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Procedia Engineering 120 (2015) 175 – 179

EUROSENSORS 2015

Application of optical pH sensors in the microfluidic free-flow

isoelectric focusing of biomolecules
Elisabeth Poehlera, Christin Herzoga, Simon A. Pfeiffera, Carsten Lottera, Andrea J.
Peretzkia, Daniel Aignerb, Torsten Mayrb, Erik Beckertc, Stefan Nagla,*
a
Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, 04103 Leipzig, Germany
b
Institut für Analytische Chemie und Lebensmittelchemie, Technische Universität Graz, Stremayrgasse 9, 8010 Graz, Austria
c
Fraunhofer-Institut für Angewandte Optik und Feinmechanik (IOF), Albert-Einstein-Straße 7, 07745 Jena, Germany

Abstract

We present methods for fabrication and application of microfluidic chips for free-flow electrophoresis (FFE) that
allow integrated pH sensing. Monitoring of the pH gradient in microfluidic free-flow isoelectric focusing (FFIEF)
allows on-line adjustment of the parameters for electrophoresis and evaluation of the isoelectric point (pI) of focused
analyte bands almost in real time. The fabrication of these assemblies is demonstrated via multistep
photopolymerization or a laser cutting and lamination technique. Integration of pH sensing layers is accomplished
via photopolymerization or inkjet printing of hydrogels that contain covalently attached pH probes. These
microchips were characterized and employed for the FFIEF of proteins, peptides and plasma. Multispectral
microscopic imaging was used to separate the pH sensor and the biomolecule fluorescence. We also show pH
sensing in FFE based on time-domain dual lifetime referencing (t-DLR), a microreactor for integrated labelling prior
to the FFIEF and the isoelectric focusing of unlabeled compounds.
©
© 2015
2015The TheAuthors. Published
Authors. by Elsevier
Published Ltd. This
by Elsevier is an open access article under the CC BY-NC-ND license
Ltd.
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the organizing
Peer-review under responsibility of the organizing committeecommittee of EUROSENSORS
of EUROSENSORS 2015 2015.

Keywords: free-flow isoelectric focusing; miniaturized luminescent pH sensor; microfluidic analytical systems; photopolymerization; protein
isoelectric points; multispectral imaging; pH gradient observation; time domain dual lifetime referencing

* Corresponding author. Tel.: +49-341-97-36066, http://research.uni-leipzig.de/nagl

E-mail address: nagl@chemie.uni-leipzig.de

1877-7058 © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the organizing committee of EUROSENSORS 2015
doi:10.1016/j.proeng.2015.08.603
176 Elisabeth Poehler et al. / Procedia Engineering 120 (2015) 175 – 179

Microfluidic free-flow electrophoresis is a mild separation method for biological species and enables the
continuous separation of biomolecules from complex matrices on a micropreparative scale. In the FFIEF analytes
are separated according to their isoelectric point (pI) in a pH gradient. Integration of optical fluorescent or
luminescent sensors into micro-FFIEF platforms allows spatially resolved on-chip observation of the pH gradient
and pI determination of focused analyte bands in real time.

1. Fabrication of microfluidic chips for free-flow isoelectric focusing with an integrated pH sensor layer

We demonstrated the fabrication and application of micro-FFIEF chips that contained integrated pH sensing
areas by various methods. First we presented a multistep procedure that allowed to form a pH sensing layer from a
fluorescein-based probe in oligoethylene glycol acrylates that were photopolymerized on glass. On top of this layer
a microfluidic FFIEF structure was constructed using a different short-chain hydrophobic oligoethylene glycol
acrylate that did not mix with the sensing layer. [1] Then we demonstrated arrays manufactured via inkjet printing of
modified pHEMA hydrogels on glass. The region of pH sensitivity could be adjusted via choice of the pH probe and
the chips were applied for IEF of small molecule markers and proteins (Fig. 1). [2]

Figure 1: a-h) Fabrication of microfluidic FFIEF chips with inkjet-printed sensor areas and sensor arrays via
multistep photopolymerization. k, l) exemplary inkjet-printed pH sensor areas m) on-chip pH sensitivity, Reprinted
and adapted with permission from [2]. Copyright 2014, American Chemical Society.

Later we demonstrated the assembly of FFIEF chips via a multistep laser cutting and lamination technique,
inkjet-printed a pH sensor matrix and built a microscopic setup for time-domain dual lifetime referencing (t-DLR)
(Fig. 2). Here the microfluidic structure is realized in an acrylate-based adhesive foil on top of a powder-blasted
glass cover plate. The pH sensing area was inkjet-printed on glass and the microfluidic and support layers were
pressed together. We built various measurement modalities for microfluidic pH sensors. Shown in Fig. 2B is a setup
for microscopic time-domain dual lifetime referencing for pH sensing in the continuous electrophoretic separation of
proteins [3]. All setups are based on the spectral separation of analyte fluorescence and pH sensor luminescence. In
the work presented at the conference we will also show the microfluidic chip integration of a novel near-infrared pH
sensing layer based on a modified perylene bismide containing a prepolymer backbone that may be readily
photopolymerized [4]. This sensor layer allows for background-free pH sensing in the NIR while leaving the UV
and visible range for monitoring of the electrophoretic separations.
 Elisabeth Poehler et al. / Procedia Engineering 120 (2015) 175 – 179 177

Figure 2. (A) Fabrication of µFFIEF chips with an integrated inkjet-printed pH sensor layer via laser cutting and
lamination. A cover plate with powder blasted cavities is subjected to laser cutting of microfluidic structure in an
acrylate-based adhesive foil, followed by jet dispensing of the polymeric coating solution for pH sensing on the
bottom plate. Finally three layers were pressed together with a force of 10 kN. (B) Schematic of the employed
microscopic measurement setup for the dual lifetime referencing method in time domain with online
observation of the pH gradient in a µFFIEF chip. Revised from [3]. Copyright Wiley-VCH Verlag GmbH
KGaA. Reproduced with permission.

2. Application in the free-flow isoelectric focusing with integrated pH gradient monitoring and pI detection

We have employed these FFIEF chips with integrated pH sensor layers already in the separation of proteins,
peptides, small molecule markers, antibiotics, tryptic digests and plasma fractions. In the already published work
displayed in Fig. 3 we focused small molecule markers and proteins and monitored the pH with sensor layers and
arrays based on fluorescein isothiocyanate covalently coupled to an amino-modified polyhydroxyethylmethacylate.
Table 1. pI determination results of µFFIEF separated analytes with an FITC-pHEMA pH sensor layer

compound literature pI µFFIEF-pI deviation

IEF marker 4.0 4.0 5.02 ± 0.23 +1.0

IEF marker 5.5 5.5 5.75 ± 0.17 +0.3

IEF marker 7.2 7.2 6.71 ± 0.14 -0.5

IEF marker 7.6 7.6 7.56 ± 0.16 +0.0

IEF marker 9.0 9.0 8.32 ± 0.13 -0.7

Į-lactalbumin type III
4.5-4.8 4.83 ± 0.04 +0.0
(bovine milk)
bovine serum albumin 4.7-4.9 5.16 ± 0.34 + 0.2
conalbumin type I
6.0-6.3 5.99 ± 0.33 +0.0
(chicken egg white)
myoglobin (horse heart) 6.5-7.2 7.04 ± 0.06 +0.0
Į-chymotrypsin type II
8.4-8.8 7.62 ± 0.33 - 0.8
(bovine pancreas)
178 Elisabeth Poehler et al. / Procedia Engineering 120 (2015) 175 – 179

Figure 3: IEF of small molecule markers and proteins (fluorescently labeled with P503) with online pH monitoring
via FITC-pHEMA pH sensors. top: false-colored fluorescence image of the analyte channel, bottom:
electropherogram and corresponding pH readout. (a) small molecule markers, (b) comparison of obtained pH via
immobilized pH sensor and pI markers (extract from (a)), (c) the proteins bovine serum albumin (BSA), conalbumin
and chymotrypsin and (d) proteins bovine serum albumin (BSA), conalbumin and chymotrypsin. Reprinted with
permission from [2]. Copyright 2014, American Chemical Society.

Table 1 summarizes the results obtained from determining the pI of focused analyte bands via the integrated pH
sensor layers. Determination of biomolecular isoelectric points is possible within a few seconds in microfluidic
FFIEF chips and the determined pI’s are generally in good agreement with literature values with bigger deviations
outside the dynamic range of the respective pH sensor.
To overcome the need for conventional off-chip fluorescence labelling prior to chip-FFIEF we have introduced
two approaches. In the first, native biomolecules are detected via their intrinsic UV fluorescence, which is
applicable to many proteins, peptides and other compounds. Using a microscopic setup and chip materials that
allowed for deep UV laser excitation and a separate optical path for pH monitoring in the near infrared (NIR) native
proteins, plasma and antibiotics could be separated and their pI determined almost in real time. [5]
In another work we introduced an on-chip micro flow reactor prior to the FFIEF and labelled proteins and
peptides with the green fluorescent Atto 425 dye. The labelled compounds were directly introduced into the
electrophoretic separation bed where FFIEF was performed and the pH gradient was monitored with a chip-
integrated pH sensor. [6]

Acknowledgements

Financial support of this work by the German Research Foundation (DFG, NA 947/1-2) and the German Ministry
of Education and Research (BMBF, V4KMU10/126) is gratefully acknowledged.
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References

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