An increasing demand in performance from electronic devices has resulted in continuous shrinking of electronic components. This shrinkage has demanded that the primary integration platform, the printed circuit board (PCB), follow this same trend. Today, PCB companies offer ~100 micron sized features (depth and width) which mean they are becoming suitable as physical platforms for Lab-on-a-Chip (LOC) and microfluidic applications. Compared to current lithographic based fluidic approaches; PCB technology offers several advantages that are useful for this technology. These include: Being easily designed and changed using free software, robust structures that can often be reused, chip layouts that can be ordered from commercial PCB suppliers at very low cost (1 AUD each in this work), and integration of electrodes at no additional cost. Here we present the application of PCB technology in connection with microfluidics for several biomedical applications. In case of commercialization the costs for each device can be even further decreased to approximately one tenth of its current cost.
Here we present a technique to integrate bottom-up nanostructures for optoelectronic and chemoresistive sensing using an AC electrical field. The work focuses mainly on two types of nanostructured materials: gold nanoparticle and silicon nanowire. In terms of electrical microintegration of these structures, it is especially important to apply a reliable electrical contact with low contact-resistance, in order to be able to use them as optoelectronic or chemo resistive sensors. To achieve this, a micro integration process was developed to achieve this goal. The contacted nanostructures were characterized electrically to optimize the integration procedure and acquire best possible sensing capabilities. Silicon nanowires were demonstrated to work as wavelength sensitive optical sensors and gold nanoparticle as marker free chemo resistive sensor.
Surface Plasmon Resonance (SPR) in metallic nanostructures is an optical effect that can be exploited for the detection of small molecules. There is a broad range of metallic nanostructures supporting different SPR modes, and nanostructures can be even geometrically combined leading to the creation of new hybridised SPR modes. In our study, we investigated the properties of a hybridised SPR mode (gap modes GM) created by the placement of metallic nanoparticles onto metallic layers and its use as a sensitive sensor. A tunneling current passing through a metal-insulator-semiconductor structure can generate supported SPR modes that can be scattered through GM, which was experimentally confirmed. Moreover, we were able to experimentally follow the degradation of anisotropic (silver nanoprism) nanoparticles under ambient conditions in real time. Using atomic force microscopy and optical spectroscopy we observed an anisotropic corrosion that is starting from the tips of the nanoparticles.
The authors present a novel in-situ method of fabricating crystalline gold nanoparticles by self-organization. This
nanoparticles are grown and modified in a surrounding thin film matrix using two different host materials (YBa2Cu3O7-δ and SrTiO3) prepared by a pulsed laser deposition technique. The crystalline Au nanoparticles are formed out of a gold seed layer whereby the thickness of the initial seed layer influences the particle size and their distribution density. As we will show, using a matrix based preparation technique offers several advantages over conventional preparation methods. On the one hand, nanoparticle size and the distribution density can be controlled individually. On the other hand, by choosing an appropriate matrix material as well as suitable growth conditions also the shape of the resulting particles can
be modified. Thus, also anisotropic nanoparticles can be prepared without using highly sophisticated methods like
electron beam lithography or focused ion beam techniques. As one might have to extract the nanoparticles or at least
theirs tips from the surrounding matrix material to realize photonic applications, we will show that an extraction is easily
possible by selectively etching the matrix. This extraction process does not influence the particle distribution, i.e.
particles can be prepared and extracted at distinct positions on the substrate utilizing a patterning of the Au seed layer. A
spectral characterization of extracted as well as embedded particles will be presented based on microspectroscopy as well
as on measurements using an integrating sphere.
Plasmonic nanostructures promise to provide sensing capabilities with the potential for sensitive and robust assays in a high parallelization. We present here the use of individual nanostructures for the detection and manipulation of biomolecules such as DNA based on optical approaches [1]. The change in localized surface plasmon resonance of individual metal nanoparticles is utilized to monitor the binding of DNA directly or via DNA-DNA interaction. The influence of different size (length) as well as position (distance to the particle surface) is thereby studied [2]. Holes in a Cr layer present another interesting approach for bioanalytics. They are used to detect plasmonic nanoparticles as labels or to sense the binding of DNA on these particles. This hybrid system of hole and particle allows for simple (just using RGB-signals of a CCD [3]) but a highly sensitive (one nanoparticle sensitivity) detection. On the other hand, the binding of molecular layers around the particles can be detected using spectroscopic features of just an individual one of these systems. Besides sensing, individual plasmonic nanostructures can be also used to manipulate single biomolecular structures such as DNA. Attached particles can be used for local destruction [4] or cutting as well as coupling of energy into (and guiding along) the molecular structure [5].
Sensors based on the localized surface plasmon resonance (LSPR) effect are known as sensitive methods for refractive
index measurements or for detection of specific binding reactions in biosensing due to a variation in layer thickness.
Fiber based arrangements are able to perform such measurements with very small analyte volumina. A generalization of
this concept for a multiplexed measurement of different biomolecules in a single fiber could be an attractive approach.
We discuss the possibility of selectively sensitizing the inner surfaces of a suspended core fiber with two different
metallic nanoparticle types in monolayers for such multiplexed measurements.
We use the nonlinear optical property of GaAs to directly visualize the path of the near infrared incident laser light
coupled into individual nanowires. We fully illuminate with near infrared pulse laser untapered and tapered GaAs
nanowires grown via the Au-assisted vapor-liquid-solid mechanism. We record second-harmonic generation (SHG)
signals in the visible spectrum. In some nanowires, an interference pattern is observed and investigated in terms of
distances between the maxima of the SHG signal taking into account the effective refractive index in such sub
wavelength structures with radius below 90 nm. We propose a model to explain the periodicity of the maxima in the
SHG interference pattern. The theoretical model includes the waveguiding and the Mie scattering theories for obtaining
the 2π periodicity fitting well the experiments. Moreover, we also measure interferences in tapererd nanowires with a
radius down to 76 nm. The possible effect of the gold in non radiative recombination and the presence of the gold
particle at the tip of some nanowires are also discussed.
Noble metal nanoparticles interacting with electromagnetic waves exhibit the effect of localized surface plasmon resonance (LSPR) based on the collective oscillation of their conduction electrons. Local refractive index changes by a (bio) molecular layer surrounding the nanoparticle are important for a variety of research areas like optics and life sciences. In this work we demonstrate the potential of two applications in the field of molecular plasmonics, single nanoparticle sensors and nanoantennas, situated between plasmonics effects and the molecular world.
A newly emerging field in bioanalytics based on biomolecular binding detected label-free at single metal
nanoparticles is introduced. Thereby particles which show the effect of localized surface plasmon resonance
(LSPR) are used as plasmonic transducers. They change their spectroscopic properties (a band in the UV-VIS
range) upon binding of molecules. This effect is even observable at the single nanoparticle level using micro
spectroscopy and presents the base for a new field of single particle bioanalytics with the promise of highly
parallel and miniaturized sensor arrays. The paper describes this approach and shows first result from our work regarding the detection of DNA binding at single nanoparticle sensors.
Microstructured optical fibers (MOFs) represent a promising platform technology for new biosensing devices. Using
MOFs with adapted cavity diameters of about 20 to 30 μm, they can be used to carry the biofluids of analytical interest.
Such cavities with their walls coated by transducer material form in combination with adequate microfluidic chips a
platform for fully integrated next generation plasmonic devices. This paper describes the use of a dynamic chemical
nanoparticle layer deposition (NLD) technique to demonstrate the wet chemical deposition of gold and silver
nanoparticles (NP) within MOFs with longitudinal, homogenously-distributed particle densities. The plasmonic
structures were realized on the internal capillary walls of a three-hole suspended core fiber. Electron micrographs, taken
of the inside of the fiber holes, confirm the even distribution of the NP. With the proposed procedure fiber lengths of
several meters can be coated and afterwards cut up into small pieces of desired lengths. Accordingly, this procedure is
highly productive and makes the resulting MOF-based sensors potentially cost efficient. In proof-of-principle
experiments with liquids of different refractive indices, the dependence of the localized surface plasmon resonance
(LSPR) on the surroundings was confirmed. Comparing Raman spectra of NP coated and uncoated MOFs, each filled
with crystal violet, a significant signal enhancement demonstrates the usability of such functionalized MOFs for surfaceenhanced
Raman spectroscopy (SERS) experiments.
Metal nanoparticles exhibit a large potential for the development of innovative and cost-effective sensing devices. They
fulfill key requirements for biosensors such as the potential for miniaturization as well as for high parallelization, and they
are compatible with the molecular world for the required biofunctionalization approaches. Their optical properties based on
the localized surface plasmon resonance (LSPR) are well adjustable from the UV- to the infrared spectral range using
chemical synthesis. Due to the strong influence of the surrounding dielectrics on the resonant properties these particles offer
a high potential for sensing of minimal changes in the surrounding media. Additionally, plasmon nanoparticles can induce a
local field-enhancement and so a signal amplification such as for fluorescence or Raman-spectroscopy. In general, plasmon
nanoparticles are well suited as label or as transducer for different optical detection techniques. We will give an overview
about recent developments in this field, and will present different sensing strategies at single particle or ensemble level and
based on planar or fiber-based systems aiming for ultrasensitive point-of care applications in bioanalytics.
Charge carrier distribution changes in solid substrates induced by the presence of biomolecules have the potential as
sensoric principle. For a high surface-to-bulk ratio as in the case of nanostructures, this effect can be used for highly
sensitive bioanalytics.
Plasmonic nanosensors represent one possible implementation: The resonance wavelength of the conductive electron
oscillation under light irradiation is changed upon molecular binding at the structure surface. This change can be detected
by spectroscopic means, even on a single nanoparticle level using microspectroscopy.
Other examples are nanowires in electrodes gaps, either by metal nanoparticles arranged in a chain-like geometry or by
rod-like semiconductor nanowires directly bridging the gap. Molecules binding at the surface will lead to changes in the
electrical conductivity which can be easily converted into an electrical readout. The various geometries will be discussed
and their sensoric potential for an electrical detection demonstrated.
Nanoscale sensors have the potential for ultrasensitive and highly parallel bioanalytical applications. Bottom up methods
like gas-phase self assembly allow for the controlled and cost-efficient preparation of numerous functional units with
nanometer dimensions. Their use in sensoric instruments, however, requires the defined integration into sensoric setups
such as electrode arrays.
We show here how to use alternating electrical fields (dielectrophoresis DEP) in order to address this micro nano
integration problem. Nanoscale units such as metal nanoparticles or semiconductor nanowires are thereby polarized and
moved into the direction of higher electrical field gradients. As result, these particles bridge an electrode gap and can so
be used for electrical sensoric using the electrical resistance through this structure as value correlated to the presence of
molecules at the sensor surface. In order to achieve high selectivity, capture molecules (such as complementary DNA or
antibodies) are used.
The broad application of surface-enhanced Raman spectroscopy (SERS) is greatly hampered by the lack of reliable and
reproducible substrates; usually the activity of a given substrate has to be determined by time-consuming experiments
such as calibration studies or ultramicroscopy. To use SERS as a standard analytical tool, cheap and reproducible
substrates are required, preferably characterizable with a technique that does not interfere with the subsequent
measurements. Here, we introduce an innovative approach to produce low cost and large scale reproducible substrates
for SERS applications, which allows an easy and economical production of micropatterned SERS active surfaces based
on an enzyme induced growth of silver nanostructures. The special structural feature of the enzymatically deposited
silver nanoparticles prevents the breakdown of SERS activity even at high particle densities and exhibits a relationship
between electrical conductivity and resulting SERS activity of a given spot. This enables the prediction of the SERS
activity of the nanostructure ensemble and therewith the controllable and reproducible production of SERS substrates of
enzymatic silver nanoparticles on a large scale. Furthermore, the presented substrate shows a high reproducibility and is
appropriate for various applications.
Within this contribution, we demonstrate a combination of microarrays, microfluidics and SERS to enable a sequence
specific detection of DNA. In this combination, the microarray allows for the immobilisation of DNA sequences as well
as the removal of unbound DNA, microfluidics permit the automation of the process and SERS provides a highly
sensitive detection by means of an interaction between an analyte molecule and the enhanced electromagnetic field in the
proximity of metallic nanostructured surfaces such as spherical nanoparticles. With this setup, we are able to distinguish
between different complementary and non-complementary target sequences in one sample solution.
Within this contribution we convincingly demonstrate that the enhancement of the intrinsically weak Raman signals
through an interaction between an analyte molecule and enhanced electromagnetic fields in the vicinity of metallic
nanostructured surfaces is an extremely potent tool in bioanalytical science because such a SERS approach comprises
high sensitivity with molecular specificity. In particular innovative approaches to realize reproducible plasmonic
nanostructures i.e. SERS substrates like e.g. lithographically produced nanostructured gold surfaces or the defined
deposition of silver nanoparticles through an enzymatic reaction are introduced.
Localized surface plasmons (LSPs) are charge density oscillations caused by an interaction of the external
electromagnetic waves with the interface between metallic nanostructures (e.g. noble metal nanoparticles) and a
dielectric medium. Intensity and frequency of the resulting SP absorption bands are characteristic for the type of material
and depends on the size, shape and surrounding environments of the nanostructures. We have designed core/shellnanostructures
with a defined Au-core and increasing Ag-shell thickness as previously described [17]. We have used
AFM measurement and dark-field microscopy to characterize the nanoparticles, which were immobilized via silane
chemistry on glass substrates. The plasmon band of selected particles was investigated by single particle spectroscopy
(SPS) in transmission and reflection mode. Their potential as optical biosensor was demonstrated by immobilization of a
protein and a protein specific antibody leading to a refractive index change in the local environment of metal
nanoparticles, which causes a characteristic shift of the SP absorption band maximum.
The connection of biomolecules like DNA to a micro scale environment such as microarrays and Lab-on-a-chip systems
is an imminent task in biochip technology. Especially in Lab-on-a-chip systems microscopic forces are used to separate
the analyte from a complex mixture for further analysis [1]. In this contribution the sorting and manipulation of DNA
using dielectrophoresis (DEP) on micro structured chips was investigated [2]. DEP represents an interesting approach to
manipulate and control objects at the micro- [3, 4] and nanoscale range [5-7], and especially to position them at
controlled locations in microelectrode arrangements. It could be shown that DNA can be reversible arranged but also
permanently immobilized in micro scale electrode gaps. It was also demonstrated that it is possible to stretch and align
DNA from a single molecule level to high DNA concentration in a parallel manner between microelectrodes [8].
Furthermore DNA was stretched between moveable electrodes.
Although functional molecular constructs promise a variety of interesting properties in combination with parallel
realization and molecular precision, the utilization requires usually integration into the macroscopic world such as
electrodes or other technical environments. Dielectrophoresis (DEP) represents an interesting approach to manipulate
and control objects at the nanoscale, and especially to position them at controlled locations in microelectrode
arrangements. Over the years this technique was established in our group and is now able to arrange either metal
nanoparticles and/or DNA into these gaps in a highly reproducible manner. Microscopic tools were optimized in order to
be able to follow single particles/molecules during the process. This ability greatly improves the potential, because now
the key parameters can be easily tuned during live imaging of the controlled objects and their behavior. It was possible to
realize bridges of nanoparticles as well as of a few stretched DNA molecules on gold microelectrode structures at chip
surfaces. Moreover, DNA positioned by DEP in electrode gaps was metallized and the resulting metal nanostructure
characterized. Work is in process to combine the various units as well as processes in order to access more complex
functionalities.
Use of gold nanoparticles (NPs) as a contrast agent for medical imaging is shown to improve the efficiency of optoacoustic signal generation; this signal enhancement allows differentiation between different tissue types. This aspect of medical imaging is important when concerned early cancer detection. The present paper presents the results on the interaction process between the laser light and gold NPs, providing valuable information necessary for improved and more efficient NP synthesis. The attenuation of laser is studied for NP solutions of different geometrical characteristics and concentrations where the study is based on both optical and optoacoustic characterization techniques. First results show that the absorption and scattering are correlated by increasing the size of the nanoparticles between 5nm and 60nm. The optoacoustic signals we have been obtained demonstrate similar behavior for gold NP diameters of 5nm to 12nm.
KEYWORDS: Nanoparticles, Particles, Atomic force microscopy, Scanning electron microscopy, Spectroscopy, Metals, Chromium, Nanophotonics, Near field scanning optical microscopy, Near field optics
Apertures with diameters below the wavelength of light represent a nanoscale structure with interesting novel
properties. They are usually discussed in an array setting leading to integral optical (spectroscopic) effects. We present
here results obtained by a combination of such apertures (but investigated as an individual structure) with metal
nanoparticles. These nanoparticles are known to exhibit surface plasmon resonance. The optical effect resulting from the
combination of both structures were studied by a complementary ultra structural (AFM, SEM) and spectroscopic
characterization on the single aperture level, yielding insights into this promising novel nanophotonic element.
Use of gold nanoparticles (GNPs) as a contrast agent for medical imaging is shown to improve the efficiency of
optoacoustic signal generation; signal enhancement allows differentiation between different tissue types. This aspect of
medical imaging is important when concerned with early cancer detection. The present paper presents a comparative
analysis of two different optical techniques, optical transmission and optoacoustics, to define the different components
associated with the attenuation of light in GNPs. This attenuation of light is first studied for a pure absorber where the
results are shown to be in agreement for both optical methods, thus showing the effectiveness of the measurement
technique. A comparative analysis is also carried out on spherical GNPs which have been synthesized to have peak
absorption at the laser wavelength.
Computer calculation of optical properties of core-shell metal nanoparticles was made for some laser
wavelengths. Efficiency factors of absorption, scattering and extinction by spherical core-shell gold-silver and
silver-gold nanoparticles of the radiuses in the range 5-100 nm and for laser wavelengths 400, 532 and 800 nm
were calculated. Analysis of influence of optical parameters of metals, radiuses of core and thicknesses of shell
on optical properties of nanoparticles was made.
Metal (especially gold) nanoparticles exhibit unique electronic, optical, and catalytic properties. In order to utilize these
properties, an integration of the particles into technical setups such as a chip surface is helpful. We develop techniques to
use (bio) molecular tools in order to address and control the positioning of particles on microstructured chips. These
techniques are utilized for novel DNA detection schemes using optical or electrical principles. Plasmonic properties of
the particles and the combination of nano-apertures with particles are promising fields for further bioanalytical
developments.
On the other hand, methods for defined positioning of single molecules or molecular constructs in parallel approaches
are under development, in order to provide needed defined nanostructures for applications in nanoelectronics.
Connecting DNA with nanoparticles, metallization of DNA or positioning of individual DNA-structures over
microstructured electrode gap including subsequent metal particle binding are important steps in this direction. The
utilization of (bio) molecular tools and principles based on highly specific binding and self-assembly represent a
promising development in order to realize novel nanoparticle-based devices for bioanalytics, nano-optics and - electronics.
Based on their various interesting properties metal nanoparticles show the potential as analytical tool in electronic,
optical, and catalytical applications. The different properties depending on composition, shape, and size of the single
particles were utilized in many different approaches such as optics, magnetics and laser technology1.
We present a way for enzymatic deposition of silver nanoparticles and a bioanalytical application in DNA microarray
technology for this method. The technology consists of a microstructured chip with 10&mgr;m broad electrode gaps on the
surface and specially designed readout device2. In principle we immobilize gold nanoparticle-labelled DNA in a gap
between two electrodes. Afterwards a silver deposition on the bound gold nanoparticles generates a conductive layer
between the electrodes. The measured drop in the resistance serves as signal for the chip-based electrical detection of
DNA3.
To further optimize this system the gold nanoparticles as seed are replaced by the enzyme horseradish peroxidase. For a
better understanding of the enzymatically silver deposition process the formed silver particles were analyzed by
spectroscopic characterization on a single particle level. Further investigations of these particles by AFM and SEM
should give a hint to the connection between size/shape and the plasmonic properties at individual particles.
We describe a novel label-free method to analyse protein interactions on microarrays as well as in solution. By this
technique the time resolved native protein fluorescence in the UV is probed. The method is based on alterations of the
protein upon ligand binding, and, as a consequence, of alterations of the environment of the proteins' aromatic amino
acids. These amino acids act as internal probes, and as a result, the fluorescence lifetime of the proteins change due to
binding to a ligand partner such as another protein. We were able to demonstrate the feasibility of the method with many
compounds, including protein-protein, protein-antibody, protein-nucleic acid and protein-small ligand pairs. Unlike
to many other label-free techniques, the sensitivity of the method does not depend on the size of the counterbinding
ligand and therefore is particularly suitable for drug monitoring, when small molecules are involved.
Chemical approaches allow for the synthesis of highly defined metal heterostructures, such as core-shell nanoparticles.
As the material of metal nanoparticles determines the plasmon resonance-induced absorption band, the control of
particle composition results in control of the absorption maximum position.
Metal deposition on gold or silver nanoparticles was used to prepare core-shell particles with modified optical properties
with respect to monometal nanoparticles. UV-vis spectroscopy on solution-grown and immobilized particles was
conducted as ensemble measurements, complemented by single particle spectroscopy of selected structures. Increasing
layers of a second metal, connected to a dominant contribution of the shell material to the extinction spectrum, lead to a
shift in the absorption band. The extent of shell growth could be controlled by reaction time or the concentration of
either the metal salt or the reducing agent. Additional to the optical characterization, the utilization of AFM, SEM and
TEM yielded important information about the ultrastructure of the nanoparticle complexes.
Small metal nanostructures, especially gold and silver nanoparticles, are known for their interesting optical properties
caused by plasmons. Isotropic or anisotropic, homogeneous or heterogeneous metal nanoparticles provide a platform for
different highly defined functional units with interesting optical properties for applications such as waveguides or (in
combination with molecular parts) molecular beacons. We combined such nanoparticles with sub-wavelengths apertures
in metal films, and studied the effect of the presence of particles in these nanocavities on the topography as well as on
the optical behavior. Therefore, methods were developed that allow for a correlation of topography and optical
properties. The transmission through the holes was clearly influenced by the presence of nanoparticles. Combined with
the potential of designing the plasmonic properties of particles by customized diameters as well as composition using
core-shell techniques, this approach promises an interesting novel class of plasmonic nanostructures.
Chemical approaches allow for the synthesis of highly defined metal hetero-nanostructures, such as core-shell nanospheres. Because the material of metal nanoparticles determines the plasmon resonance-induced absorption band, the control of particle composition results in control of the position of the absorption band. Metal deposition on gold or silver nanoparticles yielded core-shell particles with modified optical properties. Besides the optical characterization, the utilization of AFM and TEM yielded important information about the morphology of the nanoparticle complexes. UV-vis spectroscopy on solution-grown and surface-grown particles was conducted as ensemble measurements in solution. Increasing layers of a second metal lead to a shift in the absorption band, and a shell diameter comparable to the original particle diameter leads to a predominant influence of the shell material. The extent of shell growth could be controlled by reaction time or the concentration of either the metal salt or the reducing agent.
Manipulation of material by optical means represents an emerging field with numerous applications. Especially in biology and medicine, the flexible and powerful potential of laser utilization holds great promises. For many applications, the resolution of the induced effects is essential. Besides focusing of the beam by various means, the use of sub-wavelengths nanoantenna could overcome this problem. The optical absorption of certain nanostructures is based on plasmon effects. We present studies of the use of metal (homogeneous gold or gold/silver core/shell systems) nanoparticles as antennas that convert the incident laser light into irreversible destructive effects. Based on the established field of DNA-conjugated nanoparticles, we investigated the sequence-specific attachment of DNA-nanoparticle complexes onto DNA with complementary sequences, in the state of double-stranded either isolated or metaphase chromosomal DNA. Important points were the adjustment of the absorption properties of the nanoparticles by control of their material composition (e.g., by addition of a silver layer to a gold core) and diameter. Another group of experiments studied chromosome-conjugated particles before and after laser treatment, in order to reveal the lateral extension of damages as well as the underlying mechanism.
DNA restriction is a basic method in today’s molecular biology. Besides application for DNA manipulation, this method is used in DNA analytics for 'restriction analysis'. Thereby DNA is digested by sequence specific restriction enzymes, and the length distribution of the resulting fragments is detected by gel electrophoresis. Differences in the sequence lead to different restriction patterns. A disadvantage of this standard method is the limitation to a small set of fixed sequences, so that the assay can not be adapted to any sequence of interest (e.g. SNP). We designed a scheme for DNA restriction in order to provide access to any desired sequence, based on laser light conversion on sequence-specific positioned metal nanoparticles. Especially gold nanoparticles are known for their interesting optical properties caused by plasmon resonance. The resulting absorption can be used to convert laser light pulses into heat, resulting in nanoparticle destruction. We work on the combination of this principle with DNA-modification of nanoparticles and the sequence-specific binding (hybridization) of these DNA-nanoparticle complexes along DNA molecules. Different mechanisms of light-conversion were studied, and the destructive effect of laser light on the nanoparticles and DNA is demonstrated.
Metal nanoparticles represent an interesting tool for bioanalytics. Due to their small size, attachment to biomolecules does not interfere significantly with specific molecular binding. Therefore particles can be applied as label in affinity assays (e.g., DNA hybridization), using setups with high parallelization. Beside this rather passive use of nanoparticles, these structures can also be utilized as 'nano antenna' for the conversion of laser light pulses into heat. Using DNA-modified particles sequence-specific bound to DNA, a novel restriction technique is in development that applies this conversion for local DNA destruction. Metal nanoparticles combine the ability for highly precise positioning (due to specific molecular binding) with the possibility of optical access in a bright-field mode. They exhibit an interesting potential for spanning the gap between the macroscopic technical environment and the molecular scale, thereby enabling a true integration of nanoscale constructs with today’s technology.
Sequence specific cutting of DNA is a standard method in molecular biology. This cutting is realized with enzymes which have a defined recognition sequence and cutting sequence. Therefore one can manipulate only sequences for which an enzyme is available. With current physical methods (AFM) any sequences can be cut, but the precise sequence specific and highly parallel cutting is not possible.
Near infrared (NIR) femtosecond laser systems have been used to optically knock out genomic regions of highly condensed DNA in human chromosomes as well as of single expanded (stretched) DNA molecules. Working with 80 MHz laser pulses at 800 nm of low 2 nJ pulse energy but at high TW/cm2 light intensities, multiphoton ionization and optical breakdown (OB) resulted in highly precise material ablation with sub-100 nm cut sizes. This is far below the diffraction-limited spot size. A minimum FWHM cut size of 65 nm was achieved in the case of the nanodissection of a laser-treated stretched λ-DNA (48kb) molecule which corresponded to 200 optically knocked out bases.
By the use of metal nanoparticles as energy coupling objects for fs laser radiation we expect a specific highly local destruction effect within the DNA molecule (cut). Thereby, a sequence-specific binding of DNA nanoparticle complexes along the target DNA is a fundamental condition. The effect of laser exposure on DNA and DNA-nanoparticle complexes are presented.
We adapted the nanoparticle-labeling technique from microscopical applications for DNA-chip detection. Nanoparticles can be detected by simple optical means, and exhibit a high stability. So alternative optical readout devices can be applied for the detection of specific DNA- binding on microstructured spots of complementary, surface- immobilized capture DNA. Several devices were tested, and the binding of gold-labeled DNA to complementary and non- complementary sequences was investigated using optical detection of the gold-labeled substrates before and after silver enhancement.
Nanojoule and sub-nanojoule 80 MHz femtosecond laser pulses at 750-850 nm of a compact titanium:sapphire laser have been used for highly precise nanoprocessing of DNA as well as of intracellular and intratissue compartments. In particular, a mean power between 15 mW and 100 mW, 170 fs pulse width, submicron distance of illumination spots and microsecond beam dwell times on spots have been used for multiphoton- mediated nanoprocessing of human chromosomes, brain and ocular intrastromal tissue. By focusing the laser beam with high numerical aperture focusing optics of the laser scan system femt-O-cut and of modified multiphoton scanning microscopes to diffraction-limited spots and TW/cm2 light intensities, precise submicron holes and cuts have been processed by single spot exposure and line scans. A minimum FWHM cut size below 70 nm during the partial dissection of the human chromosome 3 was achieved. Complete chromosome dissection could be performed with FWHM cut sizes below 200 nm. Intracellular chromosome dissection was possible. Intratissue processing in depths of 50 - 100micrometers and deeper with a precision of about 1micrometers including cuts through a nuclei of a single intratissue cell without destructive photo-disruption effects to surrounding tissue layers have been demonstrated in brain and eye tissues. The femt-O-cut system includes a diagnostic system for optical tomography with submicron resolution based on multiphoton- excited autofluorescence imaging (MAI) and second harmonic generation. This system was used to localize the intracellular and intratissue targets and to control the effects of nanoprocessing. These studies show, that in contrast to conventional approaches of material processing with amplified femtosecond laser systems and (mu) J pulse energies, nanoprocessing of materials including biotissues can be performed with nJ and sub-nJ high repetition femtosecond laser pulses of turn-key compact lasers without collateral damage. Potential applications include highly precise cell and embryo surgery, gene diagnostics and gene therapy, intrastromal refractive surgery, cancer therapy and brain surgery.
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