Design and Implementation of Position-Encoded Microfluidic Microsphere-Trap Arrays

Microarray devices are useful for detecting and analyzing biological targets, such as DNAs, mRNAs, proteins, etc. Applications of microarrays range from fundamental research to clinical diagnostics and drug discovery. In this dissertation, we consider a microsphere array device with predetermined positions of the microspheres. The microspheres are conjugate on their surfaces with molecular probes to capture the targets, and the targets are identified by the microspheres\u27 positions. We implement the microsphere arrays by employing microfluidic technology and a hydrodynamic trapping mechanism. We call our device microfluidic microsphere-trap arrays. To fully realize the potential of the device in biomedical applications, we utilize statistical performance analysis, mathematical optimization, and finite element fluid dynamics simulations to optimize device design, fabrication, and implementation. Our device is promising as a cost-effective and point-of-care lab-on-a-chip system. We first analyze the statistical performance of position-encoded microsphere arrays in imaging biological targets at different signal-to-noise ratio (SNR) levels. We compute the Ziv-Zakai bound (ZZB) on the errors in estimating the unknown parameters, including the target concentrations. Through numerical examples, we find the SNR level below which the ZZB provides a more accurate prediction of the error than the posterior Cramer-Rao bound (PCRB) does. We further apply the ZZB to select the optimal design parameters, such as the distance between the microspheres, and to investigate the effects of the experimental variables such as the microscope point-spread function. We implement the arrays by using microfluidic technology and hydrodynamic trapping. We design a novel geometric structure for the device, and develop a comprehensive and robust framework to optimize its geometric parameters that maximize the microsphere arrays\u27 packing density. We also simultaneously optimize multiple criteria, such as high microsphere trapping efficiency and low fluidic and imaging errors. Microsphere-trapping experiments performed using the optimized device and an un-optimized device demonstrate easy control of the microspheres\u27 transportation and manipulation in the optimized device. They also show that the optimized device greatly outperforms the un-optimized one. We extend our optimization framework to build a device that enables simultaneous, efficient, and accurate screening of multiple targets in a single microfluidic channel, by immobilizing different-sized microspheres at different regions. Different biomolecules captured on the surfaces of the different-sized microspheres can thus be detected simultaneously by the microspheres\u27 positions. We employ finite element fluid dynamics simulations to investigate hydrodynamic trapping of microspheres, and to study the effects of the geometric parameters and critical fluid velocity. The accuracy of the time-dependent simulations is validated by experimental results. The simulations guide the device design and experimental operation. The guidelines on the simulation set-up and the openly available model will help researchers apply the simulation to similar microfluidic systems that may accommodate a variety of structured particles

Statistical Design And Imaging Of Position-Encoded 3D Microarrays

We propose a three-dimensional microarray device with microspheres having controllable positions for error-free target identification. Here targets: such as mRNAs, proteins, antibodies, and cells) are captured by the microspheres on one side, and are tagged by nanospheres embedded with quantum-dots: QDs) on the other. We use the lights emitted by these QDs to quantify the target concentrations. The imaging is performed using a fluorescence microscope and a sensor. We conduct a statistical design analysis to select the optimal distance between the microspheres as well as the optimal temperature. Our design simplifies the imaging and ensures a desired statistical performance for a given sensor cost. Specifically, we compute the posterior Cram&eacuter-Rao bound on the errors in estimating the unknown target concentrations. We use this performance bound to compute the optimal design variables. We discuss both uniform and sparse concentration levels of targets. The uniform distributions correspond to cases where the target concentration is high or the time period of the sensing is sufficiently long. The sparse distributions correspond to low target concentrations or short sensing durations. We illustrate our design concept using numerical examples. We replace the photon-conversion factor of the image sensor and its background noise variance with their maximum likelihood: ML) estimates. We estimate these parameters using images of multiple target-free microspheres embedded with QDs and placed randomly on a substrate. We obtain the photon-conversion factor using a method-of-moments estimation, where we replace the QD light-intensity levels and locations of the imaged microspheres with their ML estimates. The proposed microarray has high sensitivity, efficient packing, and guaranteed imaging performance. It simplifies the imaging analysis significantly by identifying targets based on the known positions of the microspheres. Potential applications include molecular recognition, specificity of targeting molecules, protein-protein dimerization, high throughput screening assays for enzyme inhibitors, drug discovery, and gene sequencing

Multiplexed microsphere diagnostic tools in gene expression applications: factors and futures

Microarrays have received significant attention in recent years as scientists have firstly identified factors that can produce reduced confidence in gene expression data obtained on these platforms, and secondly sought to establish laboratory practices and a set of standards by which data are reported with integrity. Microsphere-based assays represent a new generation of diagnostics in this field capable of providing substantial quantitative and qualitative information from gene expression profiling. However, for gene expression profiling, this type of platform is still in the demonstration phase, with issues arising from comparative studies in the literature not yet identified. It is desirable to identify potential parameters that are established as important in controlling the information derived from microsphere-based hybridizations to quantify gene expression. As these evolve, a standard set of parameters will be established that are required to be provided when data are submitted for publication. Here we initiate this process by identifying a number of parameters we have found to be important in microsphere-based assays designed for the quantification of low abundant genes which are variable between studies

Development of a quantum dot-encoded microsphere suspension assay for the genotyping of single nucleotide polymorphisms

This thesis describes the investigation of quantum dot-doped particle fluorescent technology commercially available for its application to analyte profiling in suspension. The first part of the thesis described the characterisation of the quantum dot-encoded microspheres, QDEMs, developed by Crystalplex (PA, USA). The multiple fluorescence signatures of QDEMs were analysed using microscopy and flow cytometry technology which provided high-content measurements with a single excitation sources and multiple emission wavelength detectors. The sensitivity and stability of the materials was evaluated under typical biomedical conditions encounter in multiple analyte suspension assays. Novel analytical parameters were defined to study QDEM stability and confocal microscopy detection system was used to provide structural and fluorescent imagines of the fluorescent microspheres under various conditions. Composition of the aqueous environment, temperature and physical forces applied to QDEM induced changes in their fluorescent codes and structural properties. Optimal conditions were then defined for the application of the material to biomedical assays. In a second stage, a conjugation method was developed to produce optimised QDEM bioconjugates for the detection of single strand DNA in suspension. The impact of the conjugation buffer, the concentration and the structure of oligonucleotides was evaluated to optimise QDEM bioconjugates. Then, a novel approach was investigated to optimise the hybridisation of ssDNA to QDEM bioconjugates. Experimental design with response surface methodology determined optimum conditions for the hybridisation of oligonucleotides to QDEM surface in suspension array. Finally, the specific hybridisation of ssDNA to QDEM bioconjugates in a small liquid format adapted to single nucleotide polymorphism detection was demonstrated. The work presented here shows the potential of QDEM bioconjugates for suspension array technology and DNA genotyping. Further, this report highlights the challenges that remain for QDEM fluorescent technology to be reliable for biomedical and suspension array applications.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Development of a sensitive diagnostic multiplex platform based on digitally encoded microcarriers

In answer to the ever-increasing need in biomolecular research and clinical diagnostics to carry out many assays simultaneously in on tube, several microcarrier-based multiplex technologies (suspension arrays) have arisen in the past few years. Simultaneous detection of different target molecules that are present in one sample is possible by incubating the sample with a mixture of differently encoded microcarriers, each carrying another probe which can specifically interact with one of the targets. This means that each target will bind to a differently encoded microcarrier. When the targets are caught, several methods exist to label those 'positive' microcarriers. By means of this label, and by means of the code, it becomes possible to verify whether a target was caught at its surface, and which target was caught, respectively. Those multiplex measurements work quantitatively, because the more a target is present in the sample, the more targets will bind to their corresponding microcarrier. Five years ago, our research group proposed the use of spatial selective photobleaching, as an alternative method for the development of digitally encoded microcarriers, which were called 'memobeads'. It was suggested that this method could overcome the multiplexing limitations of existing technologies. The present study aimed to optimize the surface characteristics of t hose memobeads, and to verify whether they could then be applied to multiplex protein tests and nucleic acid tests. Furthermore, it was investigated in which way these memobead assays (and in general the assays performed with every kind of suspension arrays) could be improved to make them more efficient and sensitive

Development of an automated identification system for nanocrystal encoded microspheres in flow cytometry

Aim: This work sets out to use haplotype-based tagSNP selection and a systematic in silico analysis for design of multiplex-compatible PCR primer and SAT probe sets to capture maximum variation with minimum tests across candidate genes IGF1, IGFBP1 and IGFBP3. Additionally, the work aims to develop a number of robust, high-efficiency, high-specificity multiplex PCR constructs for amplification of these targets and to demonstrate the applicability of these target types to suspension array genotyping for non-insulin-dependant diabetes mellitus association facilitation. Methods: Haplotypes for predominantly European Caucasian populations were constructed and tagSNP selection performed using Haploview to capture maximum variation across candidate genes IGF1, IGFBP1 and IGFBP3. Extensive in silico analysis was performed for design, evaluation and selection of robust high-specificity primer and probe pairs, suitable for downstream multiplex PCR and SAT analysis. Singleplex endpoint and real-time PCR was performed for primer pair profile determination which informed multiplex PCR set construction and optimisation. The applicability of this complex target type to suspension array-based genotyping was investigated using a model probe pair using both quantum dot-encoded and fluorophore-encoded microspheres. Results: Haploview was used for haplotype construction and linkage disequilibriumbased tagSNP selection across candidate genes, reducing the number of SNP targets from 292 to 32 with minimal information loss. Extensive evaluation of potential tagSNPs was performed and 29 SNPs, representing 29 bins across target genes were designed for multiplex analysis. Singleplex end-point and real-time PCR was performed for primer pair profile determination which allowed four multiplex PCR sets to be constructed and optimised for simultaneous amplification of 14, six, five and two targets. The applicability of this complex target type (14-plex) to suspension array-based genotyping was demonstrated using a model probe pair. Conclusion: In silico analysis techniques have been applied for successful development of four robust multiplex PCR sets (14-plex, 6-plex, 5-plex and 2-plex) which display high-efficiency and target-specific amplification of tagSNPs, capturing maximum assaycompatible variation across candidate genes IGF1, IGFBP1 and IGFBP3 for European Caucasian populations. The applicability of these multiplex PCR constructs to suspension array-based genotyping has been demonstrated, thus paving the way for development of large multiplex suspension array-based genotyping assays using probes designed during the course of this work. This work offers the potential for comprehensive association analyses to become more accessible to the wider-scientific community by facilitating reduced genotyping burdens which allow increased accessibility for powerful association.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Development of Immunoassay Using Graphene and Microfluidic Platforms.

Protein, as one of the most important functional biomolecules in the human body, plays a significant role in physiological responses and molecular diagnostics. Detecting the existence of proteins, quantifying concentration, and identifying protein types are therefore important techniques in many fields. Immunoassays are one of the major techniques relied on for protein detection. Immunoassays have been broadly applied in disease diagnosis, pharmaceutical development, food science, and environmental protection. The first part of this dissertation describes studies aimed at developing chemical vapor deposition (CVD) graphene as a large size protein biosensing platform. To utilize graphene as a biosensing platform, techniques to immobilize proteins on graphene are critical. In this dissertation work, carboxyl functional groups (-COOH) were created by graphene functionalization, and the functionalized graphene was characterized using Raman spectroscopy, X-ray photo spectroscopy (XPS), and fluorescence microscopy. The approach developed here provides information about protein coupling density and uniformity on large scale graphene (> cm2). The second and the third parts of the thesis describe the application of a microfluidic technique to two widely used protein detection methods – immunoblotting and dot blotting. The microfluidic systems were designed and fabricated to be easily interfaced with a common type of protein blotting membrane called polyvinylidene fluoride (PVDF) membrane. The microfluidic device was specifically applied to the antibody incubation step, which reduces antibody consumption and therefore also significantly reduces the cost of the assay. In microfluidic immunoblotting, an approach to activate the PVDF membrane to increase its protein binding capacity was developed. This was achieved by adding a surfactant Tween-20 to the antibody solution. The concentration of Tween-20 was optimized so that only the portion of the membrane within the channel region was activated. The system has been shown to be able to profile inflammatory signaling pathways. In microfluidic dot blotting, the influence of substrate hydrophobicity and protein concentrations on device design constraints were studied. Inflammatory cytokine detection using the developed microfluidic dot blotting system was determined. Altogether these experiments demonstrate that applying microfluidic techniques to protein immunoblotting and dot blotting improves detection efficiency, and reduces cost by utilizing less antibodies.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113501/1/huaining_1.pd

On-the-fly decoding luminescence lifetimes in the microsecond region for lanthanide-encoded suspension arrays

Significant multiplexing capacity of optical time-domain coding has been recently demonstrated by tuning luminescence lifetimes of the upconversion nanoparticles called 'τ-Dots'. It provides a large dynamic range of lifetimes from microseconds to milliseconds, which allows creating large libraries of nanotags/microcarriers. However, a robust approach is required to rapidly and accurately measure the luminescence lifetimes from the relatively slow-decaying signals. Here we show a fast algorithm suitable for the microsecond region with precision closely approaching the theoretical limit and compatible with the rapid scanning cytometry technique.We exploit this approach to further extend optical time-domain multiplexing to the downconversion luminescence, using luminescence microspheres wherein lifetimes are tuned through luminescence resonance energy transfer.We demonstrate real-time discrimination of these microspheres in the rapid scanning cytometry, and apply them to the multiplexed probing of pathogen DNA strands. Our results indicate that tunable luminescence lifetimes have considerable potential in high-throughput analytical sciences. © 2014 Macmillan Publishers Limited. All rights reserved

Porous Bead-Based Diagnostic Platforms: Bridging the Gaps in Healthcare

Advances in lab-on-a-chip systems have strong potential for multiplexed detection of a wide range of analytes with reduced sample and reagent volume; lower costs and shorter analysis times. The completion of high-fidelity multiplexed and multiclass assays remains a challenge for the medical microdevice field; as it struggles to achieve and expand upon at the point-of-care the quality of results that are achieved now routinely in remote laboratory settings. This review article serves to explore for the first time the key intersection of multiplexed bead-based detection systems with integrated microfluidic structures alongside porous capture elements together with biomarker validation studies. These strategically important elements are evaluated here in the context of platform generation as suitable for near-patient testing. Essential issues related to the scalability of these modular sensor ensembles are explored as are attempts to move such multiplexed and multiclass platforms into large-scale clinical trials. Recent efforts in these bead sensors have shown advantages over planar microarrays in terms of their capacity to generate multiplexed test results with shorter analysis times. Through high surface-to-volume ratios and encoding capabilities; porous bead-based ensembles; when combined with microfluidic elements; allow for high-throughput testing for enzymatic assays; general chemistries; protein; antibody and oligonucleotide applications