Localized surface plasmon resonance for biosensing lab-on-a-chip applications

In recent times, metallic nanoparticle plasmonics coupled with applications towards biosensing has gathered momentum to the point where commercial R&D are investing large resources in developing the so-called localized surface plasmon resonance (LSPR) biosensors. Conceptually, the main motivation for the research presented within this thesis is achievement of fully-operational LSPR biosensor interfaced with the state-of-the-art microfluidics, allowing for very precise control of sample manipulation and stable read-out. LSPR sensors are specifficaly engineered by electron beam lithography nanofabrication technique, where nanoparticle interactions are optimized to exhibit increased sensitivity and higher signal-to-noise ratio. However, the overall performance of LSPR lab-on-a-chip device depends critically on the biorecognition layer preparation in combination with surface passivation. As an introduction, the principles of plasmonic biosensing are identified encompassing both Surface Plasmon Resonance (SPR) and Localized SPR. Being successfully implemented into commercial product, the governing physics of SPR is compared to LSPR in chapter 1, together with advantages and disadvantages of both. Chapter 2 describes methods necessary for LSPR biosensor development, beginning with nano-fabrication methods, the modelling tool (COMSOL Multiphisics), while the basics of micro-fabrication in microfluidics conclude this chapter, where passive and active microfluidics networks are discerned. Particularly attractive optical properties are exhibited by closely-coupled nanoparticles (dimers), with the dielectric gap of below tens of nm, which were theoretically predicted to be very suitable as LSPR biosensing substrates. Chapter 3 is subjected to optical characterization (dependence on the size of the dielectric gap) of nanofabricated dimer arrays. The acquired data demonstrate the advantages of the nanofabrication methods presented in chapter 2 and the technique for fast and reliable determination of nanoparticle characteristic parameters. The initial biosensing-like experiments presented in chapter 4 (no integration with microfluidics) proved for the first time, the theoretical predictions of higher sensitivity, yielding additionally the specific response as function of analyte size and dielectric gap between nanoparticles. The overall response of different dimer arrays (various gaps) provides information about adopted conformation of analyte protein once immobilized. Broad resonances of dimers feature higher noise when employing them for the real-time LSPR biosensing. As a way to circumvent such problem, the feasibility of employing far-field interaction within the nanoparticle array to spectrally narrow resonance is investigated in chapter 5 by optimizing the array periodicity and introducing thin waveguiding layers. Finally, the concluding chapter 6 is dedicated to a full assembly of a Lab-on-a-chip (LOC) LSPR biosensor, starting with interfacing plasmonic substrates with compatible active microfluidic networks, allowing the precise sample delivery and multiplexing. The prototype device consisting of 8 individual sensors is presented with typical modes of operation. The bulk refractive index determination of various samples demonstrates the working principle of such device. Finally, various strategies of biorecognition layer formation are discussed within the on-going research

Towards single cell proteomics

This thesis focuses on different novel ideas and concepts in the area bioanalytics in order to develop the sensitivity and liquid handling towards the level of single cell proteomics. In biochemical sensors binding events are detected, when target molecules diffuse close enough to interact with specific recognition elements. To develop a fast and sensitive immunosensor, we benefit from short diffusion times and capillarity in microchannels. We fabricated an on-chip immunochemical surface assay which is performed within a microfluidic system. Using such a chip, the concentration of CRP in human blood serum was determined within eleven minutes. We were able to detect less than 1 ng/mL of CRP using only 1 microlitre of sample. To further reduce the sample volume towards single cells, we first structured surfaces with nanometer-sized patterns to separate, handle and culture individual cells. Pillar arrays with a height of 1 micrometer, aspect ratios of 1:5 and a top diameter of 120 nm were fabricated in silicon and were used as a master to produce a PDMS intermediate on which PLLA replicas were casted. At an inter-pillar distance of 200 nm, we could show how individual cells grow along the lines of cones replicated in PLLA. To handle the liquid content of individual cells and to detect single molecules within such heterogeneous analytes, we developed a method to prepare total content sample for electron microscopy. The method combines microfluidic-based in-line negative staining for TEM as well as desalting for mass measurements by STEM. The main advantages are the lossless sample preparation by liquid contact writing of micro-patterns on EM grids and excellent staining at physiological pH. To detect low molecular weight single molecules label-free with a very high specificity, we propose to immobilize arrays of single DARPins on a very flat surface and to discriminate their bound and unbound state by height measurements using AFM. Arrays of immobilization islands for single DARPins were fabricated by EUV-IL, EBL and a newly developed direct immobilization method. By EUV-IL and a glancing angle metal deposition step 20 nm sized gold immobilization islands could be fabricated. By EBL and thermal annealing, arrays of 5 nm sized gold islands have been achieved. These islands are in the size range of single DARPin molecules. To functionalize only the gold islands with DARPins, the surrounding silicon dioxide surface has to be protected against non-specific DARPin adsorption. However PEG molecules for efficient passivation are often to long and the small immobilization islands might be buried by PEG. Therefore we used a photoresist mask and a chemical linker to directly immobilize single DARPins onto silicon dioxide. On the same chip, the pattern size of the mask was varied and besides several mm sized lines full of DARPins, arrays of single immobilized DARPins could be produced. On such arrays, single binding events between DARPins and their corresponding target proteins were detected and bound and unbound DARPins could be discriminated. The developed methodologies and the engineered surfaces are promising tools for the analysis towards single cell proteomics and their further development might result in valuable methods for systems biology. Keywords: microfluidics, pillar arrays, nano-dot array, single cell, cell growth, AFM, TEM, STEM, XIL, EBL, GLAD, thermal annealing, immunoassay, DARPin

Surface Plasmon Resonance for Biosensing

The rise of photonics technologies has driven an extremely fast evolution in biosensing applications. Such rapid progress has created a gap of understanding and insight capability in the general public about advanced sensing systems that have been made progressively available by these new technologies. Thus, there is currently a clear need for moving the meaning of some keywords, such as plasmonic, into the daily vocabulary of a general audience with a reasonable degree of education. The selection of the scientific works reported in this book is carefully balanced between reviews and research papers and has the purpose of presenting a set of applications and case studies sufficiently broad enough to enlighten the reader attention toward the great potential of plasmonic biosensing and the great impact that can be expected in the near future for supporting disease screening and stratification

Organic lasers: recent developments on materials, device geometries, and fabrication techniques

MCG acknowledges financial support through the ERC Starting Grant ABLASE (640012) and the European Union Marie Curie Career Integration Grant (PCIG12-GA-2012-334407). AJCK acknowledges financial support by the German Federal Ministry for Education and Research through a NanoMatFutur research group (BMBF grant no. 13N13522).Organic dyes have been used as gain medium for lasers since the 1960s, long before the advent of today’s organic electronic devices. Organic gain materials are highly attractive for lasing due to their chemical tunability and large stimulated emission cross section. While the traditional dye laser has been largely replaced by solid-state lasers, a number of new and miniaturized organic lasers have emerged that hold great potential for lab-on-chip applications, biointegration, low-cost sensing and related areas, which benefit from the unique properties of organic gain materials. On the fundamental level, these include high exciton binding energy, low refractive index (compared to inorganic semiconductors), and ease of spectral and chemical tuning. On a technological level, mechanical flexibility and compatibility with simple processing techniques such as printing, roll-to-roll, self-assembly, and soft-lithography are most relevant. Here, the authors provide a comprehensive review of the developments in the field over the past decade, discussing recent advances in organic gain materials, which are today often based on solid-state organic semiconductors, as well as optical feedback structures, and device fabrication. Recent efforts toward continuous wave operation and electrical pumping of solid-state organic lasers are reviewed, and new device concepts and emerging applications are summarized.PostprintPeer reviewe

Micro- and nano-devices for electrochemical sensing

Electrode miniaturization has profoundly revolutionized the field of electrochemical sensing, opening up unprecedented opportunities for probing biological events with a high spatial and temporal resolution, integrating electrochemical systems with microfluidics, and designing arrays for multiplexed sensing. Several technological issues posed by the desire for downsizing have been addressed so far, leading to micrometric and nanometric sensing systems with different degrees of maturity. However, there is still an endless margin for researchers to improve current strategies and cope with demanding sensing fields, such as lab-on-a-chip devices and multi-array sensors, brain chemistry, and cell monitoring. In this review, we present current trends in the design of micro-/nano-electrochemical sensors and cutting-edge applications reported in the last 10 years. Micro- and nanosensors are divided into four categories depending on the transduction mechanism, e.g., amperometric, impedimetric, potentiometric, and transistor-based, to best guide the reader through the different detection strategies and highlight major advancements as well as still unaddressed demands in electrochemical sensing