Graphene-polyaniline modified electrochemical droplet-based microfluidic sensor for high-throughput determination of 4-aminophenol

Accepted Manuscript Graphene-Polyaniline Modified Electrochemical Droplet-Based Microfluidic Sensor for High-throughput Determination of 4-Aminophenol Poomrat Rattanarat, Akkapol Suea-Ngam, Nipapan Ruecha, Weena Siangproh, Charles S. Henry, Monpichar Srisa-Art, Orawon Chailapakul PII:

S0003-2670(16)30327-0

DOI:

10.1016/j.aca.2016.03.010

Reference:

ACA 234460

To appear in:

Analytica Chimica Acta

Received Date: 23 September 2015 Revised Date:

29 December 2015

Accepted Date: 7 March 2016

Please cite this article as: P. Rattanarat, A. Suea-Ngam, N. Ruecha, W. Siangproh, C.S. Henry, M. Srisa-Art, O. Chailapakul, Graphene-Polyaniline Modified Electrochemical Droplet-Based Microfluidic Sensor for High-throughput Determination of 4-Aminophenol, Analytica Chimica Acta (2016), doi: 10.1016/j.aca.2016.03.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Graphene-Polyaniline Modified Electrochemical Droplet-Based Microfluidic Sensor for

2

High-throughput Determination of 4-Aminophenol

3

Poomrat Rattanarat,a Akkapol Suea-Ngam,a Nipapan Ruecha,c Weena Siangproh,d Charles S.

5

Henry,e,f, Monpichar Srisa-Art, a,b and Orawon Chailapakul a,g,*

6

RI PT

4

7

a

8

Chemistry, Faculty of Science, Chulalongkorn University, Patumwan, Bangkok 10330,

9

Thailand.

M AN U

SC

Electrochemistry and Optical Spectroscopy Research Unit (EOSRU), Department of

10

b

11

Faculty of Science, Chulalongkorn University, Patumwan, Bangkok 10330, Thailand.

12

c

13

Patumwan, Bangkok 10330, Thailand.

14

d

15

Wattana, Bangkok 10110, Thailand.

16

e

17

States.

18

f

19

United States.

20

g

21

Chulalongkorn University, Patumwan, Bangkok 10330, Thailand.

Chromatography and Separation Research Unit (ChSRU), Department of Chemistry,

TE D

Program in Macromolecular Science, Faculty of Science, Chulalongkorn University,

Department of Chemistry, Faculty of Science, Srinakharinwirot University, Sukhumvit 23,

EP

Department of Chemistry, Colorado State University, Fort Collins, CO 80523, United

AC C

School of Biomedical Engineering, Colorado State University, Fort Collins, CO 80523,

National Center of Excellent of Petroleum, Petrochemicals and Advanced Materials,

22 23 24

*Corresponding authors at: E-mail address: [email protected]

ACCEPTED MANUSCRIPT GRAPHICAL ABSTRACT

RI PT

1

2

RESEARCH HIGHLIGHTS

4

• A novel combination of G-PANI/CPE and droplet-based microfluidic device for

5

determination of 4-AP was achieved.

6

• G-PANI nanocomposite increases the surface area and improved electrochemical sensitivity

7

of the electrode.

8

• This novel droplet-based system was successfully applied for 4-AP monitoring in

9

commercial PA samples.

M AN U

TE D

EP AC C

10

SC

3

ACCEPTED MANUSCRIPT 1

ABSTRACT We report herein the first development of graphene-polyaniline modified carbon paste

3

electrode (G-PANI/CPE) coupled with droplet-based microfluidic sensor for high-throughput

4

detection of 4-aminophenol (4-AP) in pharmaceutical paracetamol (PA) formulations. A

5

simple T-junction microfluidic platform using an oil flow rate of 1.8 µL/min and an aqueous

6

flow rate of 0.8 µL/min was used to produce aqueous testing microdroplets continuously.

7

The microchannel was designed to extend the aqueous droplet to cover all 3 electrodes,

8

allowing for electrochemical measurements in a single droplet. Parameters including flow

9

rate, water fraction, and applied detection potential (Edet) were investigated to obtain optimal

10

conditions. Using G-PANI/CPE significantly increased the current response for both cyclic

11

voltammetric detections of ferri/ferrocyanide [Fe(CN)6]3-/4- (10 times) and 4-AP (2 times),

12

compared to an unmodified electrode. Using the optimized conditions in the droplet system,

13

4-AP in the presence of PA was selectively determined. The linear range of 4-AP was 50-

14

500 µM (R2 = 0.9972), limit of detection (LOD, S/N=3) was 15.68 µM, and limit of

15

quantification (LOQ, S/N=10) was 52.28 µM. Finally, the system was used to determine 4-

16

AP spiked in commercial PA liquid samples and the amounts of 4-AP were found in good

17

agreement with those obtained from the conventional capillary zone electrophoresis/UV-

18

Visible spectrophotometry (CZE/UV-Vis).

19

employed for a high-throughput screening (at least 60 samples h-1) of pharmaceutical purity

20

requiring low sample and reagent consumption.

SC

M AN U

TE D

EP

The proposed microfluidic device could be

AC C

21

RI PT

2

22

KEYWORDS:

droplet-based

microfluidics,

23

polyaniline, 4-aminophenol, paracetamol

electrochemical

detection,

graphene,

ACCEPTED MANUSCRIPT 1

1. INTRODUCTION

2 3

Since the mid-1940s, paracetamol (PA, acetaminophen or N-acetyl-p-

4

aminophenol) has been commonly used as an antipyretic and analgesic drug around

5

the world [1-2].

6

suppositories, have been available and highly effective for a variety of patients

7

including children, pregnant women, and the elderly. Unfortunately, 4-aminophenol

8

(4-AP) residue might be present as an impurity in PA formulations obtained from its

9

synthesis or as a result of degradation [3]. Consuming 4-AP unintentionally can cause

10

numerous pathologies (e.g. nephrotoxicity and hepatotoxicity) [3-5]. The European,

11

United States, British, German, and Chinese Pharmacopoeias have set the maximum

12

allowable limit of 4-AP in the PA drug formulations at 50 ppm (0.005% w/w) [5].

13

Several analytical approaches to determine 4-AP in PA formulations have been

14

developed including high performance liquid chromatography (HPLC) coupled with

15

UV-visible [6-7] or fluorescence [8] detection, electrochemical detection [9], capillary

16

electrophoresis [10-11], and flow-based analytical techniques [4, 12]. Although these

17

techniques offer highly selective, and sensitive quantification of 4-AP in PA

18

formulations, these techniques are time consuming, have high operation costs, and are

19

available only in sophisticated laboratories.

RI PT

SC

M AN U

TE D

EP

AC C

20

Many PA formulations, such as tablets, syrups, injections, and

Recently, droplet microfluidic systems have held great promise for analytical

21

measurements due to their portability, fast analysis time, low manufacturing cost, low

22

reagent/sample

23

microfluidic systems are capable of generating and manipulating small droplets

24

encapsulated by an immiscible phase. Among other microfluidic platforms, droplet

25

microfluidics has gained attention because of superior mixing efficiency and low

consumption,

and

high-throughput

screening

[13].

Droplet

ACCEPTED MANUSCRIPT dispersion and adsorption of sample within the microchannel [14]. The utility of

2

droplet-based technology has been demonstrated for many applications, such as

3

biomedical diagnosis [15-16], food safety [17-18], and environmental monitoring [19].

4

Other demonstrated applications include protein crystallization [20] and enzymatic

5

kinetic assays [21], emulsion-based polymerase chain reaction [22], chemical

6

synthesis [23-24], and single cell-based analysis [25].

7

microfluidics, various detection methods have been used for content detection

8

including

9

spectroscopy [28], absorption spectroscopy [29], and bright-field microscopy [30].

10

Although these techniques offer highly selective, sensitive and accurate quantification,

11

the main disadvantage of these techniques are high equipment cost and challenge of

12

coupling them with miniaturized systems for on-site applications.

fluorescence

[26],

mass

For detection in droplet

spectrometry

[27],

Raman

M AN U

SC

laser-induced

RI PT

1

Electrochemical detection is an attractive and alternative detection method for

14

microfluidics because it is low cost, high speed, high sensitivity, cost-effectiveness,

15

and independence of sample turbidity or optical path length [31]. Currently, there

16

have been a few reports on electrochemical detection for droplet microfluidic systems.

17

For example, Liu et al. used chronoamperometry for the detection of droplet contents

18

and characterization of droplet generation (e.g. frequency, size and velocity) [32].

19

Han et al. developed an amperometric droplet microfluidic device to investigate the

20

kinetics of enzymatic decomposition of H2O2 using catalase [33]. Gu et al. used a Pt-

21

black modified electrochemical droplet-based microfluidic sensor for enzymatic

22

glucose assays in biological fluids [34]. Itoh et al. demonstrated a novel droplet-based

23

microdevice with an electrochemical ATP-sensing function for fish freshness

24

determination [35].

AC C

EP

TE D

13

ACCEPTED MANUSCRIPT The use of nanomaterials to improve electrode performance in microfluidic

2

devices has drawn increasing attention in recent years. Numerous nanomaterials (e.g.

3

gold nanoparticles, silver nanoparticles, single-wall carbon nanotubes, and multi-wall

4

carbon nanotubes) are currently available and can increase active surface area of the

5

working electrode, leading to enhanced electrochemical sensitivity and conductivity

6

[34, 36-38]. Graphene (G) has received substantial attention for chemical modification

7

of electrodes [39], owing to its large surface area, extraordinary electrical

8

conductivity, high mechanical strength and potentially low manufacturing cost. In

9

addition, G has been also used to modify electrode surface in order to improve the

10

electrochemical properties of the electrodes. One problem associated with G-based

11

electrode modification is the potential for self-agglomeration of pure G on the

12

electrode surface. One solution is use of a nanocomposite consisting of conducting

13

polymer and G to increase the distribution of graphene [40].

14

polymers, polyaniline (PANI) has been widely used for electrode modification because

15

of its inherent electrochemical properties, biocompatibility and stability [41-42].

16

Previous reports have used G-PANI hybrid modified electrochemical sensors for

17

electrochemiluminescent detection of luminol [43], voltammetric determination of

18

dopamine [41] and dobutamine [44], and electrochemical immunoassays for estradiol

19

[45] and salbutamol [46]. To the best of our knowledge, there are no previous reports

20

combining a novel G-PANI modified electrodes and droplet microfluidics for

21

electrochemical determination of 4-AP.

Among conducting

AC C

EP

TE D

M AN U

SC

RI PT

1

22

Herein, the aim of this work is to develop a high-throughput and sensitive

23

method using G-PANI modified electrochemical droplet-based microfluidic sensor for

24

determination of 4-AP in pharmaceutical PA products. An improved selectivity for 4-

25

AP was achieved using G-PANI modified carbon-paste electrodes (G-PANI/CPE)

ACCEPTED MANUSCRIPT operating at the optimized detection potential for highly selective detection of 4-AP in

2

the presence of PA. This system offers an alternative method to measure 4-AP level in

3

acceptable concentration range. Finally, this approach was successfully applied for the

4

determination of 4-AP in commercial PA liquid samples, giving high correlation with

5

a CZE/UV-Vis method. Accordingly, measurements using this approach can provide

6

a fast and high-throughput route and could be an ideal platform for screening of purity

7

in pharmaceutical products with small sample consumption.

8

development of this microfluidic model can be further extended to various applications

9

such as medical testing, environmental monitoring and quality control of foods or

SC

beverages.

11 12

Furthermore, the

2. EXPERIMENTAL

M AN U

10

RI PT

1

2.1 Materials and chemicals

14

The following materials were used as received: poly(dimethylsiloxane)

15

(PDMS) Sylgard 184 eleastomer kit (Dow Corning, USA), mineral oil (Perkin Elmer,

16

Thailand), silver paint (SPI supplies, USA), graphite powder (≤20 µm, Sigma-Aldrich,

17

Singapore), and graphene (A.C.S Medford, USA).

18

grade: sodium chloride (NaCl: Merck, Thailand), disodium hydrogen phosphate

19

(Na2HPO4: Merck, Thailand), potassium dihydrogen phosphate (KH2PO4: Carlo

20

ERBA, Thailand), potassium chloride (KCl: Ajax Finechem, Thailand), and ethanol

21

(Merck, Thailand). Potassium ferricyanide (K3[Fe(CN)6]) and potassium ferrocyanide

22

(K4[Fe(CN)6]) were obtained from Sigma-Aldrich (Singapore).

23

camphor-10-sulfonic acid (C10H16O4S) were purchased from Merck (Darmstadt,

24

Germany). N-Methyl-2-pyrrolidone (NMP), 4-aminophenol (4-AP) and paracetamol

25

(PA) were purchased from Sigma-Aldrich (Singapore).

All chemicals were analytical

AC C

EP

TE D

13

Polyaniline and

Commercial PA drugs

ACCEPTED MANUSCRIPT consisting of Infants’ TYLENOL® (Janssen-Cilag, Ltd., Thailand), TEMPRA®

2

(Taisho Pharmaceutical Co., Ltd., Thailand), SARA® (Thai Nakorn Patana Co., LTD,

3

Thailand) were purchased from a local pharmacy in Thailand. For droplet generation,

4

a 10:2 (v/v) mixture of perfluorodecalin (mixture of cis and trans, 95%, Sigma-

5

Aldrich, Germany) and 1H, 1H, 2H, 2H-perfluoro-1-octanol (97%, Sigma-Aldrich,

6

Germany) was used as an oil solution. All aqueous solutions were prepared using

7

deionized water (18.0 MΩ cm. Milli-Q Gradient System, Millipore, Thailand).

8

Phosphate buffer saline (PBS) pH 7.4 at a concentration of 0.1 M was used as a

9

supporting electrolyte for all experiments.

SC

RI PT

1

To prepare 0.1 M PBS, the mixture

comprising of 2.0 g NaCl, 0.05 g KCl, 0.36 g Na2HPO4 and 0.06 g KH2PO4 was

11

dissolved in a 250 mL volumetric flask using deionized water. All aqueous stock

12

standards of 4-AP and PA were prepared in 0.1 M PBS.

M AN U

10

13

2.2 Fabrication of patterned PDMS

15

In this work, solutions were pumped through the device using syringe pumps

16

(Harvard Apparatus PHD 2000, Harvard Apparatus, Holliston, MA). Both aqueous

17

and oil phases were prepared and stored in 1 mL plastic syringes (Nipro, Thailand).

18

Polyethylene tubing (0.38 mm I.D., 1.09 mm O.D., PORTEX, Belgium) connecting

19

the inlets of microfluidic device with syringe pumps was secured using commercial

20

epoxy. For microfluidic experiments, a T-junction microchannel and electrode layout

21

originally published by Liu et al. was used [47].

22

photoresist was coated onto a silicon wafer using a spin coater and then baked at 95 oC

23

for 45 min. Next, the transparency film containing the microchannel and electrode

24

pattern was placed over the coated wafer, and exposed to ultra-violet (UV) light for

25

pattern. The final SU-8 template was obtained by washing with the SU-8 developer

AC C

EP

TE D

14

Briefly, the SU-8 negative

ACCEPTED MANUSCRIPT and isopropanol, respectively.

Microchannel platforms were fabricated using the

2

traditional soft lithography using a 10:1 ratio of the PDMS precursor to curing agent

3

(Sylgard 184, Dow Corning) [48-49]. The mixture was degassed under vacuum to

4

remove dissolved air. The PDMS mixture was poured onto the SU-8 master and

5

baked in the oven at 65 oC for 3 hrs. The cross-linked PDMS was peeled off the mold,

6

and inlets were created using a circular biopsy puncher.

7

containing the microchannel layer and the electrode layer were obtained. The depth of

8

all microchannels was 100 µm. For upper PDMS layer, the main channel of 500 µm

9

width (having two inlets and one outlet) was narrowed to be a confined channel (50

RI PT

1

SC

Two PDMS replicas

µm width and 1 cm length).

11

channels (100 µm depth and 500 µm width) with a spacing between each electrode of

12

500 µm were used according to previous reports [48, 50].

13

For lower electrode layer, three parallel electrode

M AN U

10

2.3 Fabrication and modification of electrode

15

Electrode fabrication was accomplished in two steps, screen-printing of the

16

carbon-paste electrode and electrode modification using G-PANI nanocomposite.

17

PDMS cross-linked carbon paste electrodes (CPEs) were created using the previously

18

published method from Sameenoi et al. [48, 50]. Briefly, a carbon paste containing

19

graphite powder, mineral oil, and PDMS mixture in a ratio of 2:1:1 was spread over

20

the electrode channels in the PDMS. Excess paste was removed using Scotch Magic

21

TapeTM until well-defined carbon paste electrodes were obtained.

AC C

EP

TE D

14

22

For electrode modification, G-PANI nanocomposite was prepared using a

23

simple physical mixing procedure [40]. G in NMP solution (2 mg mL-1) was prepared

24

and dispersed using an ultrasonic bath for 12 hrs at room temperature. The conductive

25

form of PANI was prepared by doping with camphor-10-sulfonic acid and dissolved in

ACCEPTED MANUSCRIPT chloroform. Then, the G and PANI solutions were mixed and sonicated for 12 hrs to

2

obtain a well-dispersed G-PANI nanocomposite solution. Next, 1 µL-aliquot of G-

3

PANI nanocomposite was casted onto the surface area of a carbon-paste working

4

electrode. The modified CPE was baked in the oven at 65 oC in order to evaporate

5

NMP solution. The morphology of G-PANI nanocomposite was observed using a

6

JEM-2100 transmission electron microscope (Japan Electron Optics Laboratory Co.,

7

Ltd, Japan).

8

second PDMS layer (containing 10 mm diameter hole or T-junction microchannel)

9

was sealed over PDMS layer containing G-PANI/CPE using reversible ethanol

10

bonding method. Ethanol was dropped onto both PDMS surfaces, the surfaces were

11

then carefully aligned before being baked at 65°C for 1 hour in order to evaporate the

12

NMP within the casted modifier layer. Electrical connections were made using wires

13

glued to the electrodes using silver paste (SPI supplies, West Chester, PA) and epoxy.

14

The actual working electrode area for the batch system was 5 mm2 while droplet

15

system was 0.025 mm2 (due to the confined channel). Examples of microfluidic and

16

batch devices are shown in the Fig.1b.

19

SC

M AN U

TE D

EP

18

For electrochemical detection in batch and droplet-based systems, a

2.4 Electrochemical detection using G-PANI modified CPE

AC C

17

RI PT

1

Electrochemical measurements including cyclic voltammetry, square-wave

20

voltammetry, and chronoamperometry were performed using a commercially available

21

potentiostat (eDAQ, ED410, 410-088, Australia) using standard three-electrode

22

configuration. The working electrode was either a bare CPE or G-PANI/CPE. Both

23

pseudo-reference and auxiliary electrodes were CPEs. Cyclic voltammetry was used

24

to compare electrochemical performance of each working electrode type using the

ACCEPTED MANUSCRIPT 1

working potential ranges of -0.60 to +0.60 V and -0.20 to +1.00 V vs. CPE for

2

ferri/ferrocyanode and 4-AP, respectively.

3

To study mass transfer of 4-AP on G-PANI/CPE, the dependence of scan rates

4

was performed in the range of 0.01-0.20 V s-1.

5

simultaneously, square-wave voltammetry with a potential range of –0.20 to +1.00 V

6

vs. CPE was used. Voltammograms were recorded in the positive sweep direction to

7

observe the anodic responses of both 4-AP and PA. All electrochemical experiments

8

were carried out at room temperature.

SC

RI PT

To measure 4-AP and PA

10

TE D

M AN U

9

Fig. 1 (a) Schematic of microchannels (top) and electrode patterns (bottom). (b) An

12

electrochemical batch cell (left) and a microfluidic device comprising of main channel and

13

confined channel coupled with microband electrodes (WE-working electrode, CE-counter

14

electrode, and RE-reference electrode).

AC C

15

EP

11

16

2.5 Chronoamperometric determination of 4-AP

17

For microfluidic system, droplets were generated using an oil carrier phase

18

(perfluorodecalin and 1H, 1H, 2H, 2H-perfluoro-1-octanol at a ratio of 10:2 (v/v) and

ACCEPTED MANUSCRIPT an aqueous testing solution. Chronoamperometric detection was performed for the

2

detection of 4-AP in the droplets. The oil phase flow rate was 1.8 µL min-1 while the

3

aqueous phase flow rate was 0.8 µL min-1. The water fraction (Wf) was calculated

4

from following equation [51]. Wf = Fw/(Fw + Fo)

5

7

(1)

where Wf is the water fration, Fw is aqueous- phase flow rate, and Fo is the aqueous- and oil-phase flow rates, respectively.

SC

6

RI PT

1

8

Here, the total flow rate was 2.6 µL min-1, corresponding to a linear flow

10

velocity of 0.86 mm s-1 and a water fraction of 0.31. Chronoamperometric responses

11

from droplet contents were measured while droplets passing through the three-

12

electrode system. Data was collected for 1 min and averaged currents were plotted

13

versus detection potential (Edet) to generate hydrodynamic voltammograms (HDVs) in

14

the range of 0.00–0.40 V vs. CPE.

15

detection potential as defined by the highest signal-to-noise ratio (S/N). The limits of

16

detection (LOD) and quantitation (LOQ) were obtained from LOD = 3(SDbl/S) and

17

LOQ = 10(SDbl/S), respectively, where SDbl is the standard deviation of the blank

18

measurement (n = 10) and S is the slope of the linear calibration. Finally, our method

19

was applied for 4-AP determination in PA liquid samples. Prior to analysis, the PA

20

liquid samples was diluted using 0.1 M PBS pH 7.4. The 4-AP concentration levels in

21

all samples were determined and compared with those obtained for the standard

22

CZE/UV-Vis method [52].

TE D

M AN U

9

AC C

EP

HDVs were used to determine the optimal

23

CZE separation was performed using a MDQ automated CE system coupled

24

with a diode array detector (Beckman), fluid-cooled column cartridge and automatic

ACCEPTED MANUSCRIPT injector [11]. A fused-silica capillary column of 57 cm (50 cm length to detector) × 75

2

µm i.d. and 375 µm o.d. was used. For each CE run, the capillary column was first

3

rinsed with 0.1 M sodium hydroxide for 1 min at high pressure (20 psi), followed by

4

rinsing with the running buffer (50 mM borate buffer pH 9.2) for 1 min. Samples

5

were hydrodynamically injected for 10 s and a voltage of +20 kV was applied for

6

separation. UV absorption was monitored at 250 nm.

7

SC

3. RESULTS AND DISCUSSION

3.1 Morphology and electrochemical characterization and of G-PANI/CPE.

9

M AN U

8

RI PT

1

10

Various reports have used G-PANI nanocomposites to improve the

11

electrochemical sensitivity of carbon electrodes with the resulting performance

12

improvement being attributed to the increase in electrode surface area [3, 5, 37, 39-

13

46].

14

microscopy (TEM) was used to characterize G-PANI nanocomposite (Fig.2a). The

15

TEM images clearly show a well dispersion of G with slight agglomeration; moreover,

16

the electron diffraction pattern of G sheet (the inset of Fig. 2a) is matched with the

17

previous published report [42].

EP

TE D

To observe the changes of electrode morphology, transmission electron

Next, cyclic voltammetry of standard ferri/ferrocyanide redox species was used

19

to characterize the electrochemical properties of the bare CPE, PANI/CPE, and G-

20

PANI/CPE electrodes (Fig. 2b).

21

PANI/CPE exhibited well-defined anodic and cathodic ferri/ferrocyanide current

22

responses which were 10-fold higher than those obtained from the bare CPE and 3-

23

fold greater than those of PANI/CPE and G/CPE. Moreover, peak potential separation

24

between anodic and cathodic peaks was noticeably reduced when using G-PANI/CPE

25

compared to those measured from the PANI/CPE and bare CPE, indicating that the G-

AC C

18

Compared to the PANI/CPE and bare CPE, G-

ACCEPTED MANUSCRIPT 1

PANI nanocomposite can be also used as an electrode modifier to improve the electron

2

transfer kinetics.

AC C

4

EP

TE D

M AN U

SC

RI PT

3

5

Fig. 2 SEM images of (a) bare SPCE, (b) G-PANI/SPCE, (c) A TEM image of G-PANI

6

nanocomposite with an electron diffraction pattern of G (the inset of Fig.2c) and (d) cyclic

7

voltammograms of 1 mM ferri/ferrocyanide in 0.1 M KCl at bare CPE (black line),

8

PANI/CPE (green line), G/CPE (yellow line) and G-PANI/CPE (red line).

9 10

ACCEPTED MANUSCRIPT 3.2 Electrochemical detection of 4-AP and PA using G-PANI/CPE.

2

Next, the electrochemical behavior of 4-AP and PA using the G-PANI/CPE in

3

the batch cell was investigated using cyclic voltammetry with the comparison between

4

the G-PANI/CPE and bare CPE, which is shown in Fig. 3a. Cyclic voltammograms of

5

the G-PANI/CPE exhibited quasi-reversible redox peaks with significantly increased

6

redox current (2-fold) and reduced redox potentials (~100 mV) when compared to bare

7

CPE.

8

nanocomposite towards 4-AP, demonstrating that the nanomodifier offers improved

9

sensitivity for 4-AP, compared to the unmodified CPE.

RI PT

1

SC

These results can be ascribed to good electrocatalytic activity of G-PANI

M AN U

Therefore, the G-PANI

10

nanocomposite can be used as an excellent electrode modifier in the electrochemical

11

system.

To study the mass transfer process of 4-AP on the G-PANI/CPE surface, a current

13

dependence on scan rate was performed (Fig. 3b and c). A slight shift of both anodic

14

and cathodic peak potentials was observed owing to the adsorption of 4-AP on the

15

electrode surface. Both anodic (ip,a) and cathodic (ip,c) peak currents increased linearly

16

with the square root of scan rate (ν1/2) with correlation coefficients (R2) over 0.99.

17

These results verify that the redox process was controlled by diffusion of 4-AP

18

corresponding to the Randles-Sevcik equation (equation 1).

20

EP

AC C

19

TE D

12

ip = 2.686 × 105 n3/2 A C D1/2 ν1/2

(2)

where ip is the peak current of 4-AP in A, n is the number of electrons involved

21

(to reduce/oxidize one molecule of 4-AP), A is the electrode area in cm2, D is the

22

diffusion coefficient of 4-AP in cm2 s-1, C is the concentration of 4-AP in mol cm-3,

23

and ν1/2 is the scan rate in V s-1.

24

2

EP

1

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 3 (a) Comparison of cyclic voltammograms of 1 mM 4-AP obtained from the G-

4

PANI/CPE and bare CPE at a scan rate of 0.1 V/s, (b) cyclic voltammograms of 1 mM

5

4-AP at the G-PANI/CPE with various scan rates in the range of 0.01-0.2 V/s, (c) peak

6

currents of 4-AP as a function of the square root scan rate (ν1/2), and (d) square-wave

7

voltammograms of 4-AP and PA (0-0.2 mM) at the G-PANI/CPE with the scanning

8

potential range of -0.2 to 0.8 V vs. CPE.

9

AC C

3

ACCEPTED MANUSCRIPT Subsequently, square-wave voltammetry was used to determine detection limits

2

and linearity. Fig. 3d displays representative square-wave voltammograms of 4-AP

3

and PA at concentrations between 50 and 200 µM. For the response from both 4-AP

4

and PA, anodic peak currents are linearly proportional to their concentrations with an

5

LOD of 4.33 µM and 2.12 µM for 4-AP and PA, respectively. The peak potential

6

separation between 4-AP and PA is clearly observed with an anodic potential

7

difference (EPA-E4-AP) of 380 mV. As a result, the selection of potential to use in

8

further chronoamperometric experiments would be particularly useful for selective

9

electrochemical detection of 4-AP in the presence of PA.

SC

RI PT

1

M AN U

10

3.3 Chronoamperometric detection in droplets

12

Chronoamperometric detection has been broadly used in the various flow-based

13

systems due to its ease of operation and low background current. Fig. 4 shows a

14

representative chronoamperogram of 300 µM 4-AP in droplets in which each discrete

15

peak corresponds to an individual droplet. When the droplet front began to pass

16

through the electrode array, the current increased sharply. With the electrooxidation

17

reaction of 4-AP proceeded, its concentration region at the nearby electrode surface

18

exponentially decreased, resulting that the current slowly decayed with time

19

corresponding to Cottrell equation (equation 2). Subsequently, the current approaches

20

a steady state level of 5.14±0.11 nA (n=10) which can be assigned to the diffusion-

21

limited current of 4-AP. For quantification, the current was measured during this

22

regime.

AC C

EP

TE D

11

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

TE D

1

Fig. 4 (a) A chronoamperogram of droplets containing 0.3 mM 4-AP in 0.1 M PBS pH

3

7.4. The optimal conditions: a detection potential (Edet) of +0.2 V vs. CPE, a total flow

4

rate of 2.6 µL min-1 (0.016 mm s-1) and Wf = 0.31.

6 7

AC C

5

EP

2

3.3.1 Selection of the optimal detection potential (Edet).

Selective detection of 4-AP in the presence of PA in the flow-based system was

8

assessed. According to Fig. 3d, the selection of Edet lower than +0.3 V vs. CPE should

9

achieve selective detection of 4-AP in the presence of PA. To select an optimal Edet

10

for chronoamperometric detection of 4-AP and PA in the droplet-based flow system,

11

hydrodynamic voltammetry (Fig. 5a) experiments were conducted. The anodic peak

12

current of 4-AP increased with Edet.

There is no signal response from PA and

ACCEPTED MANUSCRIPT 1

background (BG) solution (0.1 M PBS pH 7.4) in the range of 0.0-0.2 V vs. CPE

2

while both anodic current signals of 300 µM PA and BG solution also increased with

3

Edet over +0.2 V vs. CPE.

4

Thus, the Edet for chronoamperometric detection in the range between 0.0-0.2 V

6

vs. CPE can be used for selective detection of 4-AP without an interfering response

7

from PA. To optimize the Edet for chronoamperometric detection of droplet contents,

8

the signal-to-background (S/B) ratio of 4-AP and PA was plotted as a function of Edet.

9

As seen from Fig. 5b, an Edet of +0.2 V vs. CPE showed the highest S/B ratio for 4-AP

10

detection;

+0.2

11

chronoamperometric Edet for quantitative measurements of 4-AP for further

12

experiment.

vs.

CPE

was

chosen

as

the

optimal

AC C

EP

TE D

13

V

M AN U

accordingly,

SC

RI PT

5

14 15 16

Fig. 5 (a) Hydrodynamic voltammogram and (b) signal to background ratio of 0.3 mM 4-AP

17

and 0.3 mM PA. The measurements were carried out using a total flow rate of 2.6 µL min-1

18

(0.016 mm s-1) and Wf = 0.31.

ACCEPTED MANUSCRIPT 1

3.3.2 Effect of water fraction and total flow rate The influence of water fraction (Wf) and total flow rate on chronoamperometric

3

measurements were thenstudied. Wf is a well-known factor strongly affecting droplet

4

size with droplet size increasing with Wf. In this work, the droplet sizes at Wf values in

5

the range of 0.15-0.46 were generated by adjusting the flow rate ratio between oil and

6

aqueous solutions while the total flow rate was kept constant at 2.6 µL min-1. As

7

shown in Fig. 6a, when the water fraction was lower than 0.31, unstable droplets with

8

small sizes were produced, resulting in low anodic current. For Wf values over 0.38,

9

droplet length was too long, resulting that the anodic steady-state current decreased

10

due to amperometrically exponential decay. The highest current was obtained at a

11

water fraction of 0.31. Accordingly, the water fraction of 0.31 was found to be the

12

optimal Wf to produce stable droplets and all subsequent experiments were performed

13

using this conditions.

M AN U

SC

RI PT

2

AC C

EP

TE D

14

15 16

Fig. 6 Effects of (a) total flow rate and (b) water fraction on chronoamperometric

17

detection of 0.3 mM 4-AP. Conditions: an detection potential (Edet) of +0.2 V vs. CPE,

18

total flow rate of 2.6 µL min-1 (0.016 mm s-1) and Wf = 0.31.

ACCEPTED MANUSCRIPT 1

Another important factor influencing on chronoampermetric response is total

2

flow rate. The effect of total flow rate was investigated as shown in Fig. 6b. While the

3

water fraction was kept at a constant of 0.31, total flow rate was varied between 1.30-

4

3.90 µL min-1, corresponding to a linear flow velocity in the range of 0.43-1.29 mm s-

5

1

6

steady-state current was obtained due to the long residence time of a single droplet

7

passing over the electrode surface. When the total flow rate was >3.25 µL min-1

8

(linear velocity > 1.08 mm/s), the current also reduced significantly. We believe that

9

this high total flow rates provided short residence time which was not sufficient for the

SC

RI PT

. Results show that for flow rates <2.6 µL min-1 (linear velocity < 0.43 mm/s), a low

electrochemical process to occur.

11

obtained from the total flow rates at 2.6 and 3.25 µL min-1. In addition, use of high

12

flow rates to produce droplets increases reagent/sample consumption. Therefore, an

13

optimal total flow rate of 2.6 µL min-1 (linear velocity = 0.86 mm s-1) was selected to

14

use in further experiments.

TE D

15

The highest steady-state current values were

M AN U

10

3.4 Analytical performance.

17

The analytical performance of the droplet microfluidics coupled with G-

18

PANI/CPE was investigated. Chronoamperometric detection of 4-AP at Edet of +0.2 V

19

vs. CPE was performed using the optimal conditions as mentioned above. Calibration

20

curve of 4-AP was plotted between anodic current as a function of the standard 4-AP

21

concentration as shown in Fig. 7. A linear range was found to be 50-500 µM with

22

correlation coefficient (R2) of 0.998. The limit of detection (LOD, S/N=3) was found

23

to be 15.68 µM while limit of quantitation (LOQ, S/N=10) was found to be 52.28 µM.

24

The European, United States, British, German, and Chinese Pharmacopoeias report the

25

maximum allowable limit of 4-AP in PA products is 50 ppm (0.005% w/w); therefore,

AC C

EP

16

ACCEPTED MANUSCRIPT 1

our system can determine 4-AP at the relevant concentration in commercial PA

2

samples.

TE D

M AN U

SC

RI PT

3

4

Fig. 7 A linear calibration plot of 4-AP in the concentration range of 0-500 µM. The optimal

6

conditions: an detection potential (Edet) of +0.2 V vs. CPE, total flow rate of 2.6 µL min-1

7

(0.016 mm s-1) and Wf = 0.31. (Inset) A chronoamperogram of 4-AP current vs. time.

9

AC C

8

EP

5

In this work, a chronoamperometric measurement within droplet volume of

10

0.625 nL was performed.

This volume of testing solution was lower than those

11

required from conventional electrochemical batch cells (normally require sample

12

volumes at least 0.5 mL), corresponding to a high limit of detection of our proposed

13

system.

Although the LOD and LOQ values reported here are higher than those

ACCEPTED MANUSCRIPT 1

obtained from the previous publications [2-3, 5, 7, 10], our approach offers simplicity

2

and inexpensive operation.

3

sample/reagent was required to operate the droplet generation within the microfluidic

4

system, leading to a fast and high-throughput analysis of 4-AP.

In addition, small aliquot (<1 mL) of testing

In addition to assess the stability of this system, chronoamperometric detection

6

of standard 4-AP at different concentrations by 20 successive measurements was

7

performed for intra-day and inter-day precisions as shown in the Fig.8. For intra-day

8

and inter-day results, relative standard deviations (RSDs) were found to be less than

9

5% (n = 20). Therefore, these results verify that our proposed system had the good

10

stability. Moreover, the reproducibility of the microfluidic sensor was evaluated using

11

chronoamperometric detection of 0.3 mM 4-AP. The %RSD of seven different devices

12

was found to be 3.5%, indicating that the fabricated microfluidic sensor had a good

13

precision.

M AN U

SC

RI PT

5

15

AC C

EP

TE D

14

16

Fig. 8 Intra-day measurements of (a) 4-AP at concentrations of 0.1, 0.2 and 0.3 mM and (b)

17

Inter-day measurements of 0.1 and 0.3 mM 4-AP for three days (n = 3).

18

ACCEPTED MANUSCRIPT 3.5 Interference study and Determination of 4-AP in commercial PA samples.

2

The effect of various foreign species on the detection of 0.3 mM 4-AP was

3

studied. The 5% tolerable ratio of foreign species is shown in Table 1. The tolerable

4

ratio was defined as the level of foreign species, generating a change in intensity of the

5

peak current of ±5%. From the results, these species were tolerated at a high level,

6

demonstrating the good selectivity of the proposed method.

RI PT

1

Additionally, since 4-AP is produced from the degradation of pharmaceutical

8

PA samples, the selectivity of this method towards the determination of 4-AP in the

9

presence of PA in commercial samples was evaluated. The tolerance concentration is

10

defined as the concentration of 4-AP, generating a change in signal response of ±5%.

11

Results show that 5000 µM PA gave a 5% change in the detection of 50 µM 4-AP;

12

therefore, the PA sample was diluted to a lower concentration. Next, the application

13

of our method was tested by measuring the spiked 4-AP concentrations (100, 200, 300,

14

400, and 500 µM) in each diluted PA sample.

M AN U

Table 1 Effect of foreign ions on the determination of 0.3 mM 4-AP

EP

16

TE D

15

SC

7

5% tolerable

AC C

Foreign species

17

molar ratio

Na+,K+,Mg2+, Ca2+, NO3-, SO42-, PO43-, Cl- ,Glucose, Sucrose

>10000

Cu2+, Fe2+, Fe3+

300

Acetylsalicilic acid, Glutathione

1000

Uric acid, Dopamine

500

Ascorbic acid, Cysteine

200

Paracetamol

100

ACCEPTED MANUSCRIPT As shown in the table 2, the % recovery of this system was found in the range

2

of 91.21%-105.34% (n=20). Moreover, to validate our method, the results obtained

3

from the proposed system was compared to those obtained from the conventional

4

capillary zone electrophoresis/UV-Visible spectrophotometric technique (CZE/UV-

5

Vis) [11] using a paired t-test at 95% confidential interval. The experimental t-values

6

(tcalculated) obtained by this proposed method was found to be -2.20 which were

7

significantly lower than the critical t-values of 1.76. Results show that there is no

8

significant difference between the results obtained from our method and CZE/UV-Vis

9

method. As a result, our electrochemical droplet system is an alternative and high-

10

throughput method (at least 60 samples h-1) for determining the 4-AP contamination in

11

commercial PA formulations.

AC C

EP

TE D

12

M AN U

SC

RI PT

1

ACCEPTED MANUSCRIPT 1

Table 2 The % recovery of the our proposed method (n=20) for determination of 4-AP

2

in PA samples vs. conventional CZE/UV-Vis method (n=3).

3

This proposed method

CZE/UV-Vis method

(n=20)

(n=3)

Sample

concentration %Recovery (µM)

%Recovery

%RSD

2.75

99.74±1.01

1.01

200

91.29±3.29

3.60

102.95±3.29

3.20

300

101.12±4.12

4.07

98.89±2.11

2.13

400

97.34±3.93

4.04

99.19±1.71

1.72

500

101.25±4.19

4.14

101.91±2.41

2.36

100

99.97±3.21

3.21

99.24±1.31

1.32

200

98.08±2.94

3.00

98.93±1.92

1.94

95.25±3.17

3.33

102.25±1.27

1.24

105.34±2.94

2.78

103.04±2.14

2.08

M AN U

®

300

EP 500

91.93±2.74

2.98

99.91±0.24

0.24

100

97.51±3.19

3.27

102.01±2.45

2.40

200

101.12±1.92

1.90

99.92±1.29

1.29

300

102.12±2.25

2.20

101.01±2.01

1.99

400

103.34±1.89

1.83

103.34±1.71

1.65

500

101.25±2.97

2.93

103.15±1.31

1.27

AC C

4 5

SC

91.21±2.51

400

SARA®

(±SD)

100

Infants’

TEMPRA®

%RSD

TE D

(±SD)

TYLENOL

RI PT

Spiked 4-AP

ACCEPTED MANUSCRIPT 1

CONCLUSIONS In this work, we report a novel electrochemical droplet microfluidic device for

3

determination of 4-AP in commercial PA formulations. The G-PANI/CPE was first

4

coupled with the droplet system to perform chronoamperometric detection of 4-AP.

5

The improvement in electrochemical sensitivity was attributed to an enhancement of

6

the active surface area of electrode and increased electrochemical conductivity. Our

7

proposed method can perform a high-throughput and rapid measurement of 4-AP in

8

commercial PA products with required small consumption of reagent/sample.

9

Eventually, this system was successfully applied for the determination of 4-AP in PA

10

liquid samples with high precision and good recovery; therefore, this system might be

11

an alternative analytical device for routine analysis of 4-AP residue in pharmaceutical

12

products.

M AN U

SC

RI PT

2

13

ACKNOWLEDGEMENTS

TE D

14

P. R. wishes to thank the financial support from Chulalongkorn University and

16

the Thailand Research Fund, through the Royal Golden Jubilee Ph.D. Program (Grant

17

No. PHD/0251/2552). Moreover, this research has been supported by the Thailand

18

Research Fund through Research Team Promotion Grant (RTA578005) and National

19

Research University project, Office of Higher Education Commission (WCU-032-

20

AM-57).

AC C

21

EP

15

ACCEPTED MANUSCRIPT 1

REFERENCES

2

[1]

N. Sarkar, Environmental Toxicology and Pharmacology 37 (2014) 438. [2]

L. Y. Shiroma, M. Santhiago, A. L. Gobbi, L. T. Kubota, Analytica Chimica Acta 725 (2012) 44.

5

RI PT

3 4

K. Vijayakaran, K. Kannan, M. Kesavan, S. Suresh, P. Sankar, S. K. Tandan, S.

[3]

H. Yin, Q. Ma, Y. Zhou, S. Ai, L. Zhu, Electrochimica Acta 55 (2010) 7102.

7

[4]

M. S. Bloomfield, Talanta 58 (2002) 1301.

8

[5]

Y. Fan, J.-H. Liu, C.-P. Yang, M. Yu, P. Liu, Sensors and Actuators B: Chemical 157 (2011) 669.

[6]

A. Brega, P. Prandini, C. Amaglio, E. Pafumi, Journal of Chromatography A

M AN U

9 10

SC

6

535 (1990) 311.

11

[7]

C.-M. Zou, H. Yu, M.-Y. Wang, Chinese Chemical Letters 25 (2014) 201.

13

[8]

B. Schultz, Journal of Chromatography A 299 (1984) 484.

14

[9]

B. C. Lourenção, R. A. Medeiros, R. C. Rocha-Filho, L. H. Mazo, O. Fatibello-

TE D

12

Filho, Talanta 78 (2009) 748.

15

[10]

Q. Chu, L. Jiang, X. Tian, J. Ye, Analytica Chimica Acta 606 (2008) 246.

17

[11]

T. Pérez-Ruiz, C. Martínez-Lozano, V. Tomás, R. Galera, Journal of

[12]

21

[13]

22

[14]

Y. Zhu, Q. Fang, Analytica Chimica Acta 787 (2013) 24. C.-G. Yang, Z.-R. Xu, J.-H. Wang, TrAC Trends in Analytical Chemistry 29

(2010) 141.

23

25

A. B. Vishnikin, M. K. E. A. Al-Shwaiyat, G. A. Petrushina, L. P. Tsiganok, V.

Andruch, Y. R. Bazel, H. Sklenářová, P. Solich, Talanta 96 (2012) 230.

20

24

AC C

Pharmaceutical and Biomedical Analysis 38 (2005) 87.

18 19

EP

16

[15]

S. Park, Y. Zhang, S. Lin, T.-H. Wang, S. Yang, Biotechnology Advances 29 (2011) 830.

ACCEPTED MANUSCRIPT 1

[16]

Chemical 180 (2013) 21.

2 3

B. Ramaswamy, Y.-T. T. Yeh, S.-Y. Zheng, Sensors and Actuators B:

[17]

Y. T. Atalay, S. Vermeir, D. Witters, N. Vergauwe, B. Verbruggen, P. Verboven, B. M. Nicolaï, J. Lammertyn, Trends in Food Science & Technology

5

22 (2011) 386.

6

[18]

RI PT

4

M. Safavieh, S. Nahar, M. Zourob, M. U. Ahmed, in High Throughput Screening for Food Safety Assessment (Eds.: A. K. Bhunia, M. S. Kim, C. R.

8

Taitt), Woodhead Publishing, 2015, pp. 327. [19]

Z.-X. Guo, Q. Zeng, M. Zhang, L.-Y. Hong, Y.-F. Zhao, W. Liu, S.-S. Guo, X.Z. Zhao, Sensors and Actuators A: Physical 172 (2011) 546.

10

M AN U

9

SC

7

11

[20]

S. Teychené, B. Biscans, Chemical Engineering Science 77 (2012) 242.

12

[21]

M.-P. N. Bui, C. A. Li, K. N. Han, J. Choo, E. K. Lee, G. H. Seong, Analytical Chemistry 83 (2011) 1603.

14

[22]

H. Tsuchiya, M. Okochi, N. Nagao, M. Shikida, H. Honda, Sensors and

TE D

13

Actuators B: Chemical 130 (2008) 583.

15

[23]

J. Ji, Y. Zhao, L. Guo, B. Liu, C. Ji, P. Yang, Lab on a Chip 12 (2012) 1373.

17

[24]

I. Lignos, L. Protesescu, S. Stavrakis, L. Piveteau, M. J. Speirs, M. A. Loi, M.

[25]

[26]

24

S.-W. Lin, C.-H. Chang, C.-H. Lin, Genomic Medicine, Biomarkers, and

Health Sciences 3 (2011) 27.

22 23

T. Konry, M. Dominguez-Villar, C. Baecher-Allan, D. A. Hafler, M. L.

Yarmush, Biosensors and Bioelectronics 26 (2011) 2707.

20 21

AC C

V. Kovalenko, A. J. deMello, Chemistry of Materials 26 (2014) 2975.

18 19

EP

16

[27]

J. Lee, H. K. Musyimi, S. A. Soper, K. K. Murray, Journal of the American Society for Mass Spectrometry 19 (2008) 964.

ACCEPTED MANUSCRIPT [28]

Edel, Analytical Chemistry 83 (2011) 3076.

2 3

[29]

G. Aubry, S. Méance, L. Couraud, A. M. Haghiri-Gosnet, Q. Kou, Microelectronic Engineering 86 (2009) 1368.

4 5

M. P. Cecchini, J. Hong, C. Lim, J. Choo, T. Albrecht, A. J. deMello, J. B.

[30]

J. Clausell-Tormos, D. Lieber, J.-C. Baret, A. El-Harrak, O. J. Miller, L. Frenz,

RI PT

1

6

J. Blouwolff, K. J. Humphry, S. Köster, H. Duan, C. Holtze, D. A. Weitz, A. D.

7

Griffiths, C. A. Merten, Chemistry & Biology 15 (2008) 427. [31]

J. Wang, Talanta 56 (2002) 223.

9

[32]

S. Liu, Y. Gu, R. B. Le Roux, S. M. Matthews, D. Bratton, K. Yunus, A. C.

SC

8

M AN U

Fisher, W. T. S. Huck, Lab on a Chip 8 (2008) 1937.

10 11

[33]

Z. Han, W. Li, Y. Huang, B. Zheng, Analytical Chemistry 81 (2009) 5840.

12

[34]

S. Gu, Y. Lu, Y. Ding, L. Li, H. Song, J. Wang, Q. Wu, Biosensors and Bioelectronics 55 (2014) 106.

[35]

Actuators B: Chemical 171–172 (2012) 619.

15

[36]

Acta 690 (2011) 10.

17 18

[37]

M. Arvand, T. M. Gholizadeh, Colloids and Surfaces B: Biointerfaces 103 (2013) 84.

19 20

W. Siangproh, W. Dungchai, P. Rattanarat, O. Chailapakul, Analytica Chimica

EP

16

D. Itoh, F. Sassa, T. Nishi, Y. Kani, M. Murata, H. Suzuki, Sensors and

AC C

14

TE D

13

[38]

H. Ghadimi, R. M.A.Tehrani, A. S. M. Ali, N. Mohamed, S. Ab Ghani,

Analytica Chimica Acta 765 (2013) 70.

21 22

[39]

S. Wu, Q. He, C. Tan, Y. Wang, H. Zhang, Small 9 (2013) 1160.

23

[40]

N.

24

Thammasoontaree,

P.

Rattanarat,

N.

Ruecha,

Rodthongkum, O. Chailapakul, Talanta 123 (2014) 115.

W.

Siangproh,

N.

ACCEPTED MANUSCRIPT [41]

Analytica Chimica Acta 804 (2013) 84.

2

[42]

Bioelectronics 52 (2014) 13. [43]

(2013) 240.

6 7

[44]

[45]

J. Li, S. Liu, J. Yu, W. Lian, M. Cui, W. Xu, J. Huang, Sensors and Actuators B: Chemical 188 (2013) 99.

10 11

E. Asadian, S. Shahrokhian, A. I. zad, E. Jokar, Sensors and Actuators B: Chemical 196 (2014) 582.

8 9

Y.-P. Dong, J. Zhang, Y. Ding, X.-F. Chu, J. Chen, Electrochimica Acta 91

RI PT

4 5

N. Ruecha, R. Rangkupan, N. Rodthongkum, O. Chailapakul, Biosensors and

[46]

SC

3

N. Rodthongkum, N. Ruecha, R. Rangkupan, R. W. Vachet, O. Chailapakul,

M AN U

1

J. Huang, Q. Lin, X. Zhang, X. He, X. Xing, W. Lian, M. Zuo, Q. Zhang, Food Research International 44 (2011) 92.

12

[47]

H. Liu, R. M. Crooks, Lab on a Chip 13 (2013) 1364.

14

[48]

Y. Sameenoi, M. M. Mensack, K. Boonsong, R. Ewing, W. Dungchai, O.

TE D

13

Chailapakul, D. M. Cropek, C. S. Henry, Analyst 136 (2011) 3177.

15

[49]

N. Ruecha, W. Siangproh, O. Chailapakul, Talanta 84 (2011) 1323.

17

[50]

A. Suea-Ngam, P. Rattanarat, O. Chailapakul, M. Srisa-Art, Analytica Chimica

19

[51]

20

[52]

21

22 23

AC C

Acta.

18

EP

16

J. D. Tice, A. D. Lyon, R. F. Ismagilov, Analytica Chimica Acta 507 (2004) 73. T. Németh, P. Jankovics, J. Németh-Palotás, H. Kőszegi-Szalai, Journal of

Pharmaceutical and Biomedical Analysis 47 (2008) 746.

ACCEPTED MANUSCRIPT

RESEARCH HIGHLIGHTS •

A novel combination of G-PANI/CPE and droplet-based microfluidic device for determination of 4-AP was achieved. G-PANI nanocomposite increases the surface area and improved electrochemical sensitivity of the electrode.

EP

TE D

M AN U

monitoring in commercial PA samples.

SC

This novel droplet-based system was successfully applied for 4-AP

AC C

•

RI PT

•