Digital detection of exosomes by interferometric imaging

Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics. The small size (30–100 nm) along with low refractive index contrast of exosomes makes direct characterization and phenotypical classification very difficult. In this work we present a method based on Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows multiplexed phenotyping and digital counting of various populations of individual exosomes (>50 nm) captured on a microarray-based solid phase chip. We demonstrate these characterization concepts using purified exosomes from a HEK 293 cell culture. As a demonstration of clinical utility, we characterize exosomes directly from human cerebrospinal fluid (hCSF). Our interferometric imaging method could capture, from a very small hCSF volume (20 uL), nanoparticles that have a size compatible with exosomes, using antibodies directed against tetraspanins. With this unprecedented capability, we foresee revolutionary implications in the clinical field with improvements in diagnosis and stratification of patients affected by different disorders.This work was supported by Regione Lombardia and Fondazione Cariplo through POR-FESR, project MINER (ID 46875467); Italian Ministry of Health, Ricerca Corrente. This work was partially supported by The Scientific and Technological Research Council of Turkey (grant #113E643). (Regione Lombardia; 46875467 - Fondazione Cariplo through POR-FESR, project MINER; Italian Ministry of Health, Ricerca Corrente; 113E643 - Scientific and Technological Research Council of Turkey)Published versio

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

Denoising of Fluorescence Image on the Surface of Quantum Dot/Nanoporous Silicon Biosensors

In the process of biological detection of porous silicon photonic crystals based on quantum dots, the concentration of target organisms can be indirectly measured via the change in the gray value of the fluorescence emitted from the quantum dots in the porous silicon pores before and after the biological reaction on the surface of the device. However, due to the disordered nanostructures in porous silicon and the roughness of the surface, the fluorescence images on the surface contain noise. This paper analyzes the type of noise and its influence on the gray value of fluorescent images. The change in the gray value caused by noise greatly reduces the detection sensitivity. To reduce the influence of noise on the gray value of quantum dot fluorescence images, this paper proposes a denoising method based on gray compression and nonlocal anisotropic diffusion filtering. We used the proposed method to denoise the quantum dot fluorescence image after DNA hybridization in a Bragg structure porous silicon device. The experimental results show that the sensitivity of digital image detection improved significantly after denoising

Development of nanophotonic biosensor platform towards on-chip liquid biopsy

Liquid biopsy has the potential to enable diagnosis, prognosis, and monitoring of some diseases at an early stage using body fluids from patients. This minimally invasive, label-free detection method is less likely to harm the cell’s viability through binding to the surface protein. Smart integration of liquid biopsy designs with microfluidics on a single chip will lead to a considerable reduction in the detection time (due to controlled diffusion length), and the volumes of sample, agent and reagent, and the limit of detection. Optical label-free biosensors are a powerful tool to analyze biomolecular interactions and have been widely studied in the field of biomedical and biological science and engineering. Label-free detection enables direct measurement of key characteristic properties of the chemical compound, DNA molecule, peptide, protein, virus, or cell, while eliminating experimental uncertainty induced by the effect of the label on molecular conformation, thus reducing the time and effort required for bioassay. Existing optical label-free biosensors suffer from three limitations, including low detection sensitivity, slow molecules mass transfer, and poor throughput. The goal of this dissertation is to overcome these limitations through the development of a novel and efficient modality towards liquid biopsy-based bioassay with increased detection sensitivity, speed, and throughput. To increase the detection sensitivity, we investigate the optical bound states in the continuum (BIC) of slotted high-contrast grating (sHCG) structures. We demonstrate that the sHCG support BICs and high-Q resonant modes, and the slot position can be utilized to tune and optimize the linewidth of the high-Q resonances. To overcome the mass-transfer limitation and reduce the assay time, we propose a lateral flow-through optical biosensor integrating high-contrast gratings and microfluidics on a silicon-on-insulator platform. The biosensor design allows reducing the diffusion length to a submicron scale and enhancing direct interactions between the analytes and sensing structures. Finally, we develop a high-throughput, label-free exosome vesicles (EVs) detection microarray formed on a photonic crystal (PC) biosensor surface. We design and implement a hyperspectral imaging approach to quantify the antibody and EV absorptions on the PC-based microarray consisting of a panel of seven antibodies specific to multiple membrane receptors of the target EVs. We validate that the EV microarray by adopting it to detect EVs released by macrophages for the analysis of immune responses

A nanostructured Fabry-Perot interferometer for label-free biodetection

A polymer nanostructured Fabry-Perot interferometer (FPI) based biosensor has been developed, fabricated, and tested. Different from a conventional FPI, this nanostructured FPI has a layer of Au-coated nanopores inside its cavity. The Au-coated nanostructure layer offers significant enhancement of optical transducing signals due to the localized surface Plasmon resonance (L-SPR) effect. Compared to a traditional FPI for label-free biosensing applications, the polymer nanostructured FPI based biosensor offers increased sensing surface area, extended penetration depth of the excitation light, and amplification of optical transducing signals. Using a nanostructured FPI, measurements taken had great improvements in free spectral range (FSR), finesse, and contrast of optical transducing signals over a traditional FPI without any device performance optimization. Several chemicals have been evaluated using the prototype device. Fourier Transform has been performed on the measured optical signals to facilitate the analysis of the transducing signals. Control experiments incubating immunoglobulin G (IgG) on a gold surface confirmed the small affinity of IgG to the Au-coated sensing surface. Then, using fluorescent images, shifts of interference fringes for IgG and BSA interaction were indirectly confirmed. Using this technical platform, the immobilization of capture proteins (Protein A) on the nanostructure layer and their binding with IgG was monitored in real time, resulting in the direct observation of the shift in interference fringes of the optical transducing signals. The results showed that the detection of limit (DOL) for this kind of biosensor should be lower than 10 pg/mL, which is approximately 55 fIVI of IgG, for IgG-Protein A binding. Control experiments were performed to confirm that the biodetection is only specific to Protein A and IgG recognition. After the proof-of-concept demonstration for IgG-Protein A binding, the ultrasensitive label-free detection of a cancer biomarker free prostate specific antigen (fPSA) using this kind of nanostructured FPI was carried out. Experiments found that the DOL of the fabricated nanostructured FPI microchip for f-PSA is about 5 pg/mL and the upper detection range for f-PSA can be dynamically changed by varying the amount of mAb immobilized on the sensing surface. Control experiments have also demonstrated that the immunoassay protocol used shows excellent specificity and selectivity, suggesting great potential to detect cancer biomarkers at trace levels in biofluids. Given its nature of low cost, simple operation, and batch fabrication capability, the nanostructured FPI microchip based platform could provide an ideal technical tool for point-of-care diagnostic applications and anti-cancer drug screening and discovery

BioMEMS

As technological advancements widen the scope of applications for biomicroelectromechanical systems (BioMEMS or biomicrosystems), the field continues to have an impact on many aspects of life science operations and functionalities. Because BioMEMS research and development require the input of experts who use different technical languages and come from varying disciplines and backgrounds, scientists and students can avoid potential difficulties in communication and understanding only if they possess a skill set and understanding that enables them to work at the interface of engineering and biosciences. Keeping this duality in mind throughout, BioMEMS: Science and Engineering Perspectives supports and expedites the multidisciplinary learning involved in the development of biomicrosystems. Divided into nine chapters, it starts with a balanced introduction of biological, engineering, application, and commercialization aspects of the field. With a focus on molecules of biological interest, the book explores the building blocks of cells and viruses, as well as molecules that form the self-assembled monolayers (SAMs), linkers, and hydrogels used for making different surfaces biocompatible through functionalization. The book also discusses: Different materials and platforms used to develop biomicrosystems Various biological entities and pathogens (in ascending order of complexity) The multidisciplinary aspects of engineering bioactive surfaces Engineering perspectives, including methods of manufacturing bioactive surfaces and devices Microfluidics modeling and experimentation Device level implementation of BioMEMS concepts for different applications. Because BioMEMS is an application-driven field, the book also highlights the concepts of lab-on-a-chip (LOC) and micro total analysis system (μTAS), along with their pertinence to the emerging point-of-care (POC) and point-of-need (PON) applications

The development and optimisation of a novel microfluidic immunoassay platform for point of care diagnostics

Protein biomarkers are important diagnostic tools for detection of non-communicable diseases, such as cancer and cardiovascular conditions. In order to be used as diagnostic tools they need to be detected at very low concentrations in biological samples (e.g. whole blood, serum or urine). This has been currently performed in central laboratories using expensive, bulky equipment and time consuming assays. [Continues.

Photon Management on a Photonic Crystal Platform

A multilayered dielectric structure, namely a one dimensional photonic crystal (1DPC), is proposed as a suitable platform for photon management, due to the low absorption of the dielectric materials. When properly designed, a 1DPC can sustain surface electromagnetic modes called Bloch Surface Waves (BSWs). In this PhD Thesis it is shown how light coupled to BSW can be focused or guided by means of ultrathin polymeric refractive structures directly patterned on the surface. Moreover, by patterning the surface with surface relief gratings, far-field radiation can be efficiently coupled to the surface modes, thus providing an enhanced electromagnetic field at the truncation interface of the 1DPC. By shaping the grating in a circular symmetry, light can be in-plane focused into a sub-wavelength spot. The same structure can be used to re-shape the radiation pattern of dipolar emitters. It is shown that an emitter lying on the surface of the 1DPC couples to the photonic structure and the fluorescence radiated couple with the surface modes. The so called BSW-coupled fluorescence propagates along the surface with low losses and a well-defined wavevector. By means of surface diffraction gratings properly designed, fluorescence can be extracted along any direction, thus improving the fluorescence collection with no need of high numerical aperture optics or critical alignements. A novel method for evaluating the enhancement gained with such photonic structures on the extraction efficiency is proposed. Such method is capable of providing at the same time spatial resolution, angular resolution and spectral resolution. A biosensing experiment to detect small amounts of labeled proteins is provided, in order to show the sensing capabilities of the photonic structure