Chapter 1

Introduction

1.1 A tendency towards smaller and smaller

A large number of applications used in the medical world and in molecular biology or bio-chemistry
laboratories benefit from detection methods specific for bio-sensing and molecular assays. Devices
developed in the early 1950s, such as titer plates, were the first used to fasten the preparation and
realization of the bio-chemical analysis in a simultaneous, high-throughput manner. With the tendency
towards size reduction, the assay processing time and costs can be significantly reduced by the
miniaturization of such systems because of both the reduced reagent consumption and inexpensive mass
production. In figure 1-1 an example is depicted where fabricated arrays in silicon are leaning against a
commercially available 96-reactor titer plate.

Figure 1-1. Silicon arrays leaning against a conventional 96-reactor titer plate.

Consequently, various micro-titer plates are available nowadays that work with volumes ranging between
milliliters and microliters. Miniaturizing such devices also allows many more tests to be carried out on the
same area, depending on the testing protocol and the chemical or molecular interactions implied.
The two important molecular recognitions in most of the biological assay detection methods used in array-
plates are deoxyribonucleic acid (DNA) hybridization and protein-protein immunoassay. The first method
is used for detecting one strand of DNA using the complementary strand, which is extremely useful
because the DNA contains all the information about how, when and where to produce each kind of protein
within a certain cell. The protein-protein immunoassay method is a biochemical test that determines the
presence and/or measures the concentration of a target molecule in a biological fluid based on the
specificity of the antigen-antibody interaction. This technique has various applications in medicine (e.g.
detection of different diseases, such as HIV, cancer, viruses infections), in the food industry (e.g. detection
of food allergens), in research laboratories, etc.
Over the past two decades both the use of various microfluidic structures and the designs integrated onto a
single chip for multi-purpose applications have developed significantly in the analytical processing of
biological and chemical samples. Besides the major advantage of fabrication technology in this area is the
ability to create micro/nano environments with controlled fluidic parameters for bio-chemical interactions
and detection.
For more insight into the bio-chemical processes and development of new experimental approaches, it is
essential that research directions are projected towards designing novel devices capable of handling and
detecting small volumes with low molecular concentrations, with the ultimate goal of single (bio-)molecule
manipulation and detection. Therefore, devices with nano-liter and pico-liter volume reaction wells have
been fabricated and tested for bio-processes such as enzymatic reactions [1] and DNA sequencing [2], with
the main advantage being the drastical decrease in the total reaction time compared to their actual standard
market procedures.
Lately, the surface plasmon resonance (SPR) technique, which most commonly involves the Kretschmann
configuration, represents a widespread methodology for many sorts of analyses in various laboratories [3].
The interfacial nature of the local evanescent waves confers them a high sensitivity to the near-surface
dielectric constant and makes them suitable for detecting surface binding events. This technique has been
generally implemented in the field of biosensor development, for which commercial devices are already
available [4]. The challenges associated with scaling down the structures involved in the system have made
the attempts to miniaturize these sorts of devices integrated with microfluidic platforms quite limited.
Nevertheless, surface plasmons can also be tailored via periodical corrugations in a thin metallic layer. The
possibility of patterning and fabricating such structures on a nanometer scale level opens up a new area of
opportunities for surface-based chemical and biological detections.

1.2 Motivation and objectives

The discussion above suggests that miniaturization of bio-chemical and medical devices brings many
benefits, specifically the lower analyzed sample volume, faster diagnosis time, and reduced investigation
costs. There is a great need for the use of such devices for early pre-diagnosis of various diseases and
treatment monitoring. In addition, within the molecular bio-technological laboratories, a reliable short-time
detection of the amplification and/or expression products would also be welcomed.
Therefore, in this thesis we present the research efforts and results of developing a miniaturized nano-liter
plate for optical bio-molecular detections which is a more compact and versatile device that features an
adequate optical detection of up to several hundred million reaction wells per square centimeter of chip
area; recently developed nano-liter plates usually only have 200 wells per square centimeter of chip area
[5]. The conflicting consequences of miniaturizing the device, such as limited resolution for the optical
detection and a low recorded signal, might be overcome by making use of the most important effects that
appear when light passes through periodical subwavelength holes in thin metallic layers: a reduced angle of
diffraction, selected spectra and enhanced transmitted light. Recently discovered in 1998 by Ebbesen et al
[6], these effects manifest themselves as promising options in creating novel applications in optics,
photonics and biosensors [7], [8].
Therefore, the scope of this research aims to make use of this extraordinary optical phenomenon to create a
molecular analyzing device by fabricating periodical nanocavities in thin metallic films deposited on
transparent substrates, where each cavity acts as a reaction chamber. With the new burst of technological
methods and processing techniques, there are several ways of creating these structures, such as focused ion
beam milling [9], soft-imprinting lithography [10], and self-assembling on pre-patterned macro-porous
films [11]. Nevertheless, inherent issues related to the nanoscale level of fabrication make it difficult to
exploit these methods, as they have their drawbacks materialized in long processing time, master demands
or re-deposition of the etched metal back on the sample surface. Since the structures to be fabricated are
subsequently illuminated and the transmitted light is collected and depicted in a spectrum, the requirements
of the device include clear and unobstructed nanocavities that are uniform in shape and size and posses a
stable periodicity between two neighboring centers. Hence, an approach is considered in this thesis where a
lift-off procedure is explored and a material is removed from the place of the upcoming holes, leaving them
optically clear for the transmission measurements.
The challenges to miniaturization from the fabrication point of view refer to the correct choice of photo and
electro-sensitive materials, which must have a good patterning resolution and a good adherence to the
substrate. Moreover, because they also act as masking layers, they need to feature strong selective
properties for the etching process. In addition, the type of exposure must be more accurate than
photolithography and the conditions for the lift-off procedure must be well correlated with the chosen
layers and substrates.
Therefore, the present scope focuses on developing a better procedure for fabricating periodical nanohole
arrays in thin films that is a less complicated and reliable process and which also features high
reproducibility with a good overall yield. The proposed method, i.e. making use of negative tone electron-
sensitive resist, electron-beam lithography, direct reactive ion etching, electron-gun deposition and a lift-off
procedure, is explored in this research together with the shortcomings and the improvements made along
the way. Designing, fabricating and characterizing the device in terms of optical transmission represent
further goals of this project. Moreover, a novel application of the nanohole arrays for the reconstitution of
artificial lipid membranes and electro-optical measurements of incorporated proteins is investigated from
the technological side, providing a promising tool for batch analysis of non-aqueous bio-molecular events.

1.3 Organization of the thesis

This thesis presents the technological fabrication and the optical characterization of different periodical
nanohole arrays in thin metallic films which are to be integrated into a novel atto-liter plate device for high-
speed molecular analysis, such as DNA hybridizations and protein immuno-assays.
The first chapter contains a general introduction to the thesis together with the motivation, scope and
objectives that represent the starting point of this project. Chapter 2 deals with a brief description of similar
devices being researched or already available on the market. These devices are undergoing a
miniaturization and a continuously decreasing volume of reagents used for the assays, ranging from
millilitres down to picolitres. Chapter 2 also presents an introduction to the atto-liter plate and a description
of its working principle.

Furthermore, in Chapter 3 the background of the detection method is presented. When light passes through
sub-wavelength holes made in optically thick films, almost no light is expected to be transmitted.
Nevertheless, “the extraordinary optical transmission (EOT)”, discovered not long ago [6], shows that
something different happens when these holes are displayed in a periodic arrangement in metallic materials.
The effects of this revolutionary phenomenon and the investigations realized in past years are briefly
summarized together with the two main proposed theories: one which implies surface plasmons polaritons
(SPP) and the other one which implies composite diffracted evanescence waves (CDEW). In addition,
advantages and applications of these effects in various research fields, such as near-filed optics, plasmonic
nanolithography and nano-lens fabrication, are also mentioned.

Chapter 4 exploits the design concepts and describes the complete process steps for fabricating the atto-liter
plate device. In the beginning of the chapter various alternative methods for creating the nanohole arrays
are presented, and their advantages and disadvantages are explored with regard to our device requirements
and the facility’s restrictions. Furthermore, the proposed method, i.e. using a negative tone electron-beam
sensitive resist, electron-beam exposure, reactive ion etching, directional flux deposition and a lift-off
technique, is detailed together with technological drawbacks and improvements. The fabrication of the top
part of the device where the delivery channels are situated is also presented with a brief description of the
interface to the necessary outer instruments used for the measurements.

In Chapter 5 an optical characterization of the devices is described. Using Köhler and then collimated
illumination under perpendicular incidence, light is transmitted through different configurations of
nanohole arrays and the obtained spectra are investigated in order to both select the structures that are
inclined to give a greater transmission enhancement, and to determine the specific wavelengths of the
enhanced peaks. In addition, an experiment is detailed where the fluorescence in/above the arrays is
determined in comparison to fluorescence through bare glass, after which the results are discussed.

In Chapter 6 a micro-fluidic device based on similar nano-hole arrays is proposed that has been fabricated
in free standing silicon nitride foil for simultaneous measurements of optical and electrical properties of
planar lipid bilayers and electrophysiological studies of incorporated membrane proteins. A deep ultraviolet
positive tone silicon containing resist exhibits sensitivity to electron-beam lithography, which features
adequate characteristics to be used as a direct mask in patterning narrow structures in the silicon nitride
layer.

The last chapter of the thesis offers some general concluding remarks together with a few suggestions for
future work. The thesis ends with a succinct summary, where the main points of this research are
highlighted.

References

[1] H. R. C. Dietrich et al., “Nanoarrays: A method for performing enzymatic assays”, Anal. Chem. 76, pp
4112-4117, 2004.

[2] M. Margulies et al., “Genome sequencing in microfabricated high-density picolitre reactors”, Nature
437, pp 376-380, 2005.
[3] B. Lieberg, C. Nylander and I. Lundstrom, “Biosensing with surface plasmon resonance – how it all
started”, Biosensors Bioelectron. 10, pp i-ix, 1995.

[4] W. M. Mullet, E.P.C. Lai and J. M. Yeung, Methods 22, pp 77-91, 2000.

[5] M. Seidel, D. M. Dankbar and G.Gauglitz, “A miniaturized heterogeneous fluorescence immunoassay

[6] T. W. Ebbesen, H. L. Lezec, H. F. Ghaemi, T. Thio, P. A. Wolff, “Extraordinary optical transmission

[7] R. Sambles, “More than transparent”, Nature 391, pp 641, 1998.

[8] T. Thio, H. J. Lezec and T. W. Ebbesen, “Strongly enhanced optical transmission through
subwavelength holes in metal films”, Physica B 279, pp 90-93, 2000.

[9] Principe 2005

[10] Malyarchuk 2005

[11] Abdersalam 2004