Confocal laser scanning microscopy of nanoparticles applied to immunosorbent assays

The aim of this project was to demonstrate and develop a confocal readout method for fluorescent immunosorbent assays and investigate its potential advantages in comparison to traditional immunoassays. The key point of a confocal immunosorbent assay is the ability to detect the thin layer of immunoassay in the presence of unbound fluorescent reagents without washing the overlayer. Heterogeneous and homogeneous sandwich immunoassays of human IgG model were demonstrated successfully followed by the use of an empirical decomposition method for quantitative separation of the signals of the thin fluorescent assay layer from the overlayer. The detection limits for the homogeneous and heterogeneous formats of the model were 2.2 and 5.5 ng/ml, respectively. The application of confocal microscopy in kinetic analysis of the antigen-antibody reaction of the human IgG model was studied for homogeneous and heterogeneous formats and two fluorescent labels antibodies (FITC and QDs)

Doctor of Philosophy

dissertationA majority of the functions in biological systems are mediated by specific interactions of cellular proteins. Such interactions also involve other biomolecules like antibodies, RNA and DNA, small molecules sometimes referred to as drugs, etc. A detailed understanding of functional proteomics necessitates the need for detection and quantification of such specific biochemical reactions with greater speed and precision. The primary biosensing technology that is employed for detecting these biological interactions optically and with good sensitivity and reproducibility is based on Surface Plasmon Resonance (SPR). In this work, we aim at utilization of chemical signal processing techniques in microfluidic chips to produce SPR measurements with higher signal-to-noise ratio (SNR), shorter measurement times, and lower reagent volumes than those of conventional SPR systems like BIAcore, ProteOn, etc. The drawbacks of conventional methods are discussed and schemes based on signal processing in frequency domain are applied to minimize the influence of spurious signals that affect the measurement accuracy. With the choice of applied excitation signal, a 100-fold improvement in SNR has been achieved. Similarly, with alteration of signal postprocessing methodology, we have reported a 10-fold faster dual-slope method that can be employed for a variety of methods are discussed and schemes based on signal processing in frequency domain are applied to minimize the influence of spurious signals that affect the measurement accuracy. With the choice of applied excitation signal, a 100-fold improvement in SNR has been achieved. Similarly, with alteration of signal postprocessing methodology, we have reported a 10-fold faster dual-slope method that can be employed for a variety of the microchip that uses less than a hundred nanoliter of reagent volume for bio-characterization. Discrete liquid droplets are synthesized in an ordered fashion to carry out the bioreaction that conventionally utilizes reagent volumes ranging from a few hundred microliters to a few milliliters

Micro- and Nanofluidics for Bionanoparticle Analysis

Bionanoparticles such as microorganisms and exosomes are recoganized as important targets for clinical applications, food safety, and environmental monitoring. Other nanoscale biological particles, includeing liposomes, micelles, and functionalized polymeric particles are widely used in nanomedicines. The recent deveopment of microfluidic and nanofluidic technologies has enabled the separation and anslysis of these species in a lab-on-a-chip platform, while there are still many challenges to address before these analytical tools can be adopted in practice. For example, the complex matrices within which these species reside in create a high background for their detection. Their small dimension and often low concentration demand creative strategies to amplify the sensing signal and enhance the detection speed. This Special Issue aims to recruit recent discoveries and developments of micro- and nanofluidic strategies for the processing and analysis of biological nanoparticles. The collection of papers will hopefully bring out more innovative ideas and fundamental insights to overcome the hurdles faced in the separation and detection of bionanoparticles

A Rapid and Ultra-sensitive Biosensing Platform based on Tunable Dielectrophoresis for Robust POC Applications

With the ongoing pandemic, there have been increasing concerns recently regarding major public health issues such as abuse of organophosphorus compounds, pathogenic bacterial infections, and biosecurity in agricultural production. Biosensors have long been considered a kernel technology for next-generation diagnostic solutions to improve food safety and public health. Significant amounts of effort have been devoted to inventing novel sensing mechanisms, modifying their designs, improving their performance, and extending their application scopes. However, the reliability and selectivity of most biosensors still have much to be desired, which holds back the development and commercialization of biosensors, especially for on-site and point-of-care (POC) usages. Herein, we introduce an innovative two-phase sensing strategy based on tunable AC electrokinetics and capacitive sensing. By dividing the detection process into a sensitivity-priority step and a selectivity-priority step, the specificity and sensitivity of a biosensor can be significantly improved. A capacitive POC aptasensor is fabricated for the implementation of the 2-phase detection and a quasi-single-cell level detection of limit together with an excellent selectivity is achieved simultaneously. The sensor is capable of handling real-world clinic samples without sophisticated pretreatment. Just after a simple one-step dilution, the developed sensor can detect bacteria no less than 2~3 bacteria/10 µL in raw milk samples within 100 s. Alongside whole bacteria detection, the biosensor can also detect endotoxin, the lipopolysaccharide, in bovine serum samples, with a limit of detection of 10 pg/mL. The biosensor is low-cost and easy to use. This work not only demonstrates a biosensor with significant advantages in sensitivity, selectivity and assay time but also opens up a new horizon for further research of all affinity-based biosensors

Microchips for Isothermal Amplification of RNA : Development of microsystems for analysis of bacteria, virii and cells

The goal of the present work was to develop a microchip for amplification and detection of mRNA by employing nucleic acid sequence-based amplification (NASBA) technology. The technology platform should in principle be adaptable for any clinical analysis using mRNA or ssDNA as a target. To demonstrate the microchip functionality, identification of human papillomavirus (HPV) type 16, the etiological agent for cervical cancer has been used. The work shows for the first time successful real-time amplification and detection employing NASBA in microsystem formats using custom-made instruments. The first silicon-glass chips contained reaction chambers of 10 nl and 50 nl, which decreased the NASBA reaction volume by a factor of 2000 and 400, respectively. Further, experiments employing cyclic olefin copolymer (COC) microchips for simultaneous amplification and detection, automatically distributed the sample into 10 parallel reaction channels with detection volumes of 80 nl. In order to detect the simultaneous amplification in the reaction channels, a second custom-made optical detection system with increased sensitivity, heat regulation and an automatic non-contact pumping mechanism, was made. Dilution series of both artificial HPV 16 oligonucleotides and SiHa cell lines showed that the detection limits for the microchips were comparable to those obtained for experiments performed in conventional routine-based laboratory-systems. For experiments related to the development of a self-contained microchip for NASBA, the detection volume was increased to 500 nl due to the advantage of an increased fluorescence signal. For the NASBA reaction, biocompatible surfaces are critical. It was not possible to amplify any target in microchips with native silicon or COC surfaces. Adsorption measurements indicated clearly that fluorescently labelled mouse IgG bound non-specifically to the hydrophobic native COC surfaces, while PEG coated COC surfaces showed adequate protein resistance. Of the coatings tested for the COC microchips, surfaces modified with PEG showed the best biocompatibility. Successful amplification was obtained with silicon microchips when the surfaces were modified with either SigmaCote™ or SiO2. In order to integrate the NASBA reagents on chip, a thorough evaluation of the reagents to be spotted and dried was performed. Because of the limited number of microchipsavailable, it was necessary to map the most critical parameters on macroscale prior to transfer to the microscale. The DMSO and sorbitol enclosed in the standard NASBA reaction mixture were difficult to dry, and therefore it was necessary to add these compounds to the oligonucleotides or the sample of extracted nucleic acids before the sample was applied on the amplification chip. The standard NASBA reagents consist of the two main solutions, mastermix and enzymes, in addition to the sample. Both the mastermix and the enzymes were stable only when spotted and dried separately. Protectants, such as PEG and trehalose were essential for recovery of enzymatic activity after drying on macroscale. The times for diffusion of modified molecular beacons in dried mastermix and of fluorescently labelled mouse IgG in the dried enzyme solution were ~60 seconds and ~10 minutes, respectively. So far, only dried enzymes with 0.05% PEG protectant have been successfully amplified on chip. Successful amplification using a rehydrated mastermix on microchip still remains. Optimal design and fabrication methods of the microchips were found to be crucial for chip performance. Rough surfaces do not only create background noise for the optical measurements, but it also contributes to generation of bubbles and problems related to manipulation of the sample within the channel network. The silicon microchips were manufactured with optically smooth surfaces. However, low surface roughness was not easily obtained for the COC microchips. Of the fabrication methods evaluated, it was the injection moulded chips which showed the smoothest surfaces, closely followed by the hot embossed chips. Milled and laser ablated chips produced the roughest surfaces. A novel non-contact pumping mechanism based on on-chip flexible COC membranes, combined with actuation pins in the surrounding instrument, was tested and evaluated. The mechanism enabled metering, isolation and movement of nanoliter sized sample plugs in parallel reaction channels. The COC chips with integrated pumps were able to simultaneously move parallel sample plugs along the reaction channels in four different positions. Each reaction channel contained a set of 4 actuation chambers in order to obtain metering, isolation and movement of the sample plug into the detection area. The pump accuracy depended on the evaporation of sample and the deformation of the COC membranes. The results presented in this work are promising with regard to the development of a complete integrated and self-contained mRNA amplification microchip for multi-parallel target testing of clinical samples

Engineering single-molecule, nanoscale, and microscale bio-functional materials via click chemistry

To expand the design envelope and supplement the materials library available to biomaterials scientists, the copper(I)-catalyzed azide-alkyne cycloaddition (CuCAAC) was explored as a route to design, synthesize and characterize bio-functional small-molecules, nanoparticles, and microfibers. In each engineered system, the use of click chemistry provided facile, bio-orthogonal control for materials synthesis; moreover, the results provided a methodology and more complete, fundamental understanding of the use of click chemistry as a tool for the synergy of biotechnology, polymer and materials science. Fluorophores with well-defined photophysical characteristics (ranging from UV to NIR fluorescence) were used as building blocks for small-molecule, fluorescent biosensors. Fluorophores were paired to exhibit fluorescence resonant energy transfer (FRET) and used to probe the metabolic activity of carbazole 1,9a-dioxygenase (CARDO). The FRET pair exhibited a signicant variation in PL response with exposure to the lysate of Pseudomonas resinovorans CA10, an organism which can degrade variants of both the donor and acceptor fluorophores. Nanoparticle systems were modified via CuCAAC chemistry to carry affinity tags for CARDO and were subsequently utilized for affinity based bioseparation of CARDO from crude cell lysate. The enzymes were baited with an azide-modified carbazolyl-moiety attached to a poly(propargyl acrylate) nanoparticle. Magnetic nanocluster systems were also modified via CuCAAC chemistry to carry fluorescent imaging tags. The iron-oxide nanoclusters were coated with poly(acrylic acid-co-propargyl acrylate) to provide a clickable surface. Ultimately, alternate Cu-free click chemistries were utilized to produce biohybrid microfibers. The biohybrid microfibers were synthesized under benign photopolymerization conditions inside a microchannel, allowing the encapsulation of viable bacteria. By adjusting pre-polymer solutions and laminar flow rates within the microchannel, the morphology, hydration, and thermal properties of the fibers were easily tuned. The methodology produced hydrogel fibers that sustained viable cells as demonstrated by the encapsulation and subsequent proliferation of Bacillus cereus and Escherichia coli communities

Microfluidics and Nanofluidics Handbook

The Microfluidics and Nanofluidics Handbook: Two-Volume Set comprehensively captures the cross-disciplinary breadth of the fields of micro- and nanofluidics, which encompass the biological sciences, chemistry, physics and engineering applications. To fill the knowledge gap between engineering and the basic sciences, the editors pulled together key individuals, well known in their respective areas, to author chapters that help graduate students, scientists, and practicing engineers understand the overall area of microfluidics and nanofluidics. Topics covered include Finite Volume Method for Numerical Simulation Lattice Boltzmann Method and Its Applications in Microfluidics Microparticle and Nanoparticle Manipulation Methane Solubility Enhancement in Water Confined to Nanoscale Pores Volume Two: Fabrication, Implementation, and Applications focuses on topics related to experimental and numerical methods. It also covers fabrication and applications in a variety of areas, from aerospace to biological systems. Reflecting the inherent nature of microfluidics and nanofluidics, the book includes as much interdisciplinary knowledge as possible. It provides the fundamental science background for newcomers and advanced techniques and concepts for experienced researchers and professionals