Label-free Immobilization of Nano-particles

on Silicon based Electrodes for Single-biomolecule Studies

Ch. Wenger

, X. Knigge

, M. Fraschke

, D. Wolansky

, P. Kulse

, U. Kaletta

, A. Wolff

, W. Mehr

E.-M. Laux

, F. F. Bier

and R. Hölzel

IHP GmbH – Leibniz Institute for Innovative Microelectronics, Im Technologiepark 25, 15236 Frankfurt (Oder), Germany

Fraunhofer Institute for Biomedical Engineering IBMT, Branch Potsdam-Golm, Potsdam, 14476, Germany

Keywords: Dielectrophoresis, Nanoelectrodes, Biosensing.

Abstract: Dielectrophoresis (DEP) is an established method for the spatial manipulation of microscopical particles.

We demonstrate the temporal and permanent immobilization of polystyrene nanoparticles and protein

molecules with sizes ranging from 4 nm to 500 nm. For this, regular arrays of silicon based nanoelectrodes

were developed with tip diameters of 10 nm and 50 nm. No chemical modifications of molecules, particles

or surfaces were needed. This opens up potentially important applications of DEP in biosensing and cell

research.

1 INTRODUCTION

There is a growing need in lab-on-a-chip systems

and similar biodevices for spatial manipulation of

nanoparticles like concentrating, immobilizing,

orientating and aligning. The manipulation should be

performed on a large number of objects

simultaneously. AC electrokinetic methods like DEP

have been successfully applied for some decades by

exploiting alternating electric fields between

microelectrodes. In the case of non-uniform fields,

polarizable particles get immobilized on top or at the

edges of the electrodes, as illustrated in Fig. 1.

So far, most of the research work performed on

DEP has been done by using metal electrodes

(Widdershoven 2010, Martinez-Duarte 2012). The

continuous downscaling of CMOS minimum feature

sizes provides great opportunities. By adapting the

typical electrode dimensions to the objects' size, it

has become possible to manipulate even single

objects like viruses and proteins on metal electrodes

(Yamamoto 2007, Pethig 2010, Diao 2011 Nakano

2013). Nevertheless, the dimensions of CMOS metal

electrodes are still in the range of 100 nm (ITRS

2012 update), which doesn’t fit to nanoparticles like

proteins with diameters of less than 10 nm (Hölzel

2005). A modern approach to optimize the

interaction between particles and electrodes is the

use of doped triangular shaped silicon as electrode

material.

Figure 1: Dielectrophoresis: Schematic illustration of the

experimental setup. a) Nanoobjects are suspended in water

b) By applying inhomogeneous AC electric fields,

electrical forces act on polarizable particles. These forces

lead to a local particle accumulation. Distance between top

electrode and electrode surface is about 100 µm.

2 EXPERIMENTAL

Cone-shaped nanoelectrodes were fabricated in a

176

Wenger C., Knigge X., Fraschke M., Wolansky D., Kulse P., Kaletta U., Wolff A., Mehr W., Laux E., Bier F. and Hölzel R..

Label-free Immobilization of Nano-particles on Silicon based Electrodes for Single-biomolecule Studies.

DOI: 10.5220/0004888101760180

In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2014), pages 176-180

ISBN: 978-989-758-013-0

 2014 SCITEPRESS (Science and Technology Publications, Lda.)

standard CMOS process line by using reactive ion

etching (RIE) process techniques (Mehr 1996). The

minimum tip radius is about 1.5 nm. The electrodes

are embedded in a SiO

matrix and the diameter on

top of the tips can be increased by chemical

mechanical polishing (CMP), as shown in Fig. 2.

The process flow is completed with silicidation of

the tip surface (CoSi). The total number of

electrodes amounts to about 100.000 per array (Fig.

3).

Figure 2: Cross-sectional scanning electron microscope

(SEM) image of plasma etched electrodes with a) 10 nm

and b) 50 nm diameter at the top of the silicon based

electrodes.

The DEP force acting on the particles is directed

along the gradient of the electric field. Therefore, the

implementation of a steep electric field gradient is

required to maximize the DEP force acting on the

particles.

The simulation (Fig. 4) shows the largest electric

field gradient and, hence, the strongest DEP force on

top of the 10 nm tip electrode. For the 50 nm top

surface, the strongest force is localized at the edges

of the electrode, leading to a ring-like alignment.

Obviously, for single molecule immobilization, the

tip diameter has to be minimized.

Figure 3: SEM image of the plasma etched nanoelectrode

array.

Figure 4: Simulation of the electric field gradient grad |E|

above the nanoelectrodes with a) 10 nm and b) 50 nm tip

diameter. Maximum field gradient (white) is close to the

tip.

To demonstrate the permanent immobilization of a

biomolecule on an electrode array (Fig. 5), we used

the fluorescently labeled bovine serum albumin

(BSA), which is a protein of prolate ellipsoidal

shape (14 nm x 4 nm x 4 nm (Squire 1968)).

)

Label-freeImmobilizationofNano-particlesonSiliconbasedElectrodesforSingle-biomoleculeStudies

177

Typically, these experiments were carried out at

about 10 kHz with 5 to 10 V

RMS

for periods of some

seconds to minutes. By choosing the optimum

operating conditions, the immobilization of

nanobeads with diameters of 200 nm is finalized

within a few minutes, as illustrated in Fig. 6.

Figure 5: DEP field induced immobilization of the

fluorescently labeled protein BSA on nanoelectrodes after

field application of 10 kHz and 7 V

RMS

for 10 min.

The distribution of fluorescence intensity of

immobilized nanobeads with diameters of 20 nm on

electrode tips with diameters of 50 nm is quite

broad, as shown in Fig. 7a. That means, ensembles

of numerous nanobeads were immobilized on each

electrode tip.

However, by applying this system to objects that

were slightly larger than the electrode tips, we were

able to achieve a proper 1:1 ratio between particles

and tips, as illustrated in Fig. 7b. That means,

exactly one nanobead was placed on each electrode

tip.

This opens a completely novel approach to

single-molecule investigations on large ensembles.

This deterministic control of local particle numbers

in aqueous solutions demonstrates the importance of

reducing the typical electrode dimensions to 10 nm

and less.

In addition, one has to consider that alternating

electric fields lead to Joule heating in the liquid

medium (Seger-Sauli 2005). Local temperature

raises could cause thermal stress, cell damages and

protein denaturing. Therefore, we measured the

heating of the medium close to the electrodes at

1 MHz and 9 V

RMS

as a function of electrode

diameter. The temperature variation was detected by

exposing the thermo-dependent fluorescent dye

)

Figure 6: Top view microscopic images through the

transparent top electrode of 200 nm fluorescent

polystyrene nanospheres in water on the nanoelectrode

array. a) Before field application, b) after field application

at 17 kHz and 8 V

RMS

for 12 s and c) after 60 s field

application.

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Figure 7: Fluorescence intensity distribution of dielectro-

phoretically immobilized polystyrene nanospheres. Bead

diameters are a) 20 nm and b) 200 nm. Electrode diameter

is 50 nm. a) For particles smaller than the electrodes,

accumulation of approx. 1 to 15 particles per electrode tip

occurs. b) 200 nm spheres are immobilized as singles.

rhodamine B to the test set-up. Its fluorescence

intensity decreases with temperature by about -

1.2%/K

As shown in Fig. 8, temperature rises within

220 s by 5 °C for 10 nm tip diameter and by 6 °C for

50 nm tip diameter. This improvement by smaller

electrodes can be explained by the corresponding

reduction of the volume carrying high current

densities. Above that, the increased surface-to-

volume ratio enhances heat dissipation from the

electrodes.

As illustrated in Fig. 9, the proposed silicon-

based technology for immobilization of nanometer

sized molecules by cone-shaped nanoelectrodes

opens a new CMOS compatible platform for the

analysis of single proteins and their functions.

Figure 8: Intensity characteristics of the thermo-dependent

fluorescence of rhodamine B in order to evaluate the

temperature increase in the liquid medium above the

nanoelectrodes with a) 10 nm and b) 50 nm tip diameter.

Figure 9: CMOS Metal1 half pitch roadmap (ITRS 2012)

compared to typical dimensions of proteins.

3 CONCLUSIONS

We demonstrated the electrically controlled

immobilization of biomolecules and nanospheres

using silicon based nanoelectrode arrays. The

nanoelectrodes with tip sizes of 10 and 50 nm were

Label-freeImmobilizationofNano-particlesonSiliconbasedElectrodesforSingle-biomoleculeStudies

179

fabricated by reactive ion etching (RIE) techniques

in a standard CMOS process line. Electric field

distributions were calculated. Temperature changes

within the device were determined by optical

microscopy to about 5 °C, which is well compatible

with biomedical applications.

Nanospheres with diameters of 20 nm and

200 nm suspended in water were immobilized at the

electrode tips. For particles larger than the

electrodes, immobilization of single objects was

demonstrated. The procedure was performed within

a few tens of seconds. Any chemical modifications

of suspended particles, dissolved molecules or

surfaces could be avoided.

The demonstrated use of silicon based

nanoelectrode arrays for the dielectrophoretic

immobilization of particles and molecules opens a

novel way for nanoparticle separation and for the

preparation of miniaturized biosensors.

ACKNOWLEDGEMENTS

This work was supported by the grant from the

federal state of Brandenburg and the European

Regional Development Fund.

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