Determination of Apparent Amylose Content in Rice by Using Paper

Oct 23, 2015 - Blue color of amylose–iodine complex generated on-chip was converted to gray and measured with Photoshop after the colored chip was s...
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Determination of apparent amylose content in rice by using paper-based microfluidic chips Xianqiao Hu, Lin Lu, Changyun Fang, Binwu Duan, and Zhiwei Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04530 • Publication Date (Web): 23 Oct 2015 Downloaded from http://pubs.acs.org on October 27, 2015

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Journal of Agricultural and Food Chemistry

Determination of apparent amylose content in rice by using paper-based microfluidic chips Xianqiao Hu1,2, Lin Lu1,2, Changyun Fang1,2, Binwu Duan1,2, Zhiwei Zhu1,2* 1

2

China National Rice Research Institute, Hangzhou 310006, China Laboratory of Quality & Safety Risk Assessment for Rice (Hangzhou), Ministry of

Agriculture *

Corresponding author. Tel.: +86 571 63370275; fax: +86 571 63370380. E-mail: [email protected].

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Abstract

2

Determination of apparent amylose content in rice is a key function for rice

3

research and rice industry. In this paper, a novel approach with paper-based

4

microfluidic chip was reported to determine apparent amylose content in rice. The

5

conventional color reaction between amylose and iodine was employed. Blue color of

6

amylose-iodine complex generated on-chip was converted to gray and measured with

7

Photoshop after colored chip was scanned. The method for preparation of paper chip

8

was described. In-situ generation of iodine for on-chip color reaction was designed,

9

and factors influencing color reaction were investigated in detail. Elimination of

10

yellow color interference of excess iodine by exploiting color removal function of

11

Photoshop was presented. Under the optimized conditions, apparent amylose content

12

in rice ranging from 1.5-26.4 % can be determined, and precision was 6.3 %. The

13

analytical results obtained with developed approach were in good agreement to those

14

with continuous flow analyzer method.

15 16

Keywords: apparent amylose content, paper-based microfluidic chips, rice, color

17

reaction

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1. Introduction

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Starch is the major component of cereal. The quality of cereal depends on the

20

property of contained starch to a great extent. Starch consists of amylose and

21

amylopectin. Amylose content makes great impact on the property of starch such as

22

gelatinization, retrogradation, texture, solubility, swelling ability, crystallinity and so

23

on1. Rice is one of the most important staple foods for world population. The major

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component of rice is starch, and the amylose content in rice starch ranges 0-30 %. The

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texture of cooked rice and the functional properties of rice starch are primarily

26

impacted by amylose content. Amylose content negatively correlated with stickiness

27

and positively with hardness of rice. Cooked rice with high amylose content tend to

28

be dry, fluffy, hard and separate, while those with low amylose content tend to be

29

cohesive, tender and glossy2-3. Hence, amylose content in rice is an important quality

30

control parameter, and convenient and cost-effective method for the determination of

31

amylose content in rice is required by rice research and rice industry.

32

The conventional method used to determine amylose content in rice is

33

batch-wisecolorimetry4-7. It is based on the principle that amylose form a helical

34

inclusion bind with iodine, resulting in a blue color. The absorption of blue

35

amylose-iodine complex is measured at 620 nm or 720 nm with a spectrophotometer,

36

and amylose content is quantified against a calibration curve. The batch-wise

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colorimetry consumes large amount of samples, reagents and labors, therefore, is

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environment-unfriendly. Kaufman et al8 developed a 96-well plate method base on the

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iodine binding principle for amylose content determination. It was capable of 3

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analyzing 50-100 samples of starch per day. Nowadays, automatic continuous flow

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analyzer has been employed for colorimetric determination of amylose content. The

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automatic analyzer greatly simplifies the labor-involved operation9, and improves the

43

precision of analytical results. However, the multi sample analysis is conducted in

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series sample by sample, therefore it is also time- and reagent consuming. Both the

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conventional batch-wise and continuous flow colorimetric methods require relatively

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expensive equipment, therefore, can’t be conducted in-site. In addition to the

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colorimetry, many other instrumental methods have been reported to determine the

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amylose content, such as electrochemical method10-12, concanavalin A precipitation

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method13, high performance size exclusion chromatography9,

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differential scanning calorimetry15, near-infrared reflectance (NIR) spectroscopy16-17,

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thermogravimetricmethod18, multivariate calibration of the surface plasmon resonance

52

spectra of silver nanoparticles19 and so on. Expensive equipments are needed for these

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methods. Recently, Khoomtong20 developed a portable amylose content meter for

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amylose content determination, needless of complicated and expensive equipment.

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Besides of amylose, iodine also binds with long-chain amylopectin (DP > 60),

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amylose content measured using colorimetry method and electrochemical method has

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been termed apparent amylose content. However, apparent amylose content was also

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an important index to rice quality, and apparent amylose content instead of exact

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amylose content were widely used in rice research and rice industry.

14

, modulated

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Since the first report on paper-based microfluidic analytical chip (paper chip)

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was published in 200721, the paper chips have attracted great interest22-23 because they 4

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possess advantages such as cheap, biocompatible, easy-to-use, portable, needless of

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expensive equipments including fluid-driving pumps, consuming less sample and

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reagents, etc. When paper chip based analysis relied on a color reaction, the color

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intensity can be read out by naked eye or by a computer after the image of paper chip

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was taken by a mobile-phone-equipped camera or a scanner. Thus, it is possible for

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chemical and biochemical analyses to be implemented in site or in remote places. A

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variety of paper chips have been developed for food analysis, medical diagnosis and

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environmental monitoring22-27. However, no work has been reported on the

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development of paper chips for rice quality analysis. Recently, He et al28-30 reported

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novel and facile techniques to fabricate paper chips by means of coupling

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hydrophobic silane to paper fibers followed by deep UV lithography or plasma

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treatment. Such prepared paper chips have been applied in the colorimetric assays of

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nitrite ions in food samples28 and glucose in whole blood29.

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This work reported a novel paper-chip based approach for the determination of

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apparent amylose content in rice samples. Reliable on-chip color reaction between

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amylose and iodine was realized by in-situ generation of iodine. The interference of

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excess iodine in amylose assay was eliminated via exploiting the function of

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Photoshop software. The method for preparation of paper chips for the assays was

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presented.

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2. Materials and methods

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2.1 Samples and materials 5

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Nine rice samples and four rice standards were provided by Rice Product Quality

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Supervision and Inspection Center, Ministry of Agriculture, China. The four rice

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standards contain 1.5%, 10.4 %, 16.2 % and 26.4 % apparent amylose, respectively.

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Before testing, samples were husked in according to the standard of GB/T

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5495-200831. Briefly, the sprout gain was selected out of the sample and husked with

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hands while others were husked with rice huller. After that, the husked rice was milled

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until that most of the bran and part of the embryo had been removed. Then, milled

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rice kernels were ground into flour by using a Cyclotec 1093 Sample Mill (Foss

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Tecator, Sweden). After sieved with a 0.18 mm sieve, the samples of rice flour were

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equilibrated with rice standards in air for 3 d before use.

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2.2 Fabrication of the paper chip

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The native paper chip for determination of apparent amylose content was

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prepared as described in reference 28 and 29 with some modifications. Briefly,

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Whatman quantitative filter paper (Hangzhou Whatman-Xinhua Filter Paper Co., Ltd,

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Hangzhou) was immersed in a 0.1 % (v/v) octadecyltrichlorosilane (OTS, Acros

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Organics, Springfield, NJ) solution in n-hexane for 5 min at room temperature.

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During this process, filter paper turned from hydrophilic to hydrophobic as the

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long-chain alkyl groups of OTS were coupled to cellulose fibers of paper via

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silanization reaction. The filter paper was removed from OTS solution, and rinsed

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sequentially with n-hexane and water. After dried under nitrogen stream, the

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OTS-treated paper was cut into small sheets of 2.5 cm × 2.5 cm. An OTS-treated

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paper sheet was then sandwiched in a PMMA-PDMS-hybrid mold29 with a 6

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flower-shaped channel network (Fig.1a) and clamped. The complex was put into the

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chamber of a plasma cleaner (model of PDC-32G-2, Harrick, NY, USA) and exposed

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to the plasma for 30 s. The plasma-exposed region (i.e. the channel network) of paper

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sheet resumed hydrophilic due to the conversion of long alkyl chains to hydrophilic

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moieties

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wettability-patterned paper sheet was rinsed sequentially with n-hexane and water.

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After dried in air, a portion of 10 µL indicator solution (containing30 mmol·L-1

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potassium iodide (KI) and 30 mmol·L-1 sodium carbonate (Na2CO3)) was pipetted into

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the central zone (Fig.1b). The indicator solution penetrated through hydrophilic

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channels into detection zones under the action of capillary force (Fig.1c). After dried

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in air, the paper chip with indicator precursor (KI-Na2CO3) was stored under room

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temperature and ready for use.

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2.3 Determination of apparent amylose content with the paper chip

during

plasma

treatment.

After

withdrawing

from

mold,

the

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A 100.0 mg portion of either sample flour or standard flour was weighted, mixed

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and homogenized with 1.0 mL 95 % ethanol solution. 9 mL of 1mol·L-1NaOH

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solution was then added. The suspended flour solution was boiled for 10 min. After

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cooling to room temperature, the solution was transferred to a 50 mL volumetric flask,

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and diluted with deionized water to the volume.

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During assay, 0.5 µL portions of either standard solutions or sample solutions

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were individually pipetted into detection zones of paper chip prepared as section 2.2

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(Fig.1d). After the spots on chip dried in air, 10 µL oxidant solution (containing 60

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mmol·L-1 hydrogen peroxide (H2O2), 200 mmol·L-1 acetic acid (HAc)) were pipetted 7

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into the central zone of chip (Fig.1e). The oxidant solution penetrated through

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channels into each of eight detection zones, where color reaction between apparent

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amylose and iodine was induced by the oxidant (Fig.1f). Twenty minutes later, paper

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chip was scanned with a desktop scanner. The collected image was processed with

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Adobe Photoshop to obtain the gray intensity of blue color developed in each

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detection zone. Thus, the image of paper chip was opened in the Adobe Photoshop as

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the top layer. Underneath of top layer, an image of fully white filter paper sheet was

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then opened as the bottom layer. With the tool of color selection, the yellow color on

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top layer was selected with appropriate tolerance level, then deleted. The

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yellow-color-removed image was finally converted to grayscale. The bottom fully

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white layer was used to prevent the black-white chess-board background of

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Photoshop from appearing in the yellow-color-removed region of top layer. A

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calibration curve was constructed based on measured gray intensities of detection

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zones for standards, and apparent amylose content in sample solution was read against

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the calibration curve. Three repeated measurements were performed for each sample.

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2.4 Determination of apparent amylose content with continuous-flow analyzer

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The preparation of sample solutions was almost the same as section 2.3 except

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that the amounts of both flour samples and added reagents were halved. The prepared

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standard and sample solutions were analyzed with a continuous-flow analyzer (Flow

147

solution IV, OI Analytical) according to manufacturer instruction.

148 149

3. Results and discussion 8

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3.1 Method development In preliminary tests, original paper sheets without subjecting to any treatment

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were used. A brown-colored triiodide ion (I3-) solution (containing 8 mmol·L-1 iodine,

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120 mmol·L-1KI,100 mmol·L-1HAc) was pipetted on the paper sheet (Fig.2a), leaving

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a yellow spot on the sheet. Then sample solution containing amylose was pipetted

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onto the yellow-colored spot. The yellow color was soon turned to slightly blue due to

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bonding of iodine to amylose (Fig.2b). The gray intensity of scanned image of blue

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spot was in proportion to the concentration of apparent amylose. However, the iodine

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deposited on paper sheet sublimed quickly. As shown in Fig.2c, the yellow color of

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iodine was almost invisible 5 min after triiodide ion solution pipetted onto the sheet.

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If the sample solution was pipetted onto the spot 5 min after triiodide solution had

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been applied, the intensity of final blue color was much lighter (Fig.2b and d),

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indicating that the time interval between reagent application and sample application

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was a crucial parameter that would seriously affect the accuracy and precision of

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analytical results. The sublimation property of iodine prevented triiodide ion solution

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from being pre-applied onto paper sheets before use.

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To overcome this problem, we designed a protocol that iodine was in-situ

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chemically generated on paper chip when it was required. Thus, KI solution rather

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than triiodide solution was pre-applied onto paper sheet. Then sample solution was

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pipetted onto paper sheet. To induce the color reaction, H2O2 solution serving as the

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oxidant was added onto sheet following sample addition. Upon addition of

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H2O2solution, iodide was in-situ oxidized to iodine (Reaction 1) which would 9

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immediately bind to amylose, resulting in a blue color (Reaction 2). With this protocol,

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we did detect amylose without the trouble of iodine sublimation (Fig.3). Nevertheless,

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another problem arose, the KI pre-applied on paper sheet would be gradually oxidized

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to I2 by the oxygen in the air (Reaction 3). Test showed that a KI-applied paper sheet,

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which had been soaked with 30 mmol·L-1 KI for 3 s and dried at a temperature of 45

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ºC, turned slightly yellowish after 1 d storage in the air. Reaction 1

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Amylose + I2 (yellow) = Amylose-I2 complex (blue)

Reaction 2

Reaction 3

180 181

Half-reaction 1

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Half-reaction 2

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Half-reaction 3

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The Nernst equation for Half-reaction 1 is shown by Equation 1:

185

EI

186

− 2 /I

2 0. 0592 V l g  I −  2

= EI0 /I − − 2

Eq. 1

where EI0 /I − = 0.5345 V 2

187 188 189

The potential EI

/I −

does not affected by the acidity of reaction medium. Under

the condition of 30 mmol·L-1 KI concentration, EI

2

/I −

is 0.6246 V.

The Nernst equation for Half-reaction 2 is shown by Equation 2:

EO

2 /H 2 O

= E

4 0. 0592 0. 0592 V l g  H +  + V l g p( O2 ) / p 0 4 4 0. 0592 − 0. 0592VpH + V l g p( O2 ) / p 0 4

= EO0

190 0 O2 /H 2 O

191

2

2 /H 2 O

+

where EO0 2/H2O = 1.229 V , p( O2 ) / p 0=0. 21 10

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The potential EO0

2

strongly depends on the pH value. Calculation reveals that

/H 2 O

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I- would be oxidized by O2 in the air when pH of reaction medium is less than 10.2.

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This explains the reason why KI-soaked paper sheet gradually turned yellow after a

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short period storage in air. The KI-applied paper sheet became totally invalid after one

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week storage (Fig.3b). To suppress the oxidization of KI by oxygen, 30mmol·L-1

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Na2CO3 was add into KI solution to make the solution alkaline (pH>10.8). Under this

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condition, Reaction 3 can’t proceed spontaneously, consequently, KI-applied paper

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sheet becomes stable in the air.

200

The Nernst Equation for Half-reaction 3 is:

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EH 2O 2 /H 2O = EH0 2O 2 /H 2O +

202 203

0.0592 V lg[H + ]2 = EH0 2O 2 /H 2O − 0.0592VpH 2

where EH0 2O2/H2O = 1.77 V Calculation of potential with Eq.3 indicates than EH O

2 2

204

Eq.3

/H 2 O

is no less than 0.94 V

in the pH range of 1-14. Compared with the above calculated result for EI

2

/I −

,

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Reaction 1 would proceed in the pH range of 1-14. Thus, the on-paper applied I-

206

would be oxidized to iodine by H2O2 in Na2CO3 medium. Unfortunately, no yellow

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color of iodine was observed after H2O2 solution was pipetted onto the paper sheet on

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which KI-Na2CO3 solution had been applied (Fig.4a). This might be ascribed to that

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in-situ generated iodine subjected to disproportionation (Reaction 4) in alkaline

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medium. Reaction 4

211 212

Therefore, acid should be added to oxidant solution to create an acidic reaction

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medium for depression of iodine disproportionation in one hand, and for facilitating 11

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the reaction of iodine binding to amylose (usually carried out in the pH range of 4-6)

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in the other hand. The color reaction did occur when 50 mmol L-1acetic acid was

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added to H2O2 solution. Tests also showed that on-paper color reaction of amylose

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was successfully implemented after KI-Na2CO3-applied paper sheet had been stored

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for 3 months (Fig.3c and d), indicating the high stability of KI-Na2CO3-paper sheet.

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3.2 Factors influencing the color intensity

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The concentration of iodide in indicator solution and those of hydrogen peroxide

221

and acetic acid in oxidant reagent solution would influence the color reaction. Their

222

effects were investigated with paper-based microfluidic chips.

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3.2.1 KI concentration

224

The influence of KI concentration on the color reaction was shown in Fig.4a. For

225

reagent blank, the mean gray intensity of yellow color in detection zone was steadily

226

increased with the increase of KI concentration. For rice sample, however, the mean

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intensity in detection zone increased quickly with the increase of KI concentration up

228

to 30 mmol·L-1, then slowly increased up to the tested highest KI concentration (120

229

mmol·L-1). By subtracting the mean intensity for reagent blank from that for rice

230

sample, the net mean intensity for rice sample reached a maximal value at the KI

231

concentration of 30 mmol·L-1. Therefore, 30 mmol·L-1 KI in the indicator solution was

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selected in the present work.

233

3.2.2 HAc concentration

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The influence of acetic acid concentration on the color reaction is shown in

235

Fig.4b. For reagent blank, slight yellow color appeared when HAc concentration in 12

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oxidant solution was higher than 10 mmol·L-1, indicating the formation of significant

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amount of iodine on detection zone. The mean gray intensity of yellow color in

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detection zone increased with the increase of HAc concentration and reached a

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plateau at the HAc concentration of 400 mmol·L-1. For rice sample containing 26.4%

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apparentamylose, slight blue color was observed when HAc concentration was higher

241

than 50 mmol·L-1. The mean gray intensity for rice sample sharply increased with the

242

increase of HAc concentration up to 400 mmol·L-1, then it leveled off. Subtracting of

243

mean gray intensity for reagent blank from that for rice sample resulted in the net

244

mean gray intensity for amylose-iodine complex. As showed in Fig.4b, the net mean

245

gray intensity for amylose-iodine complex quickly increased with the increase of HAc

246

concentration up to 200 mmol·L-1 then slightly decreased with the increase of HAc

247

concentration, the highest value being obtained at the HAc concentration of 200

248

mmol·L-1. At the HAc concentration higher than 200 mmol·L-1, much more iodine was

249

produced, consequently, dark yellow colored area in detection zone expanded. Thus,

250

HAc concentration of 200 mmol·L-1 in oxidant solution was adopted in the following

251

work.

252

3.2.3 H2O2 concentration

253

The H2O2 concentration in oxidant solution had little impact on the mean

254

intensities for both reagent blank and rice sample when concentration ratio of

255

H2O2-to-KI was within the range of 1:2-5:1. In this work, a H2O2 concentration of 60

256

mmol·L-1 was applied.

257

3.3 Determination of apparent amylose content in rice sample 13

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3.3.1 Data preprocessing

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A paper-based microfluidic chip with a flower shaped channel network,

260

including one central reagent zone and eight detection zones where sample solutions

261

would be individually added, was employed for the determination of apparent

262

amylose content in rice. Among eight detection zones, one was for reagent blank, four

263

for rice standards, and the rest three for rice samples. On-chip color reaction in eight

264

detection zones was simultaneously proceeded as described in Section 2.3, and the

265

mean gray intensity of each detection zone was sequentially measured via Photoshop

266

software after paper chip was scanned by a desktop scanner. As both yellow color of

267

excess iodine and blue color of amylose-iodine complex were converted gray intensity,

268

the contribution of yellow color to gray intensity would interfere with amylose

269

determination. When the measured mean gray intensities for standards were plotted

270

via apparent amylose contents in standards, the data were significantly dispersed from

271

the linear regressed (Fig.S1a), the linear correlation coefficient R was only 0.9275.

272

Thus, the interference of yellow color of excess iodine should be corrected from total

273

gray intensity. It was simply implemented by removal of yellow color in scanned

274

image via the color selection function of Adobe Photoshop (see Section 2.3). Then,

275

the corrected image without yellow color was converted to grayscale with Photoshop

276

software. After correcting the yellow color interference, the data of measured mean

277

gray intensities against apparent amylose contents gave excellent linearity (Fig.S1b,

278

R=0.9998), demonstrating the effectiveness of removal of yellow color interference

279

via Photoshop software. 14

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3.3.2 Performance of the developed paper-based microfluidic chip

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Analytical performance of the developed paper chip method was evaluated with

282

real rice samples. With a rice sample containing 16.2 % apparent amylose, precision

283

of three parallel measurements conducted in the same paper chip was 6.3 % in relative

284

standard deviation (RSD). The chip-to-chip difference was examined with three chips

285

of the same batch, and RSD of 6.0 % was observed for intra-day tests while RSD of

286

10.7 % (n=3) was observed for inter-day tests. Quite good linearity (R=0.9998) of

287

calibration curve was obtained in the apparent amylose content range of 1.5-26.4 %

288

which covers apparent amylose contents in most rice samples (Fig.S1b). The achieved

289

detection limit (3σ) for the developed paper chip method was 1.1 %.

290

The accuracy of present method was evaluated by comparing analytical results

291

obtained with paper chip approach to those obtained with continuous-flow analyzer

292

method based on NY/T 55-1987 which is the standard of Ministry of Agriculture of

293

China. The correspondence between the results of developed paper-based microfluidic

294

chip method and continuous-flow analyzer was showed in Fig.S2. The results of

295

developed method were consistent with those of continuous-flow analyzer method for

296

determining apparent amylose content in rice sample. The difference between

297

developed method and continuous-flow analyzer method were less 3 % except for the

298

sample with apparent amylose content of 17.1% whose difference was 4.5 %. The

299

signal precisions were quite good, standard deviations of less than 3 % were observed

300

for test rice samples. Despite that the precision of developed paper chip approach is

301

poorer than that of continuous flow analyzer method whose standard deviations were 15

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less than 1%, the developed method does possess such advantages as cost-effective,

303

needless of expensive equipment and skilled analysts, and capable of in-site assays.

304

Although this method could not be a direct replacement for the standard method, it’s

305

quite suitable for amylose-based rice screen assay that can be conducted in common

306

laboratories.

307 308

4. Conclusion

309

A paper-based microfluidic chip for determining apparent amylose content in

310

rice samples has been developed based on conventional color reaction between

311

amylose and iodine and measuring gray intensity of scanned image of colored paper

312

chip. To prevent iodine from sublimation away from paper chips, iodine needs to be in

313

situ produced on chip via oxidizing pre-applied KI with H2O2. Acidity control is the

314

key for on-chip iodine generation and color reaction. The KI solution is kept slight

315

alkaline by adding Na2CO3 to suppress the oxidation of I- by O2 in the air after KI

316

have been pre-applied on chip. This allows prepared paper chips to be effective after 3

317

months storage. Spiking of acetic acid into oxidant solution ensures in-situ iodine

318

generation and followed color reaction to properly conducted. Removal of yellow

319

color of excess iodine from scanned image of paper chip by using color selection

320

function of Photoshop software ensures the measured gray intensity be purely

321

contributed by blue amylose-iodine complex. The developed paper chip approach has

322

the advantages of cost-effective, needless of expensive equipment and skilled analysts,

323

and capable of in-site assays. It is most suitable for evaluation of amylose content in 16

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the screening tests for scientific research and breeding programs. It is also potential to

325

extend the developed method to assay of amylose content in other cereals.

326 327 328

Acknowledgment

329

This work was funded by the Special Fund of Chinese Central Government for

330

Basic Scientific Research Operations in Commonweal Research Institutes (project No.

331

2014RG006-4), Zhejiang Provincial Natural Science Foundation of China(grant NO.

332

LQ15C200007), National Natural Science Foundation of China (project No.

333

31201175) and National Key Project for Agro-product Quality & Safety Risk

334

Assessment, P.R.C. (project No. GJFP2014006).

335 336 337

Supporting Information

338

Figure S1 showed the calibration curves constructed with the measured gray

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intensities for four rice standards before and after removing yellow color, and Figure

340

S2 showed the comparison between the results of developed paper-based microfluidic

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chip method and continuous flow analyzer method for 9 rice samples. This material is

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available free of charge via the Internet at http://pubs.acs.org.

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References

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Shinde, Y. H.; Vijayadwhaja, A.; Pandit, A. B.; Joshi, J. B., Kinetics of cooking of

Zhu, T.; Jackson, D. S.; Wehling, R. L.; Geera, B., Comparison of amylose

Mahmooda, T.; Turner, M. A.; Stoddard, F. L., Comparison of methods for

Wang, J.; Li, Y.; Tian, Y.; Xu, X.; Ji, X.; Cao, X.; Jin, Z., A novel

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Kaufman, R. C.; Wilson, J. D.; Bean, S. R.; Herald, T. J.; Shi, Y. C., Development

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11. Duan, D. X.; Donner, E.; Liu, Q.; Smith, D. C.; Ravenelle, F., Potentiometric

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titration for determination of amylose content of starch - A comparison with

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12. Coton, L.; Lampitt, L. H.; Fuller, C. H. F., Studies in starch structure. 2. The

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Chen, M.-H.; Bergman, C. J., Method for determining the amylose content,

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scanning calorimetry. Starch-Starke 2006,58 (5), 209-214.

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Content Meter for Rapid Determination of Amylose Content in Milled Rice. Food

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paper mask for glucose assay. Analyst 2014,139 (18), 4593-4598.

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23. Liu, W.; Kou, J.; Xing, H. Z.; Li, B. X., Paper-based chromatographic 20

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chemiluminescence chip for the detection of dichlorvos in vegetables. Biosens.

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glucose and uric acid with bienzyme colorimetry on microfluidic paper-based analysis

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Corona Treater, Master Thesis, Zhejiang University (2014).

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Yield from Paddy, Standards Press of China, Beijing, 2008.

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Figure Captions

436 437

Figure 1 Schematic diagrams for the process of determining apparent amylose content

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with developed paper-based microfluidic chip. (a) the prepared paper-based

439

microfluidic chip with flower-shaped hydrophilic channel network; (b) 10 µL

440

indicator solution was pipetted into central zone;(c) the applied indicator solution

441

penetrated through hydrophilic channels into detection zones; (d) 0.5 µL portions of

442

sample solutions were individually pipetted into each of eight detection zones; (e) a

443

10 µL portion of oxidant solution was pipetted into central zone; (f) color reaction

444

occurred in detection zones.

445 446

Figure 2 The effect of iodine sublimation on color reaction carried out on paper sheets.

447

(a) 1 µL triiodide solution was dropped on the paper sheet; (b) 1 µL rice standard

448

solution was pipetted onto (a); (c) 5min after triiodide solution was dropped on the

449

paper sheet; (d) 1 µL rice standard solution was pipetted onto (c).

450 451

Figure 3 Color reaction carried out on indicator solution pre-applied paper sheets.

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(a-b) the indicator solution containing KI only; (c-d) the indicator solution containing

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KI and Na2CO3; (a, c) amylose solution was pipetted onto the paper sheets

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immediately after indicator solution had been applied; (b) amylose solution was

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pipetted onto the paper sheet 7 days after indicator solution had been applied; (d)

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amylose solution was pipetted onto the paper sheet 3 months after alkaline indicator 23

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solution had been applied.

458 459

Figure 4 Influence of KI concentration in indicator solution (a) and HAc

460

concentration in oxidant solution (b) on the measured gray intensity of detection

461

zones. (-■-) the gray intensity measured for rice sample; (-●-) the gray intensity

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measured for reagent blank; (-▲-) the net gray intensity for rice sample. H2O2

463

concentration in oxidant solution: 60 mmol·L-1; (a) HAc concentration in oxidant

464

solution: 200 mmol·L-1; (b) KI concentration in indicator solution: 30 mmol·L-1.

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