49 centrifugation, followed by vortex mixture.
USA). AuNPs were purified by centrifugation (Microfuge 22R
50 The clean-up proceeds were referred to the regulations of
centrifuge, Beckman Coulter, Fyllerton, CA, USA). TEM images 32
51 FDA with a little modification. The Oasis HLB cartridges for
were recorded with a transmission electron microscope
52 SPE were preconditioned with 3 mL of methanol and 3 mL of
(JEM1200EX, JEOL, Tokyo, Japan).
53 water. Three milliliters of clear supernatant was loaded onto
For AA extraction from potato chips, a vortex mixer (Vortex
54 the HLB cartridge, followed by elution with 6 mL of Milli-Q
Genie2, Scientific Industries, NY, USA), and a refrigerated
55 water. The cleaned-up aqueous extract was finally
centrifugation (Sigma 3K-15, Osterode am Harz, Germany)
56 concentrated on a heated-block (40 °C) under a stream of
with a rotor (12150-H) were used. Vacuum SPE manifold
57 nitrogen. Finally, AA was dissolved in 0.5 mL Milli-Q water.
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25 Standard addition method or method of standard addition alkaline environment (pH = 9.0) facilitates the dissociation of
36,37
26 (MSA) was carried out via adding different concentrations (0, sulfydryl of GSH (pKa = 8.66) to form anion.
-1
27 2, 4, 6, and 8 μmol L ) of AA standard solutions to the final GSH, a tripeptide containing thiol group, is commonly used
28 sample solutions. A set of A520/650 values were plotted as y-axis, to aggregate or stablize AuNPs. According to the pKa values of
29 and the concentrations of AA added in the portions (unspiked two carboxyl groups (2.12, 3.53) and the amino group
34,35
and four spiked portions) were plotted as x-axis. A calibration (9.12), GSH is positively charged at low pH value (less than
30 36,37
curve was linear fitted and the amount of AA in unspiked 3.60). As shown in Scheme 1(b), at acidic environment (pH
31
portion was the absolute value for x-axis obtained from the = 3.4), positively charged GSH competitively adsorbs on the
32
calibration curve when the value of y-axis was equal to zero. surface of AuNPs through Au-S chemistry and replaces
33 38
To evaluate the accuracy of the colorimetric method, UPLC- negative charged citrate ligands (pKa 3.13, 4.76, 6.40), which
34
MS/MS analysis was performed as a standard method to decreases the surface negative charge density and causes
35
detect AA in potato chips. Here, relative error (δ) was aggregation of AuNPs and a color change from red to purple
36 introduced to calculate the accuracy, as shown in formula (1). even blue. However, after the thiol-ene Michael addition
37

|  |
 % (1) reaction between GSH and AA, the thiol group of GSH is

38 consumed by AA, which hinders the followed ligand-
39 Where x and x0 are the measured concentrations of AA with
colorimetric method and UPLC-MS/MS method, respectively.
33 replacement and then the aggregation of AuNPs and color
40 change. AuNPs show different disperse state after adding GSH
41 with or without reacting with AA. Therefore, different
42 Results and discussion concentrations of AA can be quantified through measuring the
43 absorption spectra of AuNPs via UV-vis spectroscopy.
44 Mechanism of colorimetric detection
The thiol-ene Michael addition reaction between AA and
45 The detection principle of this colorimetric method is based on GSH catalyzed by TCEP was investigated using UV-vis
46 nucleophile initiated thiol-ene Michael addition reaction and absorption spectra, as shown in Fig. S1(a). In DHP buffer (pH =
47 the disperse state change of AuNPs caused by GSH. As 9.0), the mixture of AA or GSH with TCEP as the catalyst
48 depicted in Scheme 1(a), the thiol-ene Michael addition exhibited minor change in absorption compared with AA or
49 reaction takes place between the alkenyl group of AA and -SH GSH. A mixture of AA and GSH also gave no obvious different
50 group of GSH catalysed by TCEP at pH = 9.0. TCEP, as the absorption in comparison with AA or GSH after 2 h, indicating
51 nucleophile, first attacks the α, β-unsaturated electron- a real slow reaction kinetic without the catalyst. However, in
52 deficient alkene group of AA, followed by generating the the presence of TCEP, the mixture of AA and GSH presented an
53 strong (zwitterionic) enolate base. This intermediate is about 2.5-time enhancement in absorbance at 216 nm than
54 responsible for deprotonating thiol to generate the thiolate that without TCEP, demonstrating that the thiol-ene Michael
55 anion. Once the thiolate is formed, an extremely rapid anionic addition reaction between GSH and AA took place with
chain process takes place between thiolated GSH and the assistance of TCEP as a high efficient catalyst, which should
56 19
intermediate, and then GSH-AA adduct is formed. The benefit the rapid and sensitive detection of AA. In addition, the
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Analyst Accepted Manuscript

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20 Fig. 2 (a) Confirmation of the detection principle by UV-vis absorption spectra, insert:
the photograph of AuNPs with different treatments; TEM images of bare AuNPs (b),
21
AuNPs in the presence of GSH (c), and AuNPs in the presence of GSH-AA adduct (d).
22 The final concentration of GSH is 4 µmol L-1 both in (c) and (d).
23
24 mV, which indicated that the GSH induced ligand replacement
25 decreased the electrostatic repulsion interaction between
26 particles and broke the equilibrium of the colloid, leading to
27 the aggregation of AuNPs. Thorough research has been
28 discussed on the mechanism of aggregation of AuNPs induced
29 by small molecules and amino acids as well as the effect of pH
39
30 values on the aggregation. However, at the neutral or
31 Fig. 1 (a) The A520/650 values of AuNPs with and without GSH at different pH values, alkaline environment, because the zeta potential of AuNPs
32 insert: photographs of AuNPs with and without GSH at different pH values remained constant or even increased after negative charged
corresponding to A520/650 values. (b) The zeta potential of AuNPs with and without
33 GSH at different pH values.
GSH induced ligand replacement, AuNPs kept disperse state,
40
34 which was consistent with previous research. In addition, the
35 stability of GSH-AA adduct after mixing with AuNPs in CD surface charge of AuNPs at acidic environment was lower than
36 buffer was evaluated by UV-vis absorption spectra, as shown those at neutral and alkaline environment, which showed the
37 in Fig. S1(b). After adding into CD buffer (pH = 3.47), the equilibrium of AuNPs at the acidic environment was weaker
38 absorption of GSH-AA adduct showed no difference compared and the aggregation caused by positively charged GSH was
39 with that of GSH-AA adduct in basic environment, which easier than those at the other two pH surroundings. Besides, a
40 indicated that GSH-AA adduct after Michael addition reaction tripeptide (Glu-Val-Gly) without thiol group but with similar
41 is stable when they are mixed with AuNPs at acid environment. structure to GSH was used to further verify the principle of
The interaction between GSH and AuNPs at acidic detection by UV-vis absorption spectra, as showed in Fig. S2.
42
environment was carried out using UV-vis absorption spectra After Glu-Val-Gly adding into AuNPs (dissoloved in CD buffer),
43
and Zeta potential measurement. Fig. 1(a) illustrates the AuNPs remain disperse, which indicates the aggregation of
44
A520/650 values of AuNPs with and without GSH at different pH AuNPs caused by GSH is because thiol group of GSH positively
45
values. Significant change in color and decrease in the A520/650 charged adsorbs on the surface of AuNPs, leading to the
46
value of AuNPs were only observed at acidic environment. At decrease of surface charge density.
47 To confirm the detection principle, different mixtures of AA
48 neutral and alkaline environment, AuNPs remained red and
disperse state. Acidic environment prevented the dissociation and/or GSH and/or TCEP were further mixed with AuNPs
49 (average diameter of 12.83 nm, Fig. 2(b)) in CD,
36,37,41
and then
of two carboxyl groups of GSH according to its pKa values.
50 the dispersion of AuNPs was evaluated by UV-vis spectroscopy
Therefore, the surface negative charge density of AuNPs
51 and transmission electronic microscopy (TEM), as shown in Fig.
decreased after the replacement of citrate ligand by positively
52 2. The mixture of AuNPs with AA and/or TCEP showed little
charged GSH, leading to the aggregation and color change of
53 change in absorbance and color in comparison with pure
AuNPs. The measurement of zeta potential also verified the
54 mechanism of GSH triggered aggregation of AuNPs, as shown AuNPs solution. After the addition of GSH, the color of AuNPs
55 in Fig. 1(b). At the acidic environment (pH = 3.47), the surface solutions turned from red to purple in ca. 5 s, strong
56 negative charge of AuNPs was reduced from -37.0 mV to -28.8 absorption peaks at 624 nm appeared, both indicating serious
57
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Fig. 4 Selectivity of the colorimetric method (Asn: L-asparagine; MA: methylacrylamide;
15 SA: succinic acid; AC: 6-aminocaproic acid; Aa: acrylic acid; MAa: methylacrylic acid; PA:
16 Fig. 3 Linear relationship between A520/650 and different concentrations of AA (0, 0.1,
propionic acid; BA: N-buryric acid; HAc: acetic acid). The concentrations of all

Analyst Accepted Manuscript

0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 80.0 µmol L-1). Insert: the calibration curve of
17 A520/650 and low concentrations of AA (0, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 µmol L-1).
substances above were 80 µmol L-1.
18
19 addition reaction between GSH and AA. The A520/650 value of
aggregation of AuNPs, as also proved by TEM (Fig. 2(c)). After
20 AuNPs reached to the top when the reaction took place with
the addition of GSH-AA adduct solution, similar red-to-purple -1
21 catalysis of 0.05 eq (2 μmol L ) TCEP. Higher concentration of
change of color of AuNPs was observed, as well as an
22 TCEP did not favor the reaction, which was consistent with
appearance of a new peak at 624 nm. However, the intensity
23 previous research on phosphine catalytic Michael addition
of absorption at 624 nm was about 60% of those only with 28
24 reaction. Therefore, TCEP with 0.05 eq was used in the
GSH, demonstrating the maintaining of dispersion of AuNPs
25 reaction between GSH and AA. Fig. S6 shows that the A520/650
through consuming GSH by AA. Moreover, the dispersion of
26 value of AuNPs increased gradually with the reaction time until
GSH-AA-AuNPs with TCEP was much better than that of GSH-
120 min. To make sure the reaction adequately, 2.5 h was
27 AA-AuNPs without TCEP, as shown in Figs. 2(a) and 2(d),
chosen as the optimized reaction time in the sequent
28 indicating the reaction between GSH and AA was more
experiment.
29 completed with assistance of TCEP, which was in line with UV-
Sensitivity and selectivity
30 vis absorption spectra in Fig. S1 (a) (ESI), as discussed before.
31 Optimization of parameters Under the optimal conditions, the A520/650 value of AuNPs
32 increased with the increase of concentrations of AA. A linear
The value of pH plays an important role in aggregation of
33 relationship between A520/650 and AA concentrations was
AuNPs by GSH. Considering that the thiol-ene Michael addition
34 established, as shown in Fig. 3. The calibration curve of A520/650
reaction was carried out in DHP buffer (pH = 9.0), the volume
35 vs concentrations of AA was regressed into two sections.
ratio between CD buffer and DHP buffer (RC/D) should be -1
When the concentration of AA was lower than 5 μmol L , the
36 optimized to minimize the pH change of AuNPs surroundings 2
linear regression equation is y = 0.244 x + 1.151 (r = 0.98).
37 after adding the mixture of GSH-AA. Fig. S3(a) and (b) shows
When the concentration of AA was equal to or greater than 5
38 that the pH values increased from 3.37 to 4.38 with the -1
μmol L , the linear regression equation is y = 0.052 x + 2.159
39 decrease of RC/D from 10/0 to 5/5, corresponding to gradually 2
(r = 0.99). The limit of detection (LOD) was calculated based
40 decrease of absorbance at 624 nm and increase of the A650/520
on the former equation. Therefore, the linear range of
41 values of AuNPs. The changes in dispersion and color of AuNPs -1
colorimetric method for detecting AA is from 0.1 to 80 µmol L
42 accompanied with pH values further proved that GSH would -1
with a LOD of 28.6 nmol L (2.03 µg/kg, S/N = 3). According to
43 aggregate AuNPs only if the pH was lower than its pKa of one 42,43
the Derjaguin-Laudau-Verwey-Overbeck (DLVO) theory,
44 carboxyl group. Therefore, to minimize the change in pH, RC/D
the stability of gold nanoparticles is influenced by the interplay
45 at 9:1 with pH = 3.47 was chosen for the sequent experiment.
of attractive van der Waals forces, Fatt and repulsive Coulomb
46 Three other parameters including the final concentration of
forces, Frep. When Frep >> Fatt, the dispersed AuNPs is stable
47 GSH interacted with AuNPs, the level of TCEP in thiol-ene
whereas the condition of Frep << Fatt leads to aggregation.
48 Michael addition reaction, and the reaction time were also
AuNPs in solution are stable due to electrostatic repulsion of
optimized to maximize the signal. Fig. S4 shows the interaction
49 their negative charged surface. When the surface negative
between AuNPs and different concentrations of GSH. New
50 charge is reduced by adsorption of positive charged GSH,
absorption peaks of AuNPs appeared and shifted from 600 nm
51 AuNPs will attractive each other. Initially, particle dimers and
to 650 nm with the increase of GSH concentration from 0 to 10
52 -1 trimers will form, which is early stage. As the aggregation
μmol L , corresponding to the changes in color of AuNPs from
53 proceeds, AuNPs form larger clusters and the suspension
red to purple and then dark blue. Consideration of the
54 sensitivity and recovery of AuNPs from aggregation, 4 μmol L
-1 becomes unstable, which is late stage. After the formation of
55 was chosen as the optimized final concentration of GSH. Fig.
these large clusters, AuNPs become sediment quickly. With the
56 S5 illustrates the effect of TCEP on the thiol-ene Michael
decrease of concentration of AA, the concentration of free
57 GSH increase after Michael addition reaction. When the
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efficiency or even no reaction takes place. Methylacrylamide,
3
whose only difference from AA is the methyl group, also
4
triggered about 81% lower response (A520/650) than that of AA.
5 The reason may be that the methyl group of methylacrylamide
6 is the electron-donating group, which increases the difficulty
7 for the thiol group of GSH to attack. Noted that L-asparagine,
8
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the main precursor of AA production in foods processing

9 showed no obvious A520/650 response indicated the colorimetric
10 method have better selectivity than the fluorescent sensing
11 method based on quantum dots reported previously which
12 needed to minimize the interference of L-asparagine by adding
13 L-asparaginase.
15

14 Fig. 5 (a) Flow chart of pretreatment procedures for the extraction of AA in potato
Detection of AA in potato chips
chips. (b) The calibration curve and UV-vis absorption spectra (insert) for the detection
15 The established colorimetric method based on the interaction
of AA in potato chips using AuNPs based colorimetric method with MSA.
16 between AuNPs and GSH was applied to detect AA in potato

Analyst Accepted Manuscript

17 -1
concentration of AA decrease to 5 μmol L , the amount of free chips. In order to minimize the interference of complex food
18 GSH just make AuNPs colloidal solution reach to the late stage, matrices, standard addition method or method of standard
19 44
which is the critical point of AuNPs stability from early stage to addition (MSA) was introduced. The idea of MSA is to add AA
20 late stage. When the concentration of AA is higher than 5 μmol standard solutions to the sample (spiked) and monitor the
21 -1
L , AuNPs form small clusters gradually, corresponding to changes in A520/650 value. The different responses between
22 small slope in the linear regression equation (y = 0.052 x + unspiked and spiked samples are assumed to be only due to
23 -1
2.159). When the concentration of AA is lower than 5 μmol L , the changes in AA level. The concentration of AA in unspiked
24 AuNPs clusters grow larger and larger until to sediment sample is the absolute value for x-axis obtained from the linear
25 quickly, reflected by large slope in the linear regression fitted calibration curve when the value of y-axis is equal to
26 equation (y = 0.244 x + 1.151). zero. The pretreatment procedures described by Cheng et al.45
27 The sensitivity comparison between this colorimetric were used with some modifications, generally including
28 method and other methods was summarized in Table S1. trituration, extraction by 0.1% formic acid, purification and
29 Compared with standard methods (LC-MS/MS or GC-MS), this concentration, as shown in Fig. 5a and section 2.4 in details.
30 method showed comparable linear range and LOD value. The calibration curve for this colorimetric method is a linear
2
31 Compared with other rapid detection methods, this method regression equation of y = 0.16 x + 0.63 (r = 0.92) as shown in
32 showed one magnitude higher sensitivity than ELISA or Fig. 5b. The calculated concentration of AA in unspiked sample
-1 -1
33 fluorescent methods. The total detection time including the was 3.93 μmol L , meaning 0.9315 mg kg in detected potato
34 reaction time was about 2.5 h. The established colorimetric chips.
35 method has merits of easy operation, rapid response, and To evaluate the accuracy of the colorimetric method, UPLC-
36 visible results, which has promising to develop a simple, MS/MS analysis was performed as a standard method to
37 portable, and in-field device to detect AA in the further detect AA in the same sample. Accuracy, which is also called
38 research. systematic error or bias, is the difference of the value(s)
39 To evaluate the specificity of the colorimetric method, obtained from the true value(s) and always evaluated with
46,47
40 different compounds including L-asparagine, methylacrylamide, relative error. The data of UPLC-MS/MS were provided by
succinic acid, 6-aminocaproic acid, acrylic acid, methylacrylic the Research Centre of Natural Products and Human Health
41
acid, propionic acid, N-butyric acid, and acetic acid were from Zhejiang University and treated as the true concentration
42
detected as the same procedures as AA detection. Fig. 4 of AA in the same food sample, as shown in Fig. S7. In
43
illustrates the A520/650 value of AuNPs after adding the mixture comparison with the standard method, with which the
44 -1
of GSH and AA was five times higher than those after adding measured concentration of AA was 0.8535 ± 0.0450 mg kg ,
45 the colorimetric method showed a satisfactory result (relative
the mixture of GSH and other similar compounds. L-asparagine,
46
succinic acid, 6-aminocaproic acid, propionic acid, N-butyric error of 9.14%). In addition, the colorimetric method was also
47 acid, and acetic acid are common chemicals in foods without α, compared with other rapid methods including ELSIA and
48 β-unsaturated electron-deficient alkene group, resulting in no fluorescent methods, possessing comparable or better
49 Michael addition reaction with thiol group of GSH. Therefore, accuracy, as shown in Table S2, which indicates that the novel
50 GSH can still adsorb on the surface of AuNPs after the reaction, colorimetric method can be employed to detect AA in food
51 leading to the reduction of the surface charge density and such as potato chips.
52 followed aggregate AuNPs. For acrylic acid and methylacrylic
53 acid with α, β-unsaturated electron-deficient alkene groups,
54 also showed the aggregation of AuNPs after the reaction. The Conclusions
55 reason may be that their acidity changed the pH value of In this study, a novel AuNPs-colorimetric method based on
56 Michael addition condition (pH = 9.0) and caused low reaction nucleophile-initiated thiol-ene Michael addition between GSH
57
58
59
60 6 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

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DOI: 10.1039/C5AN01989C
Journal Name ARTICLE
1
2 17 J. Ren, J. Wang, J. Wang and E. Wang, Chem.- Eur. J., 2013,
and AA at acidic environment was proposed to detect AA. The
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4 -1 -1 -1 18 Q. Jiang, Z.-G. Wang and B. Ding, Small, 2013, 9, 1016-1020.
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-1 48
Compared with the 20 A. Lesniewski, M. Los, M. Jonsson-Niedziółka, A. Krajewska,
7 fluorescent sensor reported before, this new strategy showed K. Szot, J. M. Los and J. Niedziolka-Jonsson, Bioconjugate
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Published on 11 December 2015. Downloaded by University of California - San Diego on 21/12/2015 05:55:31.

higher selectivity. The new method was also employed to

9 21 Y. J. Sung, H.-J. Suk, H. Y. Sung, T. Li, H. Poo and M.-G. Kim,
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Analyst Accepted Manuscript

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