Droplet Microfluidics for Postcolumn Reactions in Capillary

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Article

Droplet microfluidics for post-column reactions in capillary electrophoresis Aemi S. Abdul Keyon, Rosanne M Guijt, Christopher J. Bolch, and Michael C. Breadmore Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5033963 • Publication Date (Web): 13 Oct 2014 Downloaded from http://pubs.acs.org on October 23, 2014

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 free 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 accessible to all readers and 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.

Analytical Chemistry 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 27

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

2

Droplet microfluidics for post-column reactions in capillary electrophoresis

3 4 5

Aemi S. Abdul Keyon1, 2, 3, 4

6

Rosanne M. Guijt2

7

Christopher J. Bolch3

8

Michael C. Breadmore1*

1

9 10 11

1

Australian Centre for Research on Separation Science, School of Physical Sciences, University of Tasmania, GPO Box 252-75, Hobart, Tasmania 7001, Australia

12 13

2

14 15

3

16 17 18 19

4

20

Prof Michael Breadmore,

21

Australian Centre for Research on Separation Science,

22

School of Chemistry,

23

University of Tasmania,

24

GPO Box 252-75, Hobart, Tasmania 7001, Australia

25

Phone: +61-3-6226-2154

26

Fax: +61-3-6226-2858

27 28

Email: [email protected]

Pharmacy School of Medicine, Australian Centre for Research on Separation Science, University of Tasmania, GPO Box 252-26, Hobart, Tasmania 7001, Australia National Centre for Marine Conservation and Resource Sustainability, Australian Maritime College, University of Tasmania, 7250, Launceston, Tasmania, Australia Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310, UTM Johor Bahru, Johor, Malaysia *Address Correspondence to:

29

ACS Paragon Plus Environment


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

30 31 32

List of abbreviations

33

CE Capillary electrophoresis

34

CZE Capillary zone electrophoresis

35

C4D Capacitively coupled contactless conductivity detection

36 37

EOF Electroosmotic flow

38

µeff Effective mobility

39 40

FLD Fluorescence detection HPLC High performance liquid chromatography

41

He-Cd Helium-Cadmium

42

LC Liquid chromatography

43

LED Light emitting diode

44

LOD Limit of detection

45

MEKC Micellar electrokinetic chromatography

46

MS Mass spectrometry

47

NEO Neosaxitoxin

48

PEEK Polyetheretherketone

49 50 51 52 53 54 55

PSTs Paralytic shellfish toxins UV Ultraviolet

AOAC Association of Official Analytical Chemists BGE Background electrolyte

dcGTX2 decarbamoylgonyautoxin2

Keywords: Droplet microfluidics, capillary electrophoresis, post-column reaction, paralytic shellfish toxins Total number of words including figure and table legends: 5100


Page 2 of 27

Page 3 of 27

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


56

ABSTRACT

57

A post-column reaction system based on droplet microfluidics was developed for capillary

58

electrophoresis (CE). Analytes were separated using capillary zone electrophoresis (CZE) and

59

electrophoretically transferred into droplets. The use of a micro cross for positioning a salt

60

bridge-electrode opposite the separation capillary outlet is the key element for maintaining

61

the electrical connection during electrophoretic separation. As the first of its kind,

62

positioning the droplets in the electric field eliminated the need for electroosmotic flow

63

(EOF) or hydrodynamic flow for droplet compartmentalisation. Depending on the total flow

64

rate of both aqueous and oil phases, droplets of water-in-oil could be formed having

65

frequencies between 0.7-3.7 Hz with a size of approximately 14 nL per droplet.

66

Compartmentalised in the droplets, analytes reacted with reagents already present in the

67

droplets to facilitate detection. The periodate oxidation of paralytic shellfish toxins (PSTs)

68

was demonstrated, overcoming the limitation of pre-column oxidation, which results in

69

multiple and sometimes identical oxidation products formed from the different PSTs.

70

Compartmentalisation allows the oxidation products for each peak to be contained and to

71

contribute to a single fluorescence signal, preserving the selectivity of CZE separation while

72

gaining the sensitivity of fluorescence detection.

73

74

75



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

76

INTRODUCTION

77

Capillary electrophoresis (CE) is a high-resolution separation technique allowing for the fast

78

separation of a wide range of analytes. With many analytes not ultraviolet (UV) absorbing,

79

fluorescent or electrochemically active, derivatisation is often required for sufficient

80

sensitivity. In pre-column derivatisation, the use of large reagents may compromise the

81

selectivity of the separation of small molecules, especially when the targets are smaller than

82

the label. This is overcome in liquid chromatography (LC) by performing post-column

83

reactions where reagents are introduced to the LC effluent, however this approach leads to

84

slight peak broadening due to mixing, dilution and diffusion during the extra time required

85

in the reaction coil.

86

Despite the experimental complications introduced by the electric field and the smaller

87

scale of CE, several post-column reaction schemes exist, including gap reactor1-3, coaxial

88

reactor4-6, sheath flow cuvette reactor7-10, membrane reactor11 and a laser drilled capillary

89

reactor12. Technically, some of these reactors require non-trivial construction, alignment or

90

interfacing procedures. Typically, hydrodynamic flow in the reactor results in zone

91

broadening and a loss of resolution, an effect more pronounced than in LC because of the

92

much sharper peaks2,4. To minimise the extent of broadening, fast kinetic reagents (i.e.

93

naphthalene-2,3-dicarboxyaldehyde, o-phthaldialdehyde or fluorescamine) are required to

94

rapidly produce fluorescent derivatives and preserve resolution, eliminating reagents with

95

relatively slow kinetics.

96

Droplet microfluidics or segmented flow of aqueous droplets in an oil phase within

97

microchannels, tubing or capillaries provides a unique way of compartmentalising aqueous

98

fractions as the non-miscible boundary restricts dispersion and limits cross-contamination13.

99

At the microscale, this has been used to facilitate the preservation of microscale


Page 4 of 27

Page 5 of 27

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


100

separations14-16, whilst in the droplets increased reaction rates can be realised because of

101

rapid mixing17,18. Combining these two features in a post-column reactor eliminates the

102

need for reagents with fast reaction kinetics. Compartmentalisation of separation effluent

103

into droplets has been applied in various separation systems including LC and microchip

104

electrophoresis. Ji et al.19 used droplets for post-column enzymatic digestion of proteins, to

105

be later subjected to mass spectrometry (MS) analysis. Nie and Kennedy20 demonstrated the

106

addition of fluorescent reagent to proteins collected into droplets from capillary LC (cLC).

107

Edgar et al.21 used the electroosmotic flow (EOF) to form aqueous droplets from the

108

background electrolyte (BGE) in a microfluidic structure filled with oil, compartmentalising

109

separated analyte bands. Following on from this, Draper et al.22 demonstrated the

110

compartmentalisation of separated bands in micro capillary gel electrophoresis (µCGE). Due

111

to the absence of EOF, an aqueous carrier was used to collect the bands and form the

112

droplets. In both cases, the electrical field was terminated after separation and prior to

113

compartmentalisation.

114

Here, we present a new approach where analytes are electrophoresed into the droplets

115

without disruption of the electric field, and used for post-column reaction. Off-the-shelf

116

components were used for droplet formation and interfacing with fused silica capillaries in a

117

commercial CE instrument. The potential application of this approach was demonstrated for

118

post-column periodate oxidation of paralytic shellfish toxins (PSTs) in CE, marrying the

119

selectivity of capillary zone electrophoresis (CZE) with the sensitivity and selectivity of

120

fluorescence detection (FLD).

121 122

MATERIALS AND METHODS



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

123

Chemicals and reagents

124

Chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise

125

specified. Decanol was used as the oil phase for all experiments. All aqueous solutions were

126

prepared with Milli-Q water (Millipore, Bedford, MA, USA). The BGE for CZE of fluorescein

127

and calcein was 3 mM disodium phosphate prepared from disodium hydrogen phosphate

128

anhydrous and adjusted to pH 7.8 with phosphoric acid (BDH, Melbourne, Australia).

129

Fluorescein and calcein were dissolved in the BGE. The BGE for CZE of PSTs was 20 mM

130

formic acid pH 2.8. Standards for PSTs (neosaxitoxin (NEO) and decarbamoylgonyautoxin 2

131

(dcGTX2)) were purchased from Institute for Marine Biosciences (National Research Council

132

of Canada, Halifax, Canada). Both PSTs standards, each having different concentration, were

133

diluted with Milli-Q water to the desired working concentration and used without further

134

purification. The PSTs mixture consisted of 10 µg/mL of NEO and 20 µg/mL dcGTX2. The

135

oxidant reagent was prepared daily by mixing 5 mM periodic acid and 100 mM phosphoric

136

acid, adjusted to pH 7.8 with 5M NaOH.

137

138

Droplet formation

139

The schematic outline of droplet formation was presented in Fig.1a. A dual syringe pump

140

(Model 33, Harvard Apparatus, United States) was used to infuse the oil and aqueous

141

phases into a Labsmith tee connector with 250 µm thru-holes (Labsmith, California, United

142

States), with the aqueous phase entering the tee at 90° to the oil flow. The reagents were

143

delivered to the tee using disposable syringes (3 mL volume equipped with luer lock,

144

Hapool, China) via two separate polyimide coated fused silica capillaries (16 cm long 150 μm

145

I.D, Polymicro-Technologies Phoenix, Arizona, United States). During optimisation, flow


Page 6 of 27

Page 7 of 27

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


146

rates for each of the aqueous and oil phases ranged from 0.6 to 8 μL/min. A 40 cm long, 150

147

μm I.D polyimide coated fused silica capillary connected the third tee arm with a polyether

148

ether ketone (PEEK™) micro cross with 150 μm thru-holes (Upchurch Scientific, United

149

States) for interfacing with the separation capillary. A manual 3-way valve (Labsmith,

150

California, United State) was connected on the 40 cm long capillary, positioned between the

151

tee and micro cross. The droplet valve port was closed during sample injection (droplet

152

flowed to waste at the same time) and rapidly opened at the end of injection to allow

153

droplet re-flow into the micro cross.

154 155

Interfacing of CE with droplets

156

Separations were performed using an Agilent HP3DCE system (Agilent Technologies,

157

Waldbronn, Germany), using a PEEK™ micro cross to interface the separation capillary with

158

the segmented flow and electrode as described above (Fig.1a). The micro cross was

159

positioned inside the capillary cassette. A photograph of the micro cross positioned in a

160

capillary cassette is shown in Fig. 1b. A polyimide coated fused silica capillary was used for

161

CZE (57 cm long, 25 μm I.D) with the outlet positioned 90° to the segmented flow. High

162

voltage was applied to a Pt ground electrode positioned opposite the separation capillary

163

outlet with electrical connection ensured using a salt bridge constructed by filling a 0.5 cm

164

long, 250 μm I.D capillary with 3% w/w agarose prepared in 3 mM disodium phosphate

165

buffer. Droplets containing the separation effluent flowed through another piece of fused

166

silica capillary (30 cm long, 150 μm I.D) to the detection window 10.5 cm from the micro

167

cross. For experiments with fluorescein and calcein, a blue light emitting diode (LED) at

168

wavelength of 470 nm having luminous intensity of 12,000 millicandela (Lucky Light, China)

169

was used in combination with 470 nm and 525 nm band pass filters (25 mm diameter,



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

170

Chroma, Vermont, United States) for excitation and emission, respectively. For detection of

171

the oxidised PSTs, a Helium-Cadmium laser (He-Cd, Melles Griot, California, United States)

172

was used at ƛex = 330 nm, ƛem = 390 nm using a 325 nm He-Cd laser clean-up filter and 390-

173

40 nm band pass filters (25 mm diameter, Semrock, New York, United States). The

174

fluorescence detector was constructed using a 5-port manifold (Upchurch Scientific, United

175

States), the blue LED or He-Cd laser spot was aligned to on-column detection window via

176

two fibre optics (400 µm for excitation, 600 µm for emission; Ocean Optics, Florida, United

177

States)23. Fluorescence was measured by a photomultiplier tube (Hamamatsu, Japan) and

178

the emitted light was collected using an Agilent 35900E analogue-to-digital convertor

179

(Agilent Technologies, Waldbronn, Germany). Separation and outlet capillaries were rinsed

180

with Milli-Q water and BGE by applying pressure of 2 bar to the inlet vial before and in

181

between analyses. Samples were electrokinetically injected at 5 kV for 25 s and separations

182

were performed at 25°C, unless otherwise specified. Fluorescein and calcein were separated

183

using a voltage of +30 kV (field strength of 530 V/cm), while the PSTs were separated using

184

+25 kV (field strength of 440 V/cm).

185

186

Droplet microfluidics post-column oxidation

187

The oxidant consisted of 5 mM periodic acid, 100 mM phosphoric acid and adjusted to pH

188

7.8 with 5 M NaOH24 was delivered as aqueous phase segmented by oil to the micro cross.

189

Droplet formation was initiated directly after electrokinetic injection of sample.

190

191

RESULTS AND DISCUSSION


Page 8 of 27

Page 9 of 27

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


192

Characterisations of droplets and CE-droplet microfluidic interface

193

The aim of the current study is to electrophorese analytes into preformed droplets for

194

compartmentalisation to maintain resolution during post-column reactions. To do this in a

195

no or low EOF environment, the electrical connection needs to be maintained across the

196

segmented flow. This is fundamentally different from all previous studies in which the

197

electric field was terminated after separation and prior to droplet generation21,22. These

198

works also required liquid flow through the microchannel or the addition of a make-up flow

199

for droplet formation. Here, we choose to terminate the electric field after

200

compartmentalisation into the droplets (illustrated in Fig.1a) with electrical connection

201

established within the CE-droplet microfluidics interface with a salt bridge connected to a Pt

202

electrode positioned directly opposite the separation capillary. This was constructed using a

203

commercial micro cross, with droplets formed upstream in a tee connector from flows of

204

decanol (the oil phase) and buffer or reagent (aqueous phase). The salt bridge-electrode and

205

separation capillary were positioned opposite to each other to provide a direct conducting

206

pathway during compartmentalisation. A micro cross with bigger through hole (i.e. 500 µm)

207

can also be used, although the larger droplets result in a greater dilution of the effluent.

208

Critical to this form of compartmentalisation is the formation of droplets with an

209

appropriate frequency and maintenance of the electric current.

210

To test this idea, the formation of droplets was examined at different flow rates and with

211

the separation capillary filled with BGE (3 mM phosphate buffer pH 7.8) whilst the droplets

212

were formed from BGE spiked with 1 µg/mL fluorescein. Fig.2a shows the fluorescence

213

signal when an electric field (field strength of 530 V/cm) was applied over the separation

214

capillary, with each peak corresponding to a droplet passing the detection window. The flow



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

215

rate for the oil was varied from 2.4-8 µL/min, for the aqueous phase from 0.6-2 µL/min,

216

resulting in a total flow rate of 3-10 µL/min, at an oil : aqueous flow rate ratio of 4. These

217

translate into droplet frequencies of approximately 0.7-3.7 Hz. At a flow rate of 3 µL/min,

218

formation of droplets was inconsistent as evidenced by irregular peak width and spacing. At

219

flow rates from 4-7µL/min, regularly spaced consistent peaks were observed. At flow rates

220

from 10 µL/min, droplet formation at this large scale was compromised, with droplets

221

partially merging, as evidenced by the irregular fluorescence signal. Consistent droplet

222

formation is desired to ensure efficient compartmentalisation, thus only droplets formed at

223

4-7 µL/min should be used. The repeatability of droplet formation at 4-7 µL/min

224

(percentage relative standard deviations (%RSD), n = 3) for intra-day variations in droplet

225

frequency were < 8.1%. The physical length of the droplets ranged from 730-810 µm (n =3,

226

< 2.7% RSD for intra-day variation) by digital microscope imaging; thus each droplet had a

227

volume of approximately 14 nL.

228

As can be seen in Fig.2b during the CE-droplet experiments using 3 mM disodium phosphate

229

(pH 7.8), a current of 2.0 µA was generated when applying a voltage of 30 kV. The current

230

was maintained at 2.0 µA throughout the experiments, indicating stable electrical

231

connection when droplets were delivered at both low and high flow rates. The reliability of

232

electrical connection at 4 µL/min calculated by %RSD (n = 5) for inter-day variation was

233

2.5%. While stable electrical connection was obtained using low conductive BGE

234

(conductivity value of 0.030 S/m), it was unstable or absent when using relatively high

235

conductivity BGEs (conductivity values > 0.10 S/m) at the same field strength. To investigate

236

this occurrence, experiments were conducted using a fluorescence microscope to visualise

237

the interface using a low and high conductivity BGE. When using a high conductivity BGE,


Page 10 of 27

Page 11 of 27

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


238

and hence high current, bubbles were generated in the CE-droplet interface once the

239

voltage was applied, followed by visual observation of liquid leakage from the micro cross.

240

We believe that the salt bridge ruptured due to the aggressive bubble formation, exposing

241

the Pt electrode to a non-conductive oil film, eventually terminating the electrical

242

connection and triggering liquid leakage. This limitation may be overcome by downscaling

243

and purpose-engineering the droplet generation and interface.

244

CE-droplet compartmentalisation

245

The performance of the CE-droplet microfluidics system to compartmentalise/collect the

246

electrophoresed analytes was examined with the CZE separation of fluorescein and calcein.

247

Droplets were delivered at 4 µL/min, giving droplet frequency of 1 Hz for

248

compartmentalisation. From the detection trace in Fig. 3a, the droplets containing just BGE

249

had a lower background signal than the oil phase, which we consider is due to the difference

250

in refractive index of the water and decanol resulting in more excitation light reaching the

251

emission fibre with decanol. The fluorescence signals for both fluorescent dyes were higher

252

than the background and resulted in positive peaks. To compare the data with on-capillary

253

detection (detection window on the separation capillary), an electropherogram was

254

reconstructed

255

electropherogram of Fig.3a is given in Fig.3b, while Fig.3c is with on-capillary detection. To

256

compensate for the different detector position between the two setups, a mobility scale

257

was used instead of a timescale25. The insert in Fig. 3a shows that the fluorescein peak was

258

compartmentalised into approximately 50 droplets, which was well over the minimum 8-10

259

droplets usually needed to preserve peak shape20. The variance in effective mobility

260

between CE-droplet microfluidics and on-capillary detection setups was less than 6.5%,

using

the

signal

obtained

in

the

droplets.


The

reconstructed


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

261

indicating stable pressure and electrical connection during injection, and successful

262

separation and compartmentalisation when compared with on-capillary detection. Peak

263

widths of the compartmentalised fluorescein and calcein increased 4 and 1.2-times,

264

respectively, when compared to the same analyte peaks with on-capillary detection,

265

indicating zone broadening. The LODs (calculated at S/N = 3) for fluorescein and calcein

266

using CE-droplet compartmentalisation were 2.2 and 45 µg/mL compared with 0.22 and 5.1

267

µg/mL using on-capillary detection. These are 10 and 9-times higher, although are lower

268

than anticipated based purely on dilution in the droplet and is due to the longer detection

269

pathlength in the droplet system (150 µm) than the on-capillary detection system (25 µm).

270

The commercially available tee connector with 250 µm thru-holes used in our droplet setup

271

formed droplets with a volume of approximately 14 nL – 20-47 times larger than would be

272

encountered in microchip formats21,22. It is envisaged that considerable improvement in

273

performance would be achieved by using smaller droplets as the effect of sample dilution

274

would be considerably smaller.

275

Droplet volume and droplet flow contribute to the dilution effect, therefore we examined

276

the relationship of total flow rate and dilution with the results shown in Fig. 4a. When the

277

total flow rate was reduced from 10 to 3 µL/min, droplet frequency decreased from 3.7 to

278

0.7 Hz, resulting in a higher proportion of the electrophoresed components in each droplet,

279

and a corresponding significant increase in detector response. Higher droplet frequencies

280

allow for maintenance of resolution: for a minimum of 10 droplets per peak, 2.7 s wide

281

peaks could be analysed at 3.7 Hz, whereas the minimal peak width increases to 14 s wide

282

when using 0.7 Hz. It can also be seen from the figure that as the flow rate was increased

283

that the effective mobility also increased. This is due to an increase in pressure from the


Page 12 of 27

Page 13 of 27

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


284

flowing liquid which counters the EOF from the capillary thus reducing the overall liquid

285

flow through the capillary and increasing the effective mobility. Comparison of the signal

286

response (Fig. 4b) indicates an increase by 14-times when changing from 10 to 3 µL/min,

287

thus the lower droplet frequency will be better for preserving analytical sensitivity.

288

To understand the broadening observed in Fig.3, experiments were performed using a

289

fluorescence microscope equipped with colour charge-coupled device camera. Images were

290

acquired during the experiment to observe the movement of droplets into the micro cross

291

during application of high voltage. Fig. 5 shows a series of screen shots of fluorescein-doped

292

droplets through the micro cross interface with an electric field strength of 530 V/cm. As

293

can be seen from Fig.5a, a fraction of the fluorescent aqueous droplet remained stationary

294

in front of the salt bridge-electrode to coalesce with a subsequent droplet (Fig.5b-c). The

295

fluorescent droplet facilitated the conducting pathway from the separation capillary to the

296

salt bridge and Pt electrode to close the electric circuit. Due to differences in wettability, the

297

aqueous phase wets the salt bridge, preventing the salt bridge from being covered with a

298

film of oil that would disrupt the electrical circuit due to oil’s insulating properties.

299

Experiments using the Pt electrode directly and no salt bridge did not produce a stable

300

current which we believe is due to the formation of an oil coating on the electrode surface.

301

Thus, the hydrophilicity of the salt bridge allows for preferential wetting with aqueous

302

phase and as such provides a continuous electrical pathway. While this assisted in achieving

303

the electrical connection required for electrophoresis, the capture and coalescence of

304

droplets visualised in Fig.5 is undesirable and attributes to the loss in resolution as part of

305

the electrophoresed analyte remains behind in the interface to leech back into the

306

subsequent droplet(s), compromising compartmentalisation. While we consider this



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

307

accounted for a major component of the decreased efficiency, there is also likely to be a

308

small hydrodynamic flow from the separation capillary outlet that will also induce

309

broadening, although this is minimised by using a 25 µm I.D capillary. Fabrication of a salt

310

bridge in a microfluidic device positioned at the end of the flow-through channel should

311

overcome the droplet capture and mixing issue and will preserve the efficiency better.

312

313

CE with droplet-based microfluidics post-column reaction: Paralytic shellfish

314

toxins as model analytes

315

To demonstrate the droplet approach for post-column reaction in CE, the detection of

316

paralytic shellfish toxins (PSTs) is chosen as a challenging application. PSTs are natural

317

marine toxins produced by dinoflagellates during algal blooms that accumulate in filter-

318

feeding shellfish, fish or plankton. Consumption of PSTs-contaminated shellfish may result in

319

respiratory arrest and cardiovascular shock that may lead to death26. PSTs are natively non-

320

fluorescent but yield fluorescent products upon oxidation24,27. Pre-column oxidation with

321

HPLC-FLD was the first official method used for PSTs detection, however, some toxins

322

produced more than one fluorescent oxidation product and identical fluorescent oxidation

323

products are formed from some of the toxins27, compromising selectivity and specificity. To

324

maintain selectivity, the alternative official method uses post-column oxidation and is

325

preferred24, although a post-column reactor setup is needed. Both methods offer detection

326

limits in the low ng/mL range for the analysis of PSTs. Recently, we evaluated four different

327

detection methods for PSTs analysis by CE28. FLD with pre-column oxidation was the most

328

sensitive, although micellar electrokinetic chromatography (MEKC) separation was required

329

to separate the neutral oxidation products. The CE method suffered from the same loss in


Page 14 of 27

Page 15 of 27

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


330

selectivity due to the multiple, overlapping oxidation products reported for the HPLC

331

method with precolumn oxidation. In this work, droplets containing periodic acid were used

332

for compartmentalisation of the electrophoretically separated analytes for oxidisation, thus

333

providing the sensitivity of FLD with the selectivity of CZE. It was however not possible to

334

use our previously developed CZE BGE because it had too high ionic strength to provide a

335

stable current.

336

In HPLC with post-column oxidation, the separation of PSTs usually takes ~30 min and

337

reaction times typically between 1 to 2 min were used depending on the flow rate of

338

oxidant and the length of the reaction coil24. Given that enhanced mixing in droplets

339

improves reaction rates18, we anticipated shorter reaction time in the droplet oxidation

340

system. We examined the effect of the reaction time on the fluorescence intensity by

341

changing the distance of the detection window from the micro cross. Distance of 10.5, 20.5,

342

25.5and 30.5 cm were used, corresponding to oxidation times of approximately 14, 16, 18

343

or 20 s when the droplets were delivered at total flow rate of 4 µL/min (1 Hz). No significant

344

difference in fluorescence was recorded, therefore the shortest time of 14 s was selected as

345

the reaction time. Shorter or longer times were not examined due to the practical difficulty

346

of positioning the detector closer or further to the micro cross. Fig.6a-b show the droplet

347

trace and reconstructed electropherograms for NEO and dcGTX2 with the detection window

348

positioned 10.5 cm from the cross. NEO and dcGTX2 have an effective mobility of 41 and

349

21 x 10-9 m2/Vs, respectively, and were separated and detected within 10 min. The

350

performance of CZE-droplet post-column oxidation method was evaluated in terms of

351

linearity of the detector response, LODs, intra-day and inter-day precisions (Table 1). The

352

linear ranges for NEO and dcGTX2 were between 670-10,000 ng/mL (regression coefficient



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

Page 16 of 27

353

(R2) > 0.982) and between 430-20,000 ng/mL (R2> 0.980), respectively. The LODs for NEO

354

and dcGTX2 were 670 ng/mL and 430 ng/mL, respectively. These values are approximately

355

11 and 7-times higher than previously reported with pre-column oxidation with on-capillary

356

detection28 (detection window on the separation capillary), and consistent with the 9-10

357

times drop in sensitivity for calcein and fluorescein as a consequence of dilution in the

358

droplets. This suggests that comparable oxidation (in term of fluorescent yield) between our

359

previous pre-column oxidation (3 min) and our current post-column oxidation (14 s) was

360

achieved, with droplet post-column oxidation oxidising only one toxin at a time, avoiding

361

the issues around multiple, overlapping and identical oxidation products complicating

362

identification following pre-column oxidation. While the detection limits were only

363

marginally improved when compared to UV detection (Fig. 6c), oxidation and fluorescence

364

increases specificity of detection allowing more reliable and accurate identification of toxin

365

peaks. To be applicable to the analysis of PST content in shellfish samples, it is necessary to

366

be able to detect toxins lower than the regulatory limit of 800 ng/mL27, thus the sensitivity

367

of the methods needs to be further improved. This could be achieved by various stacking

368

approaches, such as the transient isotachophoresis (tITP) method we have previously

369

developed for capacitively coupled contactless conductivity detection (C4D)29, and/or

370

through reduction of

371

compartmentalisation.

the

droplet

size

to reduce

the

dilution factor

upon

372

373

CONCLUSIONS

374

We demonstrate that droplet microfluidics can be used to compartmentalise

375

electrophoretically transferred analytes for post-column reaction in CE. The use of a PEEK™


Page 17 of 27

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


376

micro cross for positioning a salt bridge across the droplet flow from the separation capillary

377

outlet enables the compartmentalisation/collection of the electrophoretically transferred

378

analytes while maintaining the electrical connection. Positioning the droplets in the electric

379

field eliminated the need for EOF or hydrodynamic flow and allowed electrophoresing

380

effluents into the droplets. We show that the system could be successfully applied to the

381

post-column oxidation of PSTs, allowing for the combination of the selectivity of CZE with

382

the sensitivity of FLD. We believe this new approach for post-column droplet collection in CE

383

can be very powerful when miniaturised further to reduce the droplet size which will reduce

384

the dilution factor and enable a range of post-separation processing to be performed.

385

386

ACKNOWLEDGEMENTS

387

The authors would like to thank the Australian Research Council, ARC QEII Fellowship

388

(DP0984745) and Future Fellowship (FT130100101) awarded to M.C.B and University of

389

Tasmania for funding support. We acknowledge scholarships from the Ministry of Education

390

Malaysia (MOE) and Universiti Teknologi Malaysia for A.S.A.K. The authors would like to

391

acknowledge the expertise and support from Mr. John Davis, Mr. Paul Waller and Mr. Chris

392

Young of the Central Science Laboratory, University of Tasmania. A.S.A.K would like to

393

acknowledge Mr. Burhanuddin Mohamad for his help on post-processing fluorescence

394

images using Adobe® Photoshop® and Adobe® Lightroom® softwares.



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

395

REFERENCES

396 397

(1) Albin, M.; Weinberger, R.; Sapp, E.; Moring, S. Anal. Chem.1991, 63, 417-422.

398

(2) Zhu, R.; Kok, W. T. J. Chromatogr. A1995, 716, 123-133.

399

(3) Rezenom, Y. H.; Lancaster Iii, J. M.; Pittman, J. L.; Gilman, S. D. Anal. Chem.2002, 74, 1572-1577.

400

(4) Nickerson, B.; Jorgenson, J. W. J. Chromatogr. A1989, 480, 157-168.

401

(5) Emmer, Å.; Roeraade, J. J. Chromatogr. A1994, 662, 375-381.

402

(6) Feltus, A.; Hentz, N. G.; Daunert, S. J. Chromatogr. A2001, 918, 381-392.

403

(7) Oldenburg, K. E.; Xi, X.; Sweedler, J. V. Analyst1997, 122, 1581-1585.

404

(8) Nirode, W. F.; Staller, T. D.; Cole, R. O.; Sepaniak, M. J. Anal. Chem.1998, 70, 182-186.

405

(9) Ye, M.; Hu, S.; Quigley, W. W. C.; Dovichi, N. J. J. Chromatogr. A2004, 1022, 201-206.

406

(10) Dada, O. O.; Huge, B. J.; Dovichi, N. J. Analyst2012, 137, 3099-3101.

407

(11) Liu, F.; Zhang, L.; Qian, J.; Ren, J.; Gao, F.; Zhang, W. Analyst2013, 138, 6429-6436.

408

(12) Yamamoto, D.; Kaneta, T.; Imasaka, T. Electrophoresis2007, 28, 4143-4149.

409

(13) Xiao, Z.; Niu, M.; Zhang, B. J. Sep. Sci.2012, 35, 1284-1293.

410

(14) Edgar, J. S.; Pabbati, C. P.; Lorenz, R. M.; He, M.; Fiorini, G. S.; Chiu, D. T. Anal. Chem.2006, 78,

411

6948-6954.

412

(15) Niu, X.; Pereira, F.; Edel, J. B.; de Mello, A. J. Anal. Chem.2013, 85, 8654-8660.

413

(16) Zhao, Y.; Pereira, F.; deMello, A. J.; Morgan, H.; Niu, X. Lab Chip2014, 14, 555-561.

414

(17) Song, H.; Tice, J. D.; Ismagilov, R. F. Angew. Chem. Int. Ed.2003, 42, 768-772.

415

(18) Song, H.; Chen, D. L.; Ismagilov, R. F. Angew. Chem. Int. Ed.2006, 45, 7336-7356.

416

(19) Ji, J.; Nie, L.; Qiao, L.; Li, Y.; Guo, L.; Liu, B.; Yang, P.; Girault, H. H. Lab Chip2012, 12, 2625-2629.

417

(20) Nie, J.; Kennedy, R. T. J. Sep. Sci.2013, 36, 3471-3477.

418

(21) Edgar, J. S.; Milne, G.; Zhao, Y.; Pabbati, C. P.; Lim, D. S. W.; Chiu, D. T. Angew. Chem. Int.

419

Ed.2009, 48, 2719-2722.

420

(22) Draper, M. C.; Niu, X.; Cho, S.; James, D. I.; Edel, J. B. Anal. Chem.2012, 84, 5801-5808.


Page 18 of 27

Page 19 of 27

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


421

(23) Huo, F.; Guijt, R.; Xiao, D.; Breadmore, M. C. Analyst2011, 136, 2234-2241.

422

(24) AOAC. Ofﬁcial Method 2011.02 First Action 2011: Paralytic shellﬁsh toxins in mussels, clams,

423

scallops and oysters by liquid chromatography post-column oxidation; AOAC International:

424

Gaithersburg,MD, 2012.

425

(25) Schmitt-Kopplin, P.; Garmash, A. V.; Kudryavtsev, A. V.; Menzinger, F.; Perminova, I. V.;

426

Hertkorn, N.; Freitag, D.; Petrosyan, V. S.; Kettrup, A. Electrophoresis2001, 22, 77-87.

427

(26) Rossini, G. P.; Hess, P. EXS2010, 100, 65-122.

428

(27) AOAC. Official Method 2005.06 Paralytic shellfish poisoning toxins in shellfish.Pre-

429

chromatographic oxidation and liquid chromatography with fluorescence detection; AOAC

430

International: Gaithersburg, MD, 2005.

431

(28) Keyon, A. S. A.; Guijt, R. M.; Gaspar, A.; Kazarian, A. A.; Nesterenko, P. N.; Bolch, C. J.;

432

Breadmore, M. C. Electrophoresis2014, 35, 1496-1503.

433

(29) Keyon, A. S. A.; Guijt, R. M.; Bolch, C. J.; Breadmore, M. C. J. Chromatogr. A.

434 435

436

437

438

439



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

440

FIGURES

441

442

CE-droplet microfluidics system (A) Schematic outline of the setup used for post-

443

Fig.1

444

column compartmentalisation incorporated in a CE instrument. The aqueous phase was

445

segmented by oil in the tee connector (outside CE instrument) and passed the separation

446

capillary outlet to collect the electrophoresed effluent in a compartmentalised manner

447

(within CE instrument). (B) Photograph of the micro cross as the droplet microfluidics

448

interface positioned in a capillary cassette. Photo label: connection for (1) droplet capillary

449

(2) separation capillary (3) detection capillary and (4) salt bridge electrode.


Page 20 of 27

Page 21 of 27

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


450

451 452

Characterisations of droplets: (A) Fluorescence signal of a selected 6 s window for

453

Fig.2

454

aqueous droplets doped with1µg/mL fluorescein in the presence of electric field (30 kV,

455

field strength of 530 V/cm). Each peak corresponds to an individual droplet. Total flow rate

456

of droplet formation was varied from 4-10 µL/min. (B) Current readout during experiments

457

conducted in Fig.2a.



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

Page 22 of 27

458

Separation of fluorescein and calcein mixture; (A) Electropherogram from CE-droplet

459

Fig.3

460

microfluidics

461

electrophoresed analytes into droplets. The insert showed the zoomed in portion of

462

fluorescein peak. Mixture of 230 µg/mL fluorescein and 1900 µg/mL calcein was injected at

463

5 kV for 25 s, while droplets were delivered at4 µL/min (1 Hz). (B) Reconstructed

464

electropherogram from Fig. 3a. (C) Electropherogram from on-capillary detection. Mixture

465

of 19 µg/mL fluorescein and 190 µg/mL calcein was injected at 5 kV for 25 s. Peak

466

identification: (1) fluorescein and (2) calcein. In both experiments, BGE 3 mM disodium

467

phosphate (pH 7.8) and electric field strength of 530 V/cm were used.

system

demonstrating

separation

and


compartmentalisation

of

Page 23 of 27

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


468

469 470

Fig. 4 The relationship of droplet flow and dilution; (A) the electropherograms showing

471

fluorescein signal change as a response to droplet frequency. Insert in Fig.4a is the diluted

472

fluorescein (dilution factor of 5) compartmentalised at 3 µL/min, shown to compensate the

473

overloaded signal in main figure. (B) Plot of signal and droplet frequency as functions for

474

total flow rate of oil and aqueous phases. Experiment was conducted by injecting230 µg/mL

475

fluorescein at 5 kV for 25 s. The BGE and field strength used as indicated in Fig. 3.



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

476

Fluorescence images of droplets in the micro cross in electric field strength of 530

477

Fig.5

478

V/cm. The droplets contained BGE doped with fluorescein and were delivered at a total flow

479

rate 4 µL/min (1 Hz); (A) A fraction from the droplet emanating from the droplet capillary (1)

480

was trapped in front of salt bridge-electrode (4). (B-C) The subsequent droplet coalesced

481

with the trapped droplet when passing through the cross. (D) The droplet left the cross

482

flowing into the outlet capillary (3).

483

484

485

486


Page 24 of 27

Page 25 of 27

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


487

488

489

490

491

492

493

494

495

Separation of PSTs mixture; (A) Electropherogram from CZE-droplets microfluidics

496

Fig.6

497

system demonstrating separation and compartmentalisation of electrophoresed analytes

498

into preformed droplets for oxidation. (B) Reconstructed electropherogram from Fig. 6a. (C)

499

Electropherogram from CZE-UV. Peak identification: (1) NEO and (2) dcGTX2. In both

500

experiments, BGE 20 mM formic acid pH 2.8 and electric field strength of 440 V/cm were

501

used. Mixture of 10,000 ng/mL NEO and 20,000 ng/mL dcGTX2 was injected at 5 kV for 25 s.



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

Page 26 of 27

502

TABLES

503

Table 1 Performance of CZE-droplet post-column oxidation method for PSTs. Toxin LOD (ng/mL) NEO dcGTX2

670 430

Intra-day, (%RSD, n = 3) µeff a Height

Inter-day, (%RSD, n = 3) µeff a Height

8.0 2.5

8.8 5.5

2.9 8.6

504

505

a)

µeff is effective mobility

506


11 5.2

Page 27 of 27

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


507

508

509

Graphical Abstract

510 511


Droplet Microfluidics for Postcolumn Reactions in Capillary

Recommend Documents