Development of optical microchip sensor for biomolecule detection

Optical sensors play vital roles in many applications in today’s world. Photonic technologies used to design and engineer optical sensing platforms can provide distinctive advantages over conventional detection techniques. For instance, when compared to electronic and magnetic sensing systems, optical sensors require physically smaller equipment and have the capability for delivering more analytical information (e.g. spectroscopic signatures). In addition, demand for low-cost and portable bio-analyte detections is a growing area for applications in healthcare and environmental fields. Among other factors to achieve reliable results in terms of selectivity and sensitivity is key for the detection of bio-analytes with analytical relevance. Commonly used bio-analytical techniques (e. g. high performance liquid chromatography) have been appropriately designed based on qualitative and quantitative analysis. However, the requirement of expensive equipment, and complexity of procedures (e.g. biomolecule labelling, calibrations, etc.) restrict the board applicability and growth of these techniques in the field of biosensing. Optical sensors tackle these problems because they enable selective and sensitive detection of analytes of interest with label-free, real-time, and cost-effective processes. Among them, optical interferometry is increasingly popular due label-free detection, simple optical platforms and low-cost design. An ideal substrate with high surface area as well as biological/chemical stability against degradation can enable the development of advanced analytical tools with broad applicability. Nanoporous anodic alumina has been recently envisaged as a powerful platform to develop label-free optical sensors in combination with different optical techniques. This thesis presents a high sensitive label-free biosensor design combining nanoporous anodic alumina (NAA) photonic structures and reflectometric interference spectroscopy (RIfS) for biomedical, food and agricultural applications. NAA is a suitable optical sensing platform due to its optical properties; a high surface area; its straightforward, scalable, and cost-competitive fabrication process, and its chemical and mechanical stability towards biological environments. Our biosensor enables real-time screening of any absorption and desorption event occurring inside the NAA pores. A proper selection of bio-analytes were able to be detected using this platform which offers unique feature in terms of simplicity and accuracy. The most relevant components of this thesis are categorised as below: 1. Self-ordered NAA fabrication and detection of an enzymatic analyte as a biomarker for cancer diagnosis: Fabrication of NAA photonic films using two step electrochemical anodization and chemical functionalisation. Detection of trace levels of analyte enzyme and its quantification by selective digestion. The NAA photonic film with the enzyme acts as a promising combination for a real-time point-of-care monitoring system for early stages of disease. 2. NAA rugate filters used to establish the binding affinity between blood proteins and drugs: Design, fabrication, and optimisation of NAA anodization parameters using sinusoidal pulse anodization approach (i.e. anodization offset and anodization period) to produce rugate filter photonic crystals that provide two comparative sensing parameters. Establishment of highly sensitive and selective device capable for drug binding assessments linked to treating a wide range of medical conditions. 3. NAA bilayers and food bioactive compound detection: Design, fabrication, and optimisation of NAA anodization parameters (i.e. anodization time and number of anodization steps) to obtain NAA bilayered photonic structures that display the effective response of NAA geometry with different types of nano-pore engineering. The photonic properties of the NAA bilayer were studied at each layer of nano-structure under specific binding of human serum albumin and quercetin as target agent. 4. Single nucleotide polymorphism (SNP) detection: The design and implementation of a Ligation-Rolling Circle Amplification assay to detect a single nucleotide polymorphism associated with insecticide resistance in a pest beetle species, Tribolium castaneum. This proof-of-concept SNP detection assay has the potential to provide a method compatible with a biosensor platform such as NAA. This demonstrates the first step towards the potential development of a genotyping biosensor, and a real-world application of insect insecticide resistance monitoring. The results presented in this thesis are expected to enable innovative developments on NAA sensing technology that could result in highly sensitive and selective detection systems for a broad range of bio-analytes detections.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Chemical Engineering, 201

Review on carbon-derived, solid-state, micro and nano sensors for electrochemical sensing applications

The aim of this review is to summarize the most relevant contributions in the development of electrochemical sensors based on carbon materials in the recent years. There have been increasing numbers of reports on the first application of carbon derived materials for the preparation of an electrochemical sensor. These include carbon nanotubes, diamond like carbon films and diamond film-based sensors demonstrating that the particular structure of these carbon material and their unique properties make them a very attractive material for the design of electrochemical biosensors and gas sensors. Carbon nanotubes (CNT) have become one of the most extensively studied nanostructures because of their unique properties. CNT can enhance the electrochemical reactivity of important biomolecules and can promote the electron-transfer reactions of proteins (including those where the redox center is embedded deep within the glycoprotein shell). In addition to enhanced electrochemical reactivity, CNT-modified electrodes have been shown useful to be coated with biomolecules (e.g., nucleic acids) and to alleviate surface fouling effects (such as those involved in the NADH oxidation process). The remarkable sensitivity of CNT conductivity with the surface adsorbates permits the use of CNT as highly sensitive nanoscale sensors. These properties make CNT extremely attractive for a wide range of electrochemical sensors ranging from amperometric enzyme electrodes to DNA hybridization biosensors. Recently, a CNT sensor based fast diagnosis method using non-treated blood assay has been developed for specific detection of hepatitis B virus (HBV) (human liver diseases, such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma caused by hepatitis B virus). The linear detection limits for HBV plasma is in the range 0.5–3.0 μL−1 and for anti- HBVs 0.035–0.242 mg/mL in a 0.1 M NH4H2PO4 electrolyte solution. These detection limits enables early detection of HBV infection in suspected serum samples. Therefore, non-treated blood serum can be directly applied for real-time sensitive detection in medical diagnosis as well as in direct in vivo monitoring. Synthetic diamond has been recognized as an extremely attractive material for both (bio-) chemical sensing and as an interface to biological systems. Synthetic diamond have outstanding electrochemical properties, superior chemical inertness and biocompatibility. Recent advances in the synthesis of highly conducting nanocrystalline-diamond thin films and nano wires have lead to an entirely new class of electrochemical biosensors and bio-inorganic interfaces. In addition, it also combines with development of new chemical approaches to covalently attach biomolecules on the diamond surface also contributed to the advancement of diamond-based biosensors. The feasibility of a capacitive field-effect EDIS (electrolyte-diamond-insulatorsemiconductor) platform for multi-parameter sensing is demonstrated with an O-terminated nanocrystalline-diamond (NCD) film as transducer material for the detection of pH and penicillin concentration. This has also been extended for the label-free electrical monitoring of adsorption and binding of charged macromolecules. One more recent study demonstrated a novel bio-sensing platform, which is introduced by combination of a) geometrically controlled DNA bonding using vertically aligned diamond nano-wires and b) the superior electrochemical sensing properties of diamond as transducer material. Diamond nanowires can be a new approach towards next generation electrochemical gene sensor platforms. This review highlights the advantages of these carbon materials to promote different electron transfer reactions specially those related to biomolecules. Different strategies have been applied for constructing carbon material-based electrochemical sensors, their analytical performance and future prospects are discussed

Development of Nanopore Based Label-Free Optical Sensors

Optical sensors play an important role and are employed for more application in today’s lives than ever before. As an example, optical sensing systems have established strong footprints in quality assurance (i.e. ensuring safe levels of controlled substances in drinks and food products) and self-diagnostics (e.g. detection and quantification of glucose in blood or pregnancy assessment test). Conventional optical sensor read-out is based on colour change or signal variation (i.e. absorbance or fluorescence intensity) of the label/tag molecule (i.e. dyes) conjugated to the capture probes. However, requirement of expensive and sophisticated labels/tags and instruments, skilled personnel, and other inherent issues with the dye labels (i.e. short lift-time, concentration dependent quenching etc.) limit their broader application. Therefore, label-free sensors present a great advantage over their label based counterparts. Label-free optical sensors rely on changes in physical properties (e.g. refractive index: n) of the sensing substrate occurring during a binding event. Nanoporous substrates (i.e. porous silicon, nanoporous anodic alumina, and titania nanotubes arrays) prepared by simple and scalable electrochemical anodization process in combination with spectroscopy techniques that can be realized with miniature spectrometer (e.g. reflectometric interference spectroscopy, localized surface plasmon resonance spectroscopy etc.) can potentially overcome the limitations of label-based sensing systems. However, comprehensive and extensive fundamental research must be carried out in this field to make this technology feasible, efficient, reliable, sensitive, selective and inexpensive. In this scenario, this thesis puts forward a novel combination of nanoporous anodic alumina (NAA) and reflectometric interference spectroscopy (RIfS) for developing a highly sensitive detection system for environmental and biomedical sensing application. High surface area, modifiable surface chemistry, and optical activity make NAA a perfect substrate for highly sensitive label-free detection using RIfS platform. Moreover, the geometric features of NAA can be controlled during the fabrication process to generate more complex optical photonic structures. The simplicity and versatility of this combination (i.e. NAA and RIfS) also allows for real-time monitoring of the release of drug for the NAA pores. The most relevant features of this thesis are: 1. NAA Substrate and its Surface Chemistry: Optimization and fabrication of NAA substrate with straight pores using two step electrochemical anodization process. Optimization and modification of NAA surface chemistry with different silanes (e.g. amine terminated or thiol terminated) to impart it selectivity and specificity towards analyte molecules. 2. NAA Photonic Structures: Designing, fabrication, and optimization of NAA pore geometry (i.e. effective medium) to obtain photonic structures (i.e. Rugate filters) that display highly sensitive and selective detection capabilities in combination with RIfS. Comparison of sensing capabilities of NAA straight pores with NAA photonic structures. 3. Flow Cells for Sensing: Designing and fabrication of different types of flow cells including bulk and micro-fluidic flow cell that can accommodate NAA substrates. 4. Sensing of Heavy Metal Ions: Modification of NAA substrate with silane which specifically bind to heavy metal ions such as gold (III) and mercury (II) ions in model solvent (i.e. mili-Q water) and real-life samples (i.e. tap water and water from river Torrens in Adelaide, South Australia). 5. RIfS vs Photoluminescence using NAA Substrate: Sensing properties of NAA studied using RIfS and photoluminescence as the detection techniques, when analytes were introduced into NAA pores under non-specific and specific binding conditions. 6. Real-time Drug Release Monitoring from NAA Pores: NAA pores can act as nanocontainers which can hold substantial amounts of drug molecules that can be released over an extended period of time. NAA loaded with model drug acts as a way of measuring the drug release from its pores in real-time and under dynamic flow conditions using RIfS. The results presented in this thesis are expected to open doors for the development of more innovative and complex NAA photonic structures and surface chemistries aimed to produce highly sensitive and selective miniature, portable, and point-of-care analysis system for various industrial, environmental, and biomedical applications.Thesis (Ph.D.) -- University of Adelaide, School of Chemical Engineering, 201

Fluorescent nanoparticles for sensing

Nanoparticle-based fluorescent sensors have emerged as a competitive alternative to small molecule sensors, due to their excellent fluorescence-based sensing capabilities. The tailorability of design, architecture, and photophysical properties has attracted the attention of many research groups, resulting in numerous reports related to novel nanosensors applied in sensing a vast variety of biological analytes. Although semiconducting quantum dots have been the best-known representative of fluorescent nanoparticles for a long time, the increasing popularity of new classes of organic nanoparticle-based sensors, such as carbon dots and polymeric nanoparticles, is due to their biocompatibility, ease of synthesis, and biofunctionalization capabilities. For instance, fluorescent gold and silver nanoclusters have emerged as a less cytotoxic replacement for semiconducting quantum dot sensors. This chapter provides an overview of recent developments in nanoparticle-based sensors for chemical and biological sensing and includes a discussion on unique properties of nanoparticles of different composition, along with their basic mechanism of fluorescence, route of synthesis, and their advantages and limitations

Present and future of surface-enhanced Raman scattering

The discovery of the enhancement of Raman scattering by molecules adsorbed on nanostructured metal surfaces is a landmark in the history of spectroscopic and analytical techniques. Significant experimental and theoretical effort has been directed toward understanding the surface-enhanced Raman scattering (SERS) effect and demonstrating its potential in various types of ultrasensitive sensing applications in a wide variety of fields. In the 45 years since its discovery, SERS has blossomed into a rich area of research and technology, but additional efforts are still needed before it can be routinely used analytically and in commercial products. In this Review, prominent authors from around the world joined together to summarize the state of the art in understanding and using SERS and to predict what can be expected in the near future in terms of research, applications, and technological development. This Review is dedicated to SERS pioneer and our coauthor, the late Prof. Richard Van Duyne, whom we lost during the preparation of this article

Roadmap on semiconductor-cell biointerfaces.

This roadmap outlines the role semiconductor-based materials play in understanding the complex biophysical dynamics at multiple length scales, as well as the design and implementation of next-generation electronic, optoelectronic, and mechanical devices for biointerfaces. The roadmap emphasizes the advantages of semiconductor building blocks in interfacing, monitoring, and manipulating the activity of biological components, and discusses the possibility of using active semiconductor-cell interfaces for discovering new signaling processes in the biological world

Recent Progress in Optical Sensors for Biomedical Diagnostics

In recent years, several types of optical sensors have been probed for their aptitude in healthcare biosensing, making their applications in biomedical diagnostics a rapidly evolving subject. Optical sensors show versatility amongst different receptor types and even permit the integration of different detection mechanisms. Such conjugated sensing platforms facilitate the exploitation of their neoteric synergistic characteristics for sensor fabrication. This paper covers nearly 250 research articles since 2016 representing the emerging interest in rapid, reproducible and ultrasensitive assays in clinical analysis. Therefore, we present an elaborate review of biomedical diagnostics with the help of optical sensors working on varied principles such as surface plasmon resonance, localised surface plasmon resonance, evanescent wave fluorescence, bioluminescence and several others. These sensors are capable of investigating toxins, proteins, pathogens, disease biomarkers and whole cells in varied sensing media ranging from water to buffer to more complex environments such as serum, blood or urine. Hence, the recent trends discussed in this review hold enormous potential for the widespread use of optical sensors in early-stage disease prediction and point-of-care testing devices.DFG, 428780268, Biomimetische Rezeptoren auf NanoMIP-Basis zur Virenerkennung und -entfernung mittels integrierter Ansätz

Hybrid point-of-care devices for visual detection of biomarkers and drugs

Early diagnostics is a crucial part of clinical practice offering a rapid and convenient way to investigate and quantify the presence of key biomarkers related to specific pathologies and increasing the chance of successful treatments. In this regard, point-of-care testing (POCT) shows several advantages enabling simple and rapid analyses, allowing for real-time results, and permitting home testing. Metallic nanoparticles (NPs), like gold NPs (AuNPs), can be beneficially integrated into POC devices thanks to their tunable plasmonic properties which provide a naked-eye read-out. Moreover, the high sensitivity of NPs enables the detection of biomarkers in non-invasive fluids where the concentrations are typically low. These biofluids, like saliva and urine, are functionally equivalent to serum in reflecting the physiological state of the body, whilst they are easier to handle, collect, and store. In this thesis, I first reported the design and development of a colorimetric strategy based on the morphological change of multibranched plasmonic AuNPs, aimed at detecting glucose in saliva. The sensing approach relied on a target-induced reshaping process which involves the oxidation of the NP tips and the transformation into a spherical shape, characterized by a naked-eye detectable blue-to-pink color change. The platform proved to be beneficial in the early and non-invasive diagnosis of hyperglycemia. The successful technological transfer on a solid substrate paved the way for the realization of a dipstick prototype for home testing. Then, the strategy was adapted to other biomarkers, leading to the development of a multiplexing test for the simultaneous detection of three salivary analytes (cholesterol, glucose, and lactate). This multiplexing assay enabled to save reagents, costs, and time, whilst increasing the overall clinical value of the test. Exploiting the microfluidics applied on a paper sheet, I realized a monolithic and fully integrated POC device, through a low-cost and fast CO2 laser cutter. The platform showed excellent selectivity and multiplexing ability, with negligible interferences. The second part of my thesis was focused on the development of POC devices for the detection of anticancer drug contaminations in water solutions and urine samples. Antiblastic agents have revealed high toxicity for the exposed healthcare workers who prepare and administer these drugs in occupational environments. Hence, continuous monitoring is highly required, and POCT shows tremendous potential in this context. With this aim, I realized a lateral-flow (LF) device for the assessment of doxorubicin contamination, using the fluorescent properties of the drug for naked-eye detection. The pharmacological recognition of the DNA probe was exploited to overcome the lack of anti-doxorubicin antibodies. The highly sensitive strategy was successfully adapted to a real urine sample, without resorting to complex pretreatment procedures. Then, I developed a competitive LF device for the detection of methotrexate (MTX). AuNPs were employed as the label molecules and the pharmacological competition of folic acid and MTX for the capture enzyme was exploited as the recognition mechanism, instead of costly antibodies. Despite the sensitivity requires further improvements, the strategy showed fast and reliable results, demonstrating a high potential for workers’ safety control

The development of molecularly imprinted polymers for sensor and colorimetric assay applications

The development of devices capable of sensing the presence or absence of molecules is a staple of the modern day world, with fields such as healthcare, forensics, agriculture and industrial processing relying upon biosensors to operate. The presented work sets focus on the use of Molecularly Imprinted Polymers (MIPs) as receptor elements in biosensing applications, demonstrating how these synthetic alternatives to traditional affinity reagents are of value. The thesis initially gives a detailed overview of the current MIP landscape, before determining areas of the field that are currently underdeveloped. The ensuing research highlights how these areas can be built upon, deploying MIPs for drug analysis and antibiotic detection. To this end, the use of MIPs in conjugation with a thermal biosensing platform is presented before shifting the research towards the use of these synthetic receptors in simple colorimetric assays designed for the rapid analysis of compounds

Hydrogel microparticles for biosensing

Due to their hydrophilic, biocompatible, and highly tunable nature, hydrogel materials have attracted strong interest in the recent years for numerous biotechnological applications. In particular, their solution-like environment and non-fouling nature in complex biological samples render hydrogels as ideal substrates for biosensing applications. Hydrogel coatings, and later, gel dot surface microarrays, were successfully used in sensitive nucleic acid assays and immunoassays. More recently, new microfabrication techniques for synthesizing encoded particles from hydrogel materials have enabled the development of hydrogel-based suspension arrays. Lithography processes and droplet-based microfluidic techniques enable generation of libraries of particles with unique spectral or graphical codes, for multiplexed sensing in biological samples. In this review, we discuss the key questions arising when designing hydrogel particles dedicated to biosensing. How can the hydrogel material be engineered in order to tune its properties and immobilize bioprobes inside? What are the strategies to fabricate and encode gel particles, and how can particles be processed and decoded after the assay? Finally, we review the bioassays reported so far in the literature that have used hydrogel particle arrays and give an outlook of further developments of the field. Keywords: Hydrogel; Biosensor; Microparticle; Multiplex assayNovartis Institutes of Biomedical Research (Presidential Fellowship)Novartis Institutes of Biomedical Research (Education Office)National Cancer Institute (U.S.) (Grant 5R21CA177393-02)National Science Foundation (U.S.) (Grant CMMI-1120724)Institute for Collaborative Biotechnologies (Grant W911NF-09-0001)United States. Army Research Offic