Approach for Downscaling of Electromembrane Extraction as a Lab on

Subscriber access provided by University of Massachusetts Amherst Libraries

Article

A new approach for downscaling of electromembrane extraction as a lab on-a-chip device followed by sensitive Red-Green-Blue detection Mahroo Baharfar, Yadollah Yamini, Shahram Seidi, and Muhammad Balal Arain Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

1

A New Approach for Downscaling of Electromembrane Extraction as a Lab on-a-Chip

2

Device Followed by Sensitive Red-Green-Blue Detection

3

Mahroo Baharfara, Yadollah Yaminia,*, Shahram Seidib, Muhammad Balal Arainc a

4

Department of Chemistry, Faculty of Sciences, Tarbiat Modares University, P.O. Box: 14115-175, Tehran, Iran

b

5

Department of Analytical Chemistry, Faculty of Chemistry, K.N. Toosi University of Technology, Tehran, Iran c

6

Department of Chemistry, Abdul Wali Khan University, Mardan, K.P.K, Pakistan, 23200

7

Corresponding Author:

8

*

Email: [email protected], Phone: +98-21-82883449, Fax: +98-21-82883460.

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

24

ABSTRACT

25

A new design of electromembrane extraction (EME) as a lab on-a-chip device was

26

proposed for the extraction and determination of phenazopyridine as the model analyte. The

27

extraction procedure was accomplished by coupling EME and packing a sorbent. The analyte

28

was extracted under the applied electrical field across a membrane sheet impregnated by

29

nitrophenyl octylether (NPOE) into an acceptor phase. It was followed by the absorption of the

30

analyte on strong cation exchanger as a sorbent. The designed chip contained separate spiral

31

channels for donor and acceptor phases featuring embedded platinum electrodes to enhance

32

extraction efficiency. The selected donor and acceptor phases were 0 mM HCl and 100 mM HCl,

33

respectively. The on-chip electromembrane extraction was carried out under the voltage level of

34

70 V for 50 min. The analysis was carried out by two modes of a simple Red-Green-Blue (RGB)

35

image analysis tool and a conventional HPLC-UV system. After the absorption of the analyte on

36

the solid phase, its color changed and a digital picture of the sorbent was taken for the RGB

37

analysis. The effective parameters on the performance of the chip device, comprising the EME

38

and solid phase microextraction steps, were distinguished and optimized. The accumulation of

39

the analyte on the solid phase showed excellent sensitivity and a limit of detection (LOD) lower

40

than 1.0 µg L-1 achieved by an image analysis using a smartphone. This device also offered

41

acceptable intra- and inter-assay RSD% ( 0.9969) for HPLC-UV and RGB analysis,

43

respectively. To investigate the applicability of the method in complicated matrices, urine

44

samples of patients being treated with phenazopyridine were analyzed.

45 46 47

Keywords: Electromembrane extraction; Lab on-a-chip; Phenazopyridine; RGB analysis; Smartphone. 2 ACS Paragon Plus Environment

Page 2 of 29

Page 3 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

48


INTRODUCTION

49

Design and application of the new generation of miniaturized chemical devices is a current,

50

interesting topic in a broad range of research fields from biology and chemistry to engineering1,2.

51

The concept behind these microscale lab on-a-chip (LOC) devices is integrating one or several

52

processes into a single chip3. LOC systems have undergone extensive promotions since their

53

advent, thanks to the numerous advantages associated with these systems, including higher

54

efficiency and sample throughput, low consumption of reagents and solvents, portability,

55

feasibility of parallelization and automation, and shortening the process time4-6. Utilization of

56

LOC devices is also a superior strategy to benefit chemical analysis and integration of, for

57

instance, sample preparation, separation, and detection processes over a small platform7. Among

58

these steps, sample preparation is a critical step before a chemical analysis. This helps to reduce

59

the matrix effect, provide a suitable form of sample for instrumental analysis, concentrate

60

analytes, eliminate interferences, and warrant reliable measurements. For this purpose, plenty of

61

conventional sample preparation techniques have been proposed during the several past decades,

62

entailing excessive amounts of organic solvents and chemicals, time-consuming steps, and

63

difficult handling8-10.

64

Therefore, the necessity of developing more environmentally and user-friendly techniques

65

as well as improving sensitivity, selectivity, and efficiency has eclipsed many research incentives

66

in the field of analytical chemistry. Owing to these extensive efforts, several innovative sample

67

preparation methods have been established. Among them, membrane-based techniques are well-

68

known sample preparation methods because of their advantages over other similar techniques,

69

benefiting from high sample clean-up, favorable extraction efficiency, and low required amounts

70

of organic solvents11.

3 ACS Paragon Plus Environment


71

Electromembrane extraction (EME) is one of the relatively new membrane-based

72

extraction methods which was introduced at first by Pedersen Bjergaard et al12. The basis of this

73

electrically-driven method is related to the fact that the mass transfer of charged species is

74

enhanced by the application of an electrical field. Therefore, in this method, two platinum

75

electrodes connected to a power supply are responsible for providing an electrical field across a

76

thin layer of an organic solvent immobilized in the pores of a porous polypropylene sheet or a

77

hollow fiber. Analytes are extracted by the electrical force through the supported liquid

78

membrane into the acceptor phase in their ionized form13-15. Since the initiation of EME, much

79

advancement has been accomplished to make this technique more efficacious16-18. Due to the

80

fascinating and advantageous features of LOC devices, one of the most promising strategies for

81

further development of EME is the exploitation of this technique on chip platforms. The early

82

studies on this issue go back to 2010 and since then diverse designs of EME as downscaled LOC

83

systems have been proposed19,20. However, designing an integrated, sensitive, and portable

84

system without any special instrumental analysis still remains a challenge and few works on this

85

topic have been reported in the literature21.

86

In the present study, a new design of EME as a LOC device followed by a microextraction

87

step accomplished by a packed solid phase was proposed for extraction and preconcentration of

88

phenazopyridine (PP) as a model analyte. The chip device consisted of spiral channels for both

89

donor and acceptor phase compartments to obtain higher extraction efficiency. It is expected that

90

the higher amount of the acceptor solution prevents the early saturation of the acceptor phase and

91

improves the efficiency of the process. The desired electrical field was applied homogeneously

92

throughout the length of the spiral extraction channels. This homogeneity is achieved by the

93

constant distance between the electrodes and their fixed position.


Page 4 of 29

Page 5 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


94

In order to integrate the whole analysis process on a single platform, the EME procedure

95

was followed by the adsorption of the target analyte on a solid phase. Then, an RGB technique

96

was used as the detection method. The accumulation of the analyte on the solid phase caused the

97

color change of the sorbent due to the orange color of the target compound. To assess the

98

reliability of the RGB method, the HPLC-UV analysis was additionally carried out and the

99

obtained results were compared.

100

To do the RGB analysis, a digital image of the solid phase was taken by a smartphone and

101

its three main color components (red, green, and blue) were analyzed by an image analysis

102

software. The relations between variation in concentration and different color components were

103

determined, and the best concentration-dependent one was selected as the analytical signal.

104

Finally, the applicability of the method was evaluated by the analysis of the real urine samples of

105

patients being treated with PP, and then the results of both analysis methods, HPLC-UV system

106

and RGB analysis, were compared.

107

EXPERIMENTAL

108

Chemicals and reagents. Phenazopyridine (PP) was kindly donated by Tehran Shimi

109

Pharmaceutical Co (Tehran, Iran). The structure and physicochemical properties of PP is shown

110

in Table 1. All chemicals were of analytical grade. Nitrophenyl octylether (NPOE), tris-(2-

111

ethylhexyl) phosphate (TEHP), and di-(2-ethylhexyl) phosphate (DEHP) were supplied from

112

Fluka (Buchs, Switzerland). 1-Octanol was obtained from Merck (Darmstadt, Germany). The

113

Accurel 2E HF (R/P) polypropylene membrane sheet with a thickness of 100 µm and a pore size

114

of 0.2 µm was purchased from Membrana (Wup- pertal, Germany). The ultrapure water used in

115

this work was prepared by a Younglin 370 series aqua MAX purification instrument (Kyounggi-

116

do, Korea). The utilized sorbent was strong cation exchange (SCX) with the particle diameter of



117

50 µm and pore size of 60 Å, provided by Analytica International (New York, NY, United

118

State). The stock solution of PP was prepared at the concentration of 1.0 mg mL-1 in methanol.

119

Standard solutions were freshly prepared from the stock solution by dilution with ultrapure

120

water. All of the standard solutions were stored at 4 ºC and protected from light.

121

Real samples. To plot the calibration curves and assess figures of merit, all experiments

122

were conducted in a drug-free urine sample of a 24-year-old healthy female volunteer. For this

123

purpose, two milliliters of the urine sample was spiked with proper amounts of the standard

124

solution to obtain the desirable concentration without any pretreatment or dilution. The real urine

125

samples were collected from volunteer patients being treated with PP. One of the patients was a

126

53-year-old male volunteer, taking a regular dose of 100 mg three times a day, and the other one

127

was a 43-year-old female volunteer, taking a single dose of 100 mg. All samplings were carried

128

out based on the guidelines for research ethics, and the protocol was approved by an internal

129

review board.

130

Chromatographic apparatus. Separation and detection of PP was accomplished by an

131

Agilent 1260 HPLC system equipped with a quaternary pump, degasser, a 20 µL sample loop

132

and UV-Vis detector (Waldbornn, Germany). Elution of PP was carried out by an isocratic

133

program at the flow rate of 1.0 mL min-1. The mobile phase consisted of acetonitrile and a 10

134

mmol L-1 acetate buffer with the pH of 5.5 (60:40, v/v). For detection and quantification, the

135

detector wavelength was set at 395 nm. The results were recorded and analyzed using

136

ChemStation for the LC system software (version B.04.03). The elution was performed on an

137

ODS-3 column (150 mm × 4.6 mm, with 5 µm particle size) provided by MZ-Analysenteknik

138

(Mainz, Germany).


Page 6 of 29

Page 7 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


139

Preparation of the chip device. The chip device consisted of two polymethylmethacrylate

140

(PMMA) plates as substrates. In each part, the same pattern of spiral channels was carved with

141

the width of 2.0 mm and the depth of 500 µm. The chip device for electromembrane extraction is

142

shown in Figure 1A. The upper plate was a compartment for the stagnant acceptor phase and the

143

lower plate was dedicated to the flow pass of the sample solution. In each spiral pattern, three

144

individual holes were drilled (I.D. of 0.5 mm) to provide inlet and outlet tubes and insert the

145

platinum electrodes. As shown in Figure 1B, holes a and b were exploited to provide the inlet

146

and outlet tubes, and hole c was used to mount the platinum electrodes. These platinum

147

electrodes with the diameter of 0.2 mm were purchased from Pars Platin (Tehran, Iran). The

148

electrodes were fixed by bending and embedding along the length of the channels. To mile all

149

channels and holes, a SMG-302 CNC micromilling machine from Sadrafan Gostar Industries

150

(Tehran, Iran) was used.

151

PMMA plates were separated by a piece of NPOE impregnated polypropylene sheet, which

152

was located between the two parts. The whole structure was fixed by the aid of bolts and nuts,

153

and the membrane sheet was renewed after each extraction. The sample solution was pumped

154

into the corresponding channels by a syringe pump obtained from Fanavaran Nano-Meghyas

155

(Tehran, Iran), and the acceptor solution was injected into the dedicated channel and withdrawn

156

from that by a microsyringe. A transparent silicon tube was packed with 2 mg of SCX and both

157

ends of the tube were manually fitted by two frit pieces. Then, it was connected to the outlet tube

158

of the acceptor phase.

159

On-chip EME coupled with the solid phase microextraction step. A piece of

160

polypropylene sheet, with the dimension of 3.5 mm × 3.5 cm, was cut and impregnated by

161

NPOE. The desired organic solvent was immobilized in the pores of the porous propylene sheet,



162

and the excess amount of that was removed by a small piece of Kleenex. The resulted sheet was

163

located between the two parts of the chip. After fixing the whole device, the sample solution was

164

introduced to the dedicated channel by a syringe pump. 500 µL of 100 mM HCl was injected into

165

the upper channel by a microsyringe as the acceptor phase. After the fulfillment of the EME, the

166

acceptor phase was conducted into the solid phase by conducting the excess air at a certain flow

167

rate with the same syringe pump. Then, the digital image, captured from the solid phase, was

168

decomposed to three main color components (RGB analysis). Through another approach, the

169

tube containing the solid phase was mounted and fixed on the needle of a microsyringe, and the

170

target analyte was desorbed by the ejection and withdrawal of a 20 µL methanol solution

171

containing 40% of 0.2 mol L-1 NaOH. These steps were repeated certain times to ensure the

172

complete desorption of the analyte. The final solution was injected to the HPLC-UV system for

173

further analysis. A diagram of the whole procedure is illustrated in Figure 1. After each

174

extraction, the chip device was carefully washed and dried by ultrapure water and methanol, and

175

the packed silicon tube was replaced with a new one.

176

RGB analysis. To design a portable device capable of an in-situ analysis, an on-line RGB

177

analysis was performed by a smart phone (iPhone SE 2016, USA). For this purpose, the obtained

178

solid phase was placed in a certain position and the smart phone was mounted at a fixed place,

179

12 cm above the solid phase. A digital photo of the solid phase was taken and was analyzed via

180

an iDropper tool, which is an image analysis application available for iOS operating systems.

181

The correlation between concentration and intensity of color components, as a concentration-

182

dependent signal, was investigated by subtraction of the RGB intensities from white. All

183

experiments were carried out with respect to a blank analysis, and optical conditions were the

184

same in all cases.


Page 8 of 29

Page 9 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

185


RESULTS AND DESCUSSION

186

Composition of the supported liquid membrane. Composition of the supported liquid

187

membrane (SLM) has a crucial effect on the EME efficiency. Non-volatile organic solvents that

188

have definite conductivity, compatibility with polypropylene, less toxicity, immiscibility in

189

water, and certain viscosity are worthy of investigation as the SLM in the EME. Considering

190

these criteria and based on previous studies, NPOE and 1-octanol are among the widely utilized

191

solvents in the EME. In addition, numerous reports in the literature have revealed that

192

modification of SLM with DEHP and TEHP as carries can benefit the extraction recovery of the

193

EME, especially in the case of polar compounds11. Therefore, 1-octanol and NPOE containing a

194

known amount of DEHP and TEHP were evaluated. This effect is shown in Figure 2, according

195

to which, NPOE had the best performance and thus was selected as the SLM for further studies.

196

Effect of acceptor and donor phase composition. In EME, extraction mechanism is

197

mainly based on electrokinetic migration, and thus it is of great importance for the donor phase

198

composition to keep the target analytes in their ionized form. In addition, the high concentration

199

of HCl in the acceptor phase facilitates the release of the analytes at the interface of the SLM and

200

the acceptor phase. The ratio of HCl concentration in the donor phase to that in the acceptor

201

phase is defined as ion balance. In line with the reported studies, analyte mass transfer increases

202

with the reduction of ion balance22. This fact is attributed to the competition between proton ions

203

and target ionized compounds, feasibility of electrolysis reactions, bubble formation, Joule

204

heating phenomenon, and other instability problems23. These effects were investigated, and

205

according to Figures 3A and 3B, 0 mM (ultrapure water) and 100 mM HCl were chosen as the

206

composition of the donor and acceptor phases, respectively.



207

Effect of applied voltage. In EME, the electrical field accounts for the main driving force

208

to transfer the target analyte across the SLM into the acceptor phase. Basically, the higher the

209

applied voltage, the more the extraction efficiency in the EME. Nevertheless, at high voltage

210

values, extraction efficiency deteriorates as a result of increasing the current level across the

211

SLM. To reach the optimum level of the applied voltage, this parameter was studied within the

212

range of 50 to 90 V. The results are shown in Figure 3C. At the voltage level of 70 V, the best

213

performance was achieved and was selected as the optimum value.

214

Effect of sample solution flow rate. The main characteristic of the non-exhaustive

215

extraction methods, such as EME, is that extraction recoveries are limited by the time in which

216

the system reaches equilibrium. However, in EME, the extraction time should be adequately

217

short due to the unsustainability of the SLM when the voltage is applied. This parameter was

218

studied, and as can be seen in Figure 3D, at the optimum flow rate of 40 µL min-1, the system

219

reached the desired equilibrium. This flow rate value, regarding 2000 µL of the sample solution,

220

corresponds to an extraction time of 50 min.

221

Solid phase microextraction parameters. The effective parameters on the extraction

222

efficiency of the solid phase microextraction step, including the adsorption flow rate, the type of

223

desorption solvent, the number of draw-eject cycles for desorption, and the desorption volume,

224

were taken into account. The effect of these parameters on the extraction efficiency of the solid

225

phase microextraction step can be found in Figures S1 to S4 in the Supporting Information

226

section. Studies were carried out at the flow rate of 120 µL min-1, at which adsorption

227

equilibrium was acquired. After the RGB detection for the subsequent analysis by the HPLC-UV

228

system, the desorption step is necessary and the related parameters should be investigated. Due

229

to the ionized form of the analyte in the EME and the nature of the target drug, SCX was chosen


Page 10 of 29

Page 11 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


230

as the solid phase material. For desorption of the analyte, a basic desorption solvent was

231

required, and 20 µL of methanol containing 40% NaOH of 0.2 mol L-1 showed the best

232

performance. After 11 cycles of draw-eject, the analyte was successfully desorbed.

233

Method evaluation. To assess the analytical performance of the proposed method, figures

234

of merit were determined in the blank urine samples by the addition of known amounts of the

235

standard solution. In terms of the RGB analysis, the calibration curves were established against

236

different color components. Considering the obtained results shown in Table 2, the sum of green

237

and blue components showed the best linearity and was thus selected for further studies.

238

Linearity, repeatability, and LODs were studied under the optimal conditions. The results are

239

summarized in Table 3. In accordance with the obtained results, the level of concentration as low

240

as 1.0 µg L-1 can be detected by a simple smartphone image analysis, while the corresponding

241

LOD by HPLC-UV was 0.2 µg L-1. In comparison with the instrumental analysis, RGB detection

242

by smartphones normally lacks sensitivity, but in this work, the concentrated accumulation of the

243

analyte in the solid phase caused outstanding sensitivity. The variation in the color of the solid

244

phase at different concentration levels of the case is illustrated in Figure 4A. In all experiments,

245

the middle region, just above the first frit, in which the analyte was accumulated more

246

homogeneously, was exploited. In this region, there is the least variation in the RGB components

247

against the location of the analysis. Corresponding utilized areas for the RGB analysis are also

248

shown in Figure 4B. Calibration curves were linear within the range of 30-1000 µg L-1 (r2 =

249

0.9969) and 10-1000 µg L-1 (r2 = 0.9980) for RGB detection and HPLC-VU analysis,

250

respectively. Relative standard deviations (RSDs), indicative of method precision, were

251

evaluated as inter- and intra-assay values based on four replicate measurements. The method



252

showed acceptable repeatability with inter- and intra-assay RSD% values less than 6.3 and 7.5,

253

respectively.

254

Table 4 is provided to compare the characteristics of the proposed method with other

255

reported studies in the literature. Compared with the conventional LLE methods, which are

256

associated with large amounts of organic solvents and samples and difficult handling, the chip

257

device is much more user- and environmental-friendly. The chip-based device also needs lower

258

amounts of samples and enjoys less power consumption in comparison with conventional EME.

259

Moreover, as opposed to the conventional extraction methods, in this procedure samples can be

260

used without any pretreatment owing to the remarkable clean-up of the first membrane-based

261

step, which can be a prominent strategy to eliminate pretreatments in the case of solid phase

262

methods. More importantly, the main advantage of the proposed system is the simplicity of the

263

instrumentation and detection. Detection by the image analysis tool is capable of direct and

264

sensitive analyte quantification in complicated matrices. Additionally, elimination of the liquid

265

chromatography decreases the cost of analysis and organic solvents used for the elution. It goes

266

without saying that detection via smartphone is an available approach which allows in-field

267

analysis.

268

It is noteworthy that in the case of detecting several colorful species, the analytes can be

269

accumulated in different distinct zones of the solid phase by further elution similar to what we

270

have in the chromatographic columns. Another fascinating approach for eliminating

271

interferences or multi detection is benefiting from the curve resolution methods. This device is a

272

simple prototype, paving a new way for sensitive portable detection by LOC devices.

273

Additionally, in the case of colorless analytes, derivtization and tagging by a chromophore would

274

be helpful, and the interaction between a fluorophore and desired analytes can be even used as an


Page 12 of 29

Page 13 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


275

analytical signal for quantification. Further studies are also being conducted on this issue and the

276

results will be reported in not too distant future.

277

Analysis of real samples. The LOC system was used for the analysis of the target drug in

278

urine samples of patients being treated with PP to assess the applicability of the method. The

279

results are presented in Table 5. Accuracy was evaluated as the parameter of error%. The

280

obtained values were less than 8.2% for both analysis modes. Furthermore, RSD% values are

281

indicative of acceptable precision related to the proposed method. Figures 5A and 6A are typical

282

chromatograms, resulted from extraction of PP from patients’ urine samples before and after the

283

addition of certain concentration levels. As can be seen, excellent clean-up was acquired

284

considering the fact that the samples were used without any pretreatment. In addition, the

285

corresponding image analysis of the blank and spiked samples is illustrated below each

286

chromatogram (Figures 5B and 6B).

287

To validate the RGB method and compare the obtained results by both detection modes, the

288

extraction of each real urine sample was repeated three times. The acquired average

289

concentration level in each urine sample by the two analysis modes were assessed by a t-test at

290

the confidence level of 95%. The related results are shown in Table 6. According to the

291

performed t-test, there was no significant difference between the calculated concentration values

292

by the RGB method and the HPLC-UV system and both confirmed each other. Therefore, the

293

RGB method can be utilized as a reliable, accurate detection mode for sensitive and in-situ

294

analysis of the model analyte with the minimum cost.

295

CONCLUSIONS

296

In the present research, a new LOC device based on the combination of EME with a solid

297

phase as the medium for RGB analysis was proposed. Although RGB analysis via smartphones



298

normally suffers from poor sensitivity, in this method utilization of the solid phase as a medium

299

for concentrated accumulation of the model analyte caused remarkable sensitivity as compared

300

with the conventional chromatographic methods. It was shown that RGB intensities can be used

301

as a reliable concentration-dependent signal to quantify analytes from the complicated matrices.

302

The proposed device can be considered as an encouraging strategy for further developments in

303

smartphone-based LOC systems for in-field and inexpensive detections.

304

ACKNOWLEDGMENTS

305

This work was supported by a grant from the Program of Cooperation (PoC) in Science and

306

Technology between the Ministry of Science and Technology, Government of the Islamic

307

Republic of Pakistan and the Ministry of Science, Research, and Technology, Government of the

308

Islamic Republic of Iran for the years 2017-2019 (Project No. 962.201.19503). The authors also

309

gratefully acknowledge the support of University of Sistan and Baluchestan.

310

REFERENCES

311

(1) Zhang, Y.; Timperman, A. T. Analyst 2003, 128, 537-542.

312

(2) Maruyama, T.; Kaji, T.; Ohkawa, T.; Sotowa, K.-i.; Matsushita, H.; Kubota, F.; Kamiya, N.;

313

Kusakabe, K.; Goto, M. Analyst 2004, 129, 1008-1013.

314

(3) Volpatti, L. R.; Yetisen, A. K. Trends Biotechnol. 2014, 32, 347-350.

315

(4) Sackmann, E. K.; Fulton, A. L.; Beebe, D. J. Nature 2014, 507, 181-189.

316

(5) Date, Y.; Aota, A.; Terakado, S.; Sasaki, K.; Matsumoto, N.; Watanabe, Y.; Matsue, T.; Ohmura, N.

317

Anal. Chem. 2012, 85, 434-440.

318

(6) Kazarine, A.; Kong, M. C.; Templeton, E. J.; Salin, E. D. Anal. Chem, 2012, 84, 6939-6943.

319

(7) De Jong, J.; Lammertink, R.; Wessling, M. Lab Chip 2006, 6, 1125-1139.

320

(8) Clark, K. D.; Zhang, C.; Anderson, J. L.; ACS Pubs., 2016.

321

(9) Ebrahimpour, B.; Yamini, Y.; Seidi, S.; Tajik, M. Anal, Chim. Acta 2015, 885, 98-105.


Page 14 of 29

Page 15 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


322

(10) Tajik, M.; Yamini, Y.; Baheri, T.; Safari, M.; Asiabi, H. New J. Chem. 2017.

323

(11) Yamini, Y.; Seidi, S.; Rezazadeh, M. Anal. Chim. Acta 2014, 814, 1-22.

324

(12) Pedersen-Bjergaard, S.; Rasmussen, K. E. J. Chromatogr. A 2006, 1109, 183-190.

325

(13) Kjelsen, I. J. Ø.; Gjelstad, A.; Rasmussen, K. E.; Pedersen-Bjergaard, S. J. Chromatogr. A 2008,

326

1180, 1-9.

327

(14) Gjelstad, A.; Rasmussen, K. E.; Pedersen-Bjergaard, S. Anal. Bioanal. Chem. 2009, 393, 921-928.

328

(15) Gjelstad, A.; Pedersen-Bjergaard, S. Anal. Methods 2013, 5, 4549-4557.

329

(16) Rezazadeh, M.; Yamini, Y.; Seidi, S.; Esrafili, A. J. Chromatogr. A 2012, 1262, 214-218.

330

(17) Asl, Y. A.; Yamini, Y.; Seidi, S.; Ebrahimpour, B. Anal. Chim. Acta 2015, 898, 42-49.

331

(18) Rezazadeh, M.; Yamini, Y.; Seidi, S.; Ebrahimpour, B. J. Chromatogr. A 2013, 1280, 16-22.

332

(19) Petersen, N. J.; Jensen, H.; Hansen, S. H.; Foss, S. T.; Snakenborg, D.; Pedersen-Bjergaard, S.

333

Microfluid. Nanofluid. 2010, 9, 881-888.

334

(20) Asl, Y. A.; Yamini, Y.; Seidi, S.; Rezazadeh, M. Anal. Chim. Acta 2016, 937, 61-68.

335

(21) Seidi, S.; Rezazadeh, M.; Yamini, Y.; Zamani, N.; Esmaili, S. Analyst 2014, 139, 5531-5537.

336

(22) Gjelstad, A.; Rasmussen, K. E.; Pedersen-Bjergaard, S. J. Chromatogr. A 2007, 1174, 104-111.

337

(23) Rezazadeh, M.; Yamini, Y.; Seidi, S.; Esrafili, A. Anal. Chim. Acta 2013, 773, 52-59.

338

(24) Li, K.-j.; Chen, Q.-h.; Zhang, Z.; Zhou, P.; Li, P.; Liu, J.; Zhu, J. J. Chromatogr. Sci. 2008, 46, 686-

339

689.

340

(25) Farin, D.; Piva, G.; Kitzes-Cohen, R. Chromatographia 2000, 52, 179-180.

341

(26) Belal, F. Chromatographia 1988, 25, 61-63.

342

(27) Fotouhi, L.; Yamini, Y.; Hosseini, R.; Rezazadeh, M. Can. J. Chem. 2015, 93, 702-707.



Figures captions Figure 1. (A) Equipment used for EME chip device. (B) Schematic illustration of the whole extraction and detection procedure. Figure 2. Effect of SLM composition on extraction efficiency. PP was extracted by the voltage of 60 V, flow rate of 50 µL min-1, 0 and 100 mM HCl for the donor and acceptor phases, respectively. Figure 3. Optimization of (A) donor phase composition, (B) acceptor phase composition, (C) voltage, and (D) flow rate. Figure 4. (A) Final digital images of the solid phase after the analyte absorption as a medium for the RGB analysis at different concentration levels of the case. (B) Utilized zones for the RGB analysis. Figure 5. (A) Typical chromatogram of PP extraction from urine sample of a patient after and before the addition of 200 µg L-1. (B) Corresponding images used for the RGB analysis. Figure 6. (A) Obtained chromatograms after the LOC procedure ((a) non-spiked, (b) spiked at the concentration level of 40 µg L-1. (B) Corresponding images for the RGB analysis.


Page 16 of 29

Page 17 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1 Chemical structure and corresponding pKa and Log KO/W values of PP. Name

Chemical structure

Phenazopyridine


pka

Log KO/W

6.86

2.69


Page 18 of 29

Table 2 Evaluation of calibration curves against different RGB components Analysis component

Linear dynamic range (µg L-1)

R2

Green

50.0-1000.0

0.9686

Blue

50.0-1000.0

0.9700

Green + Blue

30.0-1000.0

0.9969

Green + Red

50.0-1000.0

0.9657

Blue + Red

50.0-1000.0

0.9649


Page 19 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 3 Analytical performance of LOC device for determination of PP from urine samples by both the detection approach of HPLC-UV and the RGB analysis Linearity R2 PFa LOQ RSD%b Analysis LOD method (µg L-1) (µg L-1) (µg L-1)

HPLC/UV 0.2 RGB a

1.0

Inter-assay

Intra-assay

5.0

5.0-1000.0

0.9980 25

5.2

7.1

30.0

30.0-1000.0

0.9969 ---

6.3

7.5

Preconcentration based on four-replicate measurements at 250 µg L-1 for HPLC. Based on four-replicated measurements.

b


Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 20 of 29

Table 4 Comparison of analytical performance of the proposed method with other reported methods for PP determination Method Matrix LOD RSD% LDR R2 (µg L-1)

(µg L-1)

Ref.

LLE/GC -MSa

Plasma

0.3

5.0-500.0

0.9992

2.7

24

LLE/HPLC-UVb

Plasma

10.0

50.0-1000.0

0.9986

8.3

25

Tablets

10.0

2000.0-20000.0

0.9999

-

26

Urine

0.5

1.0-1000.0

0.9960

4.6

27

Plasma

1.0

5.0-1000.0

0.9985

5.9

27

OC-EME/HPLC-UVd

Urine

0.2

5.0-1000.0

0.998

5.2

This work

OC-EME/RGB analysise

Urine

1.0

50.0-1000.0

0.9934

6.3

This work

EME/HPLC-UVc

a

Liquid-liquid extraction-gas chromatography mass spectrometry detection. Liquid-liquid extraction-liquid chromatography ultraviolet detection. c Electromembrane extraction. d On-chip electromembrane extraction. e On-chip electromembrane extraction-RGB image analysis detection. b

20

ACS Paragon Plus Environment

Page 21 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 5 Determination of PP in real samples Sample

Urine 1a

Urine 2b

a b

Analysis method

Creal

Cadded

Cfound

RSD%

(µg L-1)

(µg L-1)

(µg L-1)

(n=4)

HPLC/UV

237.3

200.0

433.7

5.8

-1.8

RGB

234.4

200.0

440.1

7.2

2.8

HPLC/UV

33.3

40.0

72.8

6.6

-1.2

RGB

39.2

40.0

75.9

7.6

-8.2

After 9 h. After 12 h.


Error%


Page 22 of 29

Table 6 Validation of the RGB method by t-test Urine 1 -1

PP conc. (µg L )

Average Standard deviation Spooled ttest tTable(4,0.05) = 2.776

HPLC 252.6 234.1 225.2 237.3 13.9

Urine 2 RGB 252.9 230.1 220.2 234.4 16.7

HPLC 36.2 30.6 33.1 33.3 2.8

15.4 2.9 0.23 2.44 ttets˂ tTable(4,0.05) , Not significant


RGB 35.6 40.9 41.1 39.2 3.1

Page 23 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1.



Figure 2.


Page 24 of 29

Page 25 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3.



Figure 4.


Page 26 of 29

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5.



Figure 6.


Page 28 of 29

Page 29 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For TOC only


Approach for Downscaling of Electromembrane Extraction as a Lab on

Recommend Documents