Formation and characterization of an ideal excitation beam geometry in an optofluidic device

An optimal excitation beam shape is necessary to perform reliable flow cytometric analysis but has so far not been implemented in a photonic-microfluidic integrated (i.e. optofluidic) device. We have achieved this feature by integrating a 1D lens system with planar waveguides and microfluidic channel on a substrate using one patterning material via a one-shot process. In this paper, we report the method of design and the performance of specifically formed excitation regions shaped to be ideal for reducing double detections, improving SNR, and for reliable detection in a flow cytometry application. Demonstration of different sizes via changes to lens design shows the ability to control the width of the shaped beam according to a targeted detection

Optofluidic components for biomedical applications

In the last years the term Optofluidics has popped up in a large amount of scientific publications. The goal of this thesis is to prove Optofluidics as an applicable, powerful tool for distinct biological and medical applications. In the main part of this work, an optofluidic sensing unit for the determination of liquid concentration is presented. In an experimental evaluation, the sensor was tested with different dissolved molecules including phosphate and lactose. Depending on the analyte, a smallest detectable change in concentration of 10 to 40mmol/L was determined. Besides the sensing device, a new subfield of optofluidics was introduced for the very first time. Thermo-optofluidics deals with the integration of thermal elements in optofluidic systems. Compared to solely solid based realizations more than twice the theoretical switching frequency can be achieved. In conclusion, the applicability of optofluidics for different sensor as well as actuator systems was experimentally proven. All the devices were fabricated applying low-cost, rapid-prototyping fabrication techniques which allow the realization of cost-effective hand-held devices

Development of an integrated microspectrometer using arrayed waveguide grating (AWG)

With non-invasive properties and high sensitivities, portable optical biosensors are extremely desirable for point-of-care (POC) applications. Lab-on-a-chip technology such as microfluidics has been treated as an ideal approach to integrate complex sample processing and analysis units with optical detection elements. Spectroscopic sensing (such as fluorescence, Raman and absorption spectroscopy) remains the most highly developed, widely applied, optical technique. However, conventional spectroscopic sensing systems still rely on bulky and expensive dispersive components such as spectrophotometers in a well established laboratory. The work in this thesis is to develop an integrated dispersive component in combination with a microfluidic chip, providing a portable and inexpensive platform for on-chip spectroscopic sensing. In this study, an arrayed waveguide grating (AWG) design developed for telecommunication is re-engineered and utilized to realise a compact, dispersive optical component operating in the visible spectral region. The AWG devices operating in the visible region (λ_c=680 nm) are designed and fabricated with flame hydrolysis deposited (FHD) silica waveguide material. The micro-spectrometer in this proof of concept study has a small (1 cm x 1 cm) footprint and 8 output channels centred on different wavelengths. A series of fabrication issues and challenges are investigated and discussed for the specific AWG device. Subsequently, a sample cuvette is formed by using lithographic technique and dry etching process. Following this, a PDMS chip with microfluidic channels is bonded with the AWG device, leading to an integrated AWG-microfluidic platform. To the best of the author’s knowledge, this is the first work to integrate a visible AWG device and a microfluidic chip towards spectroscopic sensing. The monolithic integrated AWG microspectrometer–microfluidic platform is demonstrated for fluorescence spectroscopic analysis. Signals from the output channels detected on a camera chip can be used to re-create the complete fluorescence spectrum of an analyte. By making fluorescence measurements of (i) mixed quantum dot solutions, (ii) an organic fluorophore (Cy5) and (iii) the propidium iodide (PI)-DNA assay, the results obtained illustrate the unique advantages of the AWG platform for simultaneous, quantitative multiplex detection and its capability to detect small spectroscopic shifts. Although the current system is designed for fluorescence spectroscopic analysis, in principle, it can be implemented for other types of analysis, such as Raman spectroscopy. Fabricated using established semiconductor industry methods, this miniturised platform holds great potential to create a handheld, low cost biosensor with versatile detection capability. Also, the AWG device design is modified with focusing properties that enable localised spectroscopic measurements. Micro-beads based, multiplexed fluorescence detection is performed with the AWG + CCD system and the results have demonstrated capabilities of using the adapted AWG device for localised, multiplexed fluorescence detections, opening up potential applications in the field of cell sorting and single cell analysis. Furthermore, the AWG-microfluidic device is investigated for absorption spectroscopy measurement. As a test system, the pH dependence of the absorption spectra of bromophenol blue is measured to illustrate how an AWG device could be used as a colorimetric pH sensor. Overall, it is believed that the AWG technology holds great potential to realise a compact, integrated spectroscopic biosensor for point-of-care applications

Dual-Mode Electro-Optical Techniques for Biosensing Applications : A Review

The monitoring of biomolecular interactions is a key requirement for the study of complex biological processes and the diagnosis of disease. Technologies that are capable of providing label-free, real-time insight into these interactions are of great value for the scientific and clinical communities. Greater understanding of biomolecular interactions alongside increased detection accuracy can be achieved using technology that can provide parallel information about multiple parameters of a single biomolecular process. For example, electro-optical techniques combine optical and electrochemical information to provide more accurate and detailed measurements that provide unique insights into molecular structure and function. Here, we present a comparison of the main methods for electro-optical biosensing, namely, electrochemical surface plasmon resonance (EC-SPR), electrochemical optical waveguide lightmode spectroscopy (EC-OWLS), and the recently reported silicon-based electrophotonic approach. The comparison considers different application spaces, such as the detection of low concentrations of biomolecules, integration, the tailoring of light-matter interaction for the understanding of biomolecular processes, and 2D imaging of biointeractions on a surface

Polymer-Based Microfluidic Devices for High Throughput Single Molecule Detection: Applications in Biological and Drug Discovery

The realization of high throughput sample processing has become a primary ambition in many research applications with an example being high throughput screening (HTS), which represents the first step in the drug discovery pipeline. Microfluidics is a viable platform for parallel processing of biochemical reactions to increase data production rates due to its ability to generate fluidic networks with a high number of processors over small footprints suitable for optical imaging. Single-molecule detection (SMD) is another technology which has emerged to facilitate the realization of high throughput data processing afforded by its ability to eliminate sample processing steps and generate results with high statistical accuracy. A combination of microfluidics and SMD with wide-field fluorescence detection provides the ability to monitor biochemical reactions in a high throughput format with single-molecule sensitivity. In this dissertation, the integration of these techniques was presented for HTS applications in drug discovery. An ultra-sensitive fluorescence detection system with a wide field-of-view (FoV) was constructed to transduce fluorescence signatures from single chromophores that were electrokinetically transported through a series of tightly packed fluidic channels poised on poly(methylmethacrylate), PMMA and contained within the FoV of a laser detection system. The system was used to monitor biochemical reactions at the single-molecule level in a continuous-flow format. Enhancement in sampling-throughput was demonstrated by constructing a high density fluidic network for parallel analysis of multiple biochemical assays. In another development, the ability to enhance single-molecule sensitivity in a flow-based biochemical assay was investigated using a novel cyclic olefin copolymer (COC) planar waveguide embedded in PMMA and situated orthogonal to multiple fluidic channels. This design allowed for fluorescence detection from multiple fluidic channels using evanescent excitation and a wide FoV fluorescence detection system for parallel readout. Results from these technologies were presented as well as their applications in drug discovery for increasing data production rates and quality. An approach toward monitoring the efficacy of therapeutic agents, which is important in clinical evaluation of drug potency in the drug discovery process, was also considered, by designing a microfluidic system with integrated conductivity sensor for label-free enumeration of isolated tumor cells from clinical samples

Silicon-Based Integrated Label-Free Optofluidic Biosensors: Latest Advances and Roadmap

By virtue of the well-developed micro- and nanofabrication technologies and rapidly progressing surface functionalization strategies, silicon-based devices have been widely recognized as a highly promising platform for the next-generation lab-on-a-chip bioanalytical systems with a great potential for point-of-care medical diagnostics. Herein, an overview of the latest advances in silicon-based integrated optofluidic label-free biosensing technologies relying on the efficient interactions between the evanescent light field at the functionalized surface and specifically bound analytes is presented. State-of-the-art technologies demonstrating label-free evanescent wave-based biomarker detection mainly encompass three device configurations, including on-chip waveguide-based interferometers, microring resonators, and photonic-crystal-based cavities. Moreover, up-to-date strategies for elevating the sensitivities and also simplifying the sensing processes are discussed. Emerging laboratory prototypes with advanced integration and packaging schemes incorporating automatic microfluidic components or on-chip optoelectronic devices lead to one significant step forward in real applications of decentralized diagnostics. Besides, particular attention is paid to currently commercialized label-free optical bioanalytical models on the market. Finally, the prospects are elaborated with several research routes toward chip-scale, low-cost, highly sensitive, multi-functional, and user-friendly bioanalytical systems benefiting to global healthcare. © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinhei

Single-Molecule Detection of Unique Genome Signatures: Applications in Molecular Diagnostics and Homeland Security

Single-molecule detection (SMD) offers an attractive approach for identifying the presence of certain markers that can be used for in vitro molecular diagnostics in a near real-time format. The ability to eliminate sample processing steps afforded by the ultra-high sensitivity associated with SMD yields an increased sampling pipeline. When SMD and microfluidics are used in conjunction with nucleic acid-based assays such as the ligase detection reaction coupled with single-pair fluorescent resonance energy transfer (LDR-spFRET), complete molecular profiling and screening of certain cancers, pathogenic bacteria, and other biomarkers becomes possible at remarkable speeds and sensitivities with high specificity. The merging of these technologies and techniques into two different novel instrument formats has been investigated. (1) The use of a charge-coupled device (CCD) in time-delayed integration (TDI) mode as a means for increasing the throughput of any single molecule measurement by simultaneously tracking and detecting single-molecules in multiple microfluidic channels was demonstrated. The CCD/TDI approach allowed increasing the sample throughput by a factor of 8 compared to a single-assay SMD experiment. A sampling throughput of 276 molecules s-1 per channel and 2208 molecules s-1 for an eight channel microfluidic system was achieved. A cyclic olefin copolymer (COC) waveguide was designed and fabricated in a pre-cast poly(dimethylsiloxane) stencil to increase the SNR by controlling the excitation geometry. The waveguide showed an attenuation of 0.67 dB/cm and the launch angle was optimized to increase the depth of penetration of the evanescent wave. (2) A compact SMD (cSMD) instrument was designed and built for the reporting of molecular signatures associated with bacteria. The optical waveguides were poised within the fluidic chip at orientation of 90° with respect to each other for the interrogation of single-molecule events. Molecular beacons (MB) were designed to probe bacteria for the classification of Gram +. MBs were mixed with bacterial cells and pumped though the cSMD which allowed S. aureus to be classified with 2,000 cells in 1 min. Finally, the integration of the LDR-spFRET assay on the cSMD was explored with the future direction of designing a molecular screening approach for stroke diagnostics

Three dimensional optofluidic devices for manipulation of particles and cells

Optical forces offer a powerful tool for manipulating single cells noninvasively. Integration of optical functions within microfluidic devices provides a new freedom for manipulating and studying biological samples at the micro scale. In the pursuit to realise such microfluidic devices with integrated optical components, Ultrafast Laser Inscription (ULI) fabrication technology shows great potential. The uniqueness and versatility of the technique in rapid prototyping of 3D complex microfluidic and optical elements as well as the ability to perform one step integration of these elements provides exciting opportunities in fabricating novel devices for biophotonics applications. The work described in this thesis details the development of three dimensional optofluidic devices that can be used for biophotonics applications, in particular for performing cell manipulation and particle separation. Firstly, the potential of optical forces to manipulate cells and particles in ULI microfluidic channels is investigated. The ability to controllably displace particles within a ULI microchannel using a waveguide positioned orthogonal to it is explored in detail. We then prototype a more complex 3D device with multiple functionalities in which a 3D optofluidic device containing a complex microchannel network and waveguides was used for further investigations into optical manipulation and particle separation. The microfluidic channel network and the waveguides within the device possess the capability to manipulate the injected sample fluid through hydrodynamic focusing and optically manipulate the individual particles, respectively. This geometry provided a more efficient way of investigating optical manipulation within the device. Finally, work towards developing a fully optimised 3D cell separator device is presented. Initial functional validation was performed by investigating the capability of the device to route particles through different outlet channels using optical forces. A proof of concept study demonstrates the potential of the device to use for cell separation based on the size of the cells. It was shown that both passive and active cell separation is possible using this device

PRINCIPLES FOR NEW OPTICAL TECHNIQUES IN MEDICAL DIAGNOSTICS FOR mHEALTH APPLICATIONS

Medical diagnostics is a critical element of effective medical treatment. However, many modern and emerging diagnostic technologies are not affordable or compatible with the needs and conditions found in low-income and middle-income countries and regions. Resource-poor areas require low-cost, robust, easy-to-use, and portable diagnostics devices compatible with telemedicine (i.e. mHealth) that can be adapted to meet diverse medical needs. Many suitable devices will need to be based on optical technologies, which are used for many types of biological analyses. This dissertation describes the fabrication and detection principles for several low-cost optical technologies for mHealth applications including: (1) a webcam based multi-wavelength fluorescence plate reader, (2) a lens-free optical detector used for the detection of Botulinum A neurotoxin activity, (3) a low cost micro-array reader that allows the performance of typical fluorescence based assays demonstrated for the detection of the toxin staphylococcal enterotoxin (SEB), and (4) a wide-field flow cytometer for high throughput detection of fluorescently labeled rare cells. This dissertation discusses how these technologies can be harnessed using readily available consumer electronics components such as webcams, cell phones, CCD cameras, LEDs, and laser diodes. There are challenges in developing devices with sufficient sensitivity and specificity, and approaches are presented to overcoming these challenges to create optical detectors that can serve as low cost medical diagnostics in resource-poor settings for mHealth