H I B I S C U S. Executive Summary

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1 Executive Summary H I B I S C U S HIBISCUS (Hybrid Integrated BIophotonic Sensors Created by Ultrafast laser Systems) is an FP6 STREP project within the IST Priority. It is coordinated by Prof. Giulio Cerullo, Physics Department, Politecnico di Milano, Italy ( giulio.cerullo@fisi.polimi.it) and involves the following contractors: University of Hannover (Germany) University of Twente (the Netherlands) University of Hull (U.K.) Lionix BV (the Netherlands) HighQLaser GmbH (Austria) Zebra Bioscience BV (the Netherlands) The project website is: Thanks to the introduction of miniaturized analysis systems, the life sciences are undergoing a revolution similar to that triggered by integrated microelectronic systems, which gave birth to the Information Society. Such systems, also known as Lab-On- Chips (LOCs), are devices aiming at squeezing on a single substrate the functionalities of an entire biological laboratory. Through a network of microfluidic channels, very small volumes (nano- to picoliters) of biological samples can be transported, mixed, separated, and analyzed with significant advantages in terms of sensitivity, speed of analysis, low sample and reagent consumption, and measurement automation and standardization. Applications of LOCs range from basic science (genomics and proteomics), to chemical synthesis and drug development, to high-throughput medical and biochemical analysis, to environmental monitoring and detection of chemical and biological threats. A key unsolved problem in LOC technology is the development of an integrated onchip optical detection system for the biomolecules flowing in the microchannels. Traditional free-space optical detection uses bulk optical components, such as lenses, mirrors and microscope objectives, which require accurate optical alignment to the microfluidic channels and frustrate many of the advantages of LOC miniaturization, in particular strongly limiting device portability and preventing field applications. The main goal of the HIBISCUS project is the development of a new technology, based on high-intensity femtosecond laser pulses, for the fabrication of optical waveguides and photonic devices in glass LOCs, to implement integrated optical sensors. This integrated approach to optical sensing has many advantages over the traditional freespace configurations. The waveguides are precisely aligned to the channels during 2

2 fabrication, resulting in stable setups with enhanced sensitivity; parallel excitation at multiple points of the microfluidic flow can be performed, enabling to follow a chemical reaction in real time; a robust and portable device can be obtained by pigtailing the waveguides to optical fibers. Femtosecond laser writing has several advantages with respect to standard techniques for optical waveguide fabrication: (i) it is a direct technique that does not require any photolithographic process or clean room environment, enabling the integration of photonic components into pre-existing LOCs; (ii) it is a three-dimensional technique, allowing the fabrication of waveguides at different depths within the sample and thus the implementation of unique sensor configurations. A sketch of the basic idea of integrating microfluidic channels with femtosecond laser written waveguides is shown in the following figure. An additional goal of HIBISCUS is the use of the femtosecond laser technology also for the direct fabrication of the microfluidic channels. This is a more ambitious objective but, if achieved, will allow a single production line for LOCs, based on an high-power femtosecond laser, that can manufacture both the microfluidic channels and the optical waveguides. Achievement of the goals of HIBISCUS critically depends on the availability of innovative femtosecond laser sources with suitable characteristics in terms of wavelength, pulse energy, average power and repetition rate. In addition these laser sources need to be sufficiently compact, reliable and user-friendly to enable industrial application in a microfabrication line. A research line of HIBISCUS is thus devoted to the development of advanced diode-pumped femtosecond laser sources. The unique integration of photonics and microfluidics, enabled by the inscription of optical waveguides on the LOCs, will pave the way to a wealth of novel functionalities. In the project we concentrate on two prototypical devices: i) Microreactors for chemical synthesis of polypeptides: such devices offer, thanks to the small reaction volumes and high surface-to-volume ratios, a level of reaction control that is not attainable in conventional bench-top reactors, resulting in higher product yield and selectivity. Integration of photonic devices on the microreactor chip will enable direct in-situ monitoring of the reaction products, thereby greatly simplifying the measurement and improving its sensitivity. 3

3 ii) Analysis of biomolecules separated by microchip capillary electrophoresis (MCE): MCE is a powerful technique for the separation of a variety of biomolecules exploiting the different transit times in a microfluidic channel. We propose to integrate the femtosecond written optical waveguide in a MCE setup, to sensitively detect in situ the separated biomolecules, in particular DNA fragments with diagnostic relevance, after suitable fluorescence labelling. During the whole duration of the project, all the partners worked synergically towards the objectives and reached most of the planned goals. The main achievements have been: Scaling the average power of femtosecond lasers: different amplification approaches have been used in order to scale the average power of femtosecond lasers; chirped pulse amplification in a rod-type fiber was found to be very promising and output powers in the 10 W range (10 J, 1 MHz) were achieved. As an alternative, regenerative amplification using an high power laser head based on bulk crystals was developed, with comparable performance, and has become a commercial product, as shown below.. Microfabrication of complex photonic devices using a spatial beam shaper: by using a liquid-crystal based spatial beam shaper, it is possible to achieve multiple foci with dynamically controlled distance and depth, allowing the fabrication of complex three-dimensional photonic devices such as couplers and splitters, as shown in the picture below. Laser fabrication of 3D Mach-Zehnder Interferometers (MZIs): optimization of the waveguide fabrication process has resulted in improved refractive index contrast, up to n This has allowed the fabrication of curved waveguides and more complex devices such as splitters and Mach-Zehnder interferometers. Exploiting the 3D capabilities of the femtosecond laser writing technique, it was possible to integrate the MZIs with microfluidic channels, achieving spatially selective label-free sensing. An important application of this device is in situ monitoring of the efficiency of chemical synthesis in a microreactor chip. 4

4 Optofluidic platform for Microchip Capillary Electrophoresis (MCE): a high sensitivity and high resolution platform has been developed using integrated optical waveguides for detection of separated biomolecules, in particular DNA fragments. The platform achieved sensitivity down to 130 fm and, thanks to modulation frequency encoding, virtually infinite resolution. Separation and integrated optical detection of diagnostically relevant DNA fragments: the optimized optofluidic platform has been applied to solving real-life problems, such as detection of multiple sequences of DNA fragments with diagnostic relevance. Such sequences, obtained by selective amplification of portions of a gene, are separated and simultaneously detected in a multiplex fashion. In particular, the genes related to breast cancer gene and Diamond- Blackfan anemia have been detected. 5

5 In conclusion, the HIBISCUS project has demonstrated a new technology for the integration of optical and microfluidic components. This promises to solve one of the main open issues of LOC technologies, i.e. integration of on-chip detection, and paves the way to a new generation of portable, low-cost devices with applications ranging from analytical science to chemical synthesis in microreactors. The results of HIBISCUS are being actively exploited by the SMEs involved in the project, which are transferring the new knowledge into commercial products: in particular HighQLaser has upgraded the performance of its line of femtosecond laser sources, introducing a new system with an order of magnitude higher average power; Lionix will produce the LOCs with integrated optical sensing for specific applications, such as chemical microreactors and bioanalyzers; Zebra will use the LOCs together with its line of innovative diagnostic products for multiplex biomarker analysis of diseases based on microchip capillary electrophoresis. HIBISCUS has also generated a large amount of basic new knowledge, which has been extensively disseminated by the university partners through publications and conference presentations. 6