ON-DEMAND DROPLET GENERATOR FOR EXTRACTION OF
ELECTROKINETICALLY FOCUSED ANALYTES
Vasileios A. Papadimitriou, Stella A. Kruit, Loes I. Segerink, and Jan C.T. Eijkel
BIOS Lab on a Chip Group, University of Twente, The Netherlands
ABSTRACT
We demonstrate the integration of a droplet-on-demand (DoD) generator that enables us to extract “packaged” concentrated analytes from ion concentration polarization focusing (ICPF) without interrupting the separation and concentration process.
KEYWORDS: Ion Concentration Polarization Focusing, Droplet-on-demand INTRODUCTION
A number of techniques exist that combine on-chip focusing and separation. Though they offer very powerful analytical tools, the focused and separated analytes become “trapped” inside the chip. A few examples [1-3] exist of on-chip combination of electrokinetic separation and/or concentration techniques with integrated droplet generators to extract and maintain the spatial concentration profile. In contrast to those previous reported works our DoD generator has minimal interference in the separation process allowing extraction of analytes with low loss of concentration factor without interrupting the focusing process. Additionally our DoD generator can be integrated with other electrokinetic separation processes.
THEORY
In the DoD generator, the continuous (oil) phase is continuously transported using such reservoir pressures (Pin, Pout) that the meniscus remains stagnant at the site of the droplet generator (Fig.1). Then actuation potentials are applied and analytes are focused via ICPF in the separation channel. Once the desired concentration is reached, the focusing location is moved to the DoD generator interface by controlling the actuation potentials. A short negative pressure pulse of the continuous phase (oil) is then applied to temporarily disturb the pressure equilibrium, creating a single droplet sampling the highly concentrated focused analytes without interrupting the ICPF process. Once the droplet is formed the pressure equilibrium is regained.
EXPERIMENTAL, RESULTS AND DISCUSSION
Polydimethylsiloxane (PDMS) chips were made with standard soft lithography methods and bonded on standard microscopy slides (Fig.1). An ion-permselective membrane (Nafion) was filled and patterned on chip via capillary forces [4]. The aqueous sample (1x PBS(Sigma) spiked with fluorescent markers Bodipy(Invitrogen) and Alexa Fluor 647(Invitrogen)) is added in the separation channel, followed by filling of the continuous phase channel with mineral oil and surfactant Span80(Sigma) (3% v/v). To form a single droplet, a pressure pulse (-10mbar, 3s) is applied to the oil inlet (Fig.2). We report a success rate of 77% for creating a single DoD by external pressure control, with all unsuccessful attempts translating to either none or two droplets formed. We were able to reach a maximum concentration factor of 310x in the extracted droplets. On average analyte concentration in the extracted droplets was 65-100% of the maximum concentration factor in the separation channel. During droplet formation we found the ICPF process continued unaffected. These results obtained using the presented channel geometry improve on the previously reported recovery rates of 20% [2] and 60% [3], where also, importantly, interruption of the focusing process was required. The concentration recovery of our device depends on the correct placement of the focused analyte with respect to the DoD generator interface. Due to the hydrophobicity of PDMS occasionally oil wicked in the separation channel affecting the stability of ICPF, a problem we think can be potentially solved with surface modification of the separation channel. In the near future higher concentration factors will be attempted and the purity of the extracted analytes will be investigated.
ACKNOWLEDGEMENTS
This work was supported and funded by Horizon 2020 Framework Programme of the European Union under the project H2020-PHC-634013 (PHOCNOSIS).
978-1-7334190-0-0/µTAS 2019/$20©19CBMS-0001 977 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences 27 - 31 October 2019, Basel, SWITZERLAND
Figure 1 - Schematic of the ICPF design with integrated droplet generator. The separation chan-nel (dispersed phase - light blue) is filled with the sample. The green channels represent the continu-ous phase channels filled with oil. Nafion mem-brane is patterned in the grey channels. The dark blue channels are filled with buffer and are electri-cally grounded. The pressure conditions for equi-librium and droplet formation are shown in the bottom figure. PLP is the oil-water interface
La-place pressure, Pd is the pressure in the disperse
phase (arising from hydrostatic pressure or any in-duced pressure of the ICPF process) and Pc is the
pressure at the interface resulting from the actua-tion pressures (Pin and Pout).
Figure 2 - Schematic of the setup and a typical pressure actuation scheme for a single droplet.
Figure 3 - Sequence of ICPF and DoD. Microscopy image - left, Fluorescent intensity profile - right 1) The initial condition 2) A poten-tial difference is applied, and the analytes are focused, with Bodipy in green and Alexa fluor 647 in red. The concentration of the focused an-alytes is that high the camera sensor is saturated making hard the dis-tinction between colors (analytes). 3) Adjusting the potential difference enables the control of the focusing location. 4) Once the focused plug has settled in front of the droplet generator, DoDs are created.
REFERENCES
[1] J. Scott Edgar, G. Milne, Y. Zhao, C.P. Pabbati, D.S.W. Lim, and D.T. Chiu. Compartmentalization of chemically separated components into droplets. Angewandte Chemie International Edition, 48(15):2719-2722, 2009.
[2] C. Chen, A. Sarkar, Y. Song, M.A. Miller, S. Jae Kim, L.G. Griffth, D.A. Lauffenburger, and J. Han. Enhancing protease activity assay in droplet-based microfluidics using a biomolecule concentrator. Journal of the American Chemical Society, 133(27): 10368-10371, 2011.
[3] X.F. van Kooten, M. Bercovici, and G.V. Kaigala. Extraction of electrokinetically separated analytes with on-demand encapsulation. Lab Chip, 18(23):3588-3597, 2018.
[4] “Capillary-valve-based fabrication of ion-selective membrane junction for electrokinetic sample preconcentration in PDMS chip,” V. Liu, Y. Song and J. Han, Lab Chip, 10, 1485, (2010).
CONTACT
* V.A. Papadimitriou; phone: +31633621802; v.papadimitriou@utwente.nl 978
