At sites of vessel injury, thrombin mediates coagulation by catalyzing fibrin clot formation and platelet activation. Conversely, plasmin facilitates fibrinolysis by catalyzing the degradation of fibrin clots. Thrombin generation is most frequently measured in plasma samples using small molecule substrates, these substrates have low free thrombin efficiency and specificity, cannot measure thrombin’s exosite interactions and have limited utility in whole blood. Plasma assays are limited because they ignore the hemostatic contributions of blood cells, require anticoagulation and the addition of supraphysiological concentrations of calcium. To overcome these limitations, we have designed and characterized a fluorescence resonance energy quenching (FREQ)-based thrombin sensor (FTS) protein. Compared to small molecule substrates, the FTS demonstrated high specificity for thrombin because it is not cleaved by thrombin inhibited by α2-macroglobulin and interacts with thrombin’s anion binding exosite I. The FTS can effectively measure thrombin generation in plasma and in whole blood. The FTS does not inhibit standard thrombin generation assays. Lastly, FTS-based thrombin generation in non-anticoagulated finger-prick blood is delayed but enhanced compared to citrated plasma. Similarly, plasmin generation is also restricted to plasma samples and measured using the fluorogenic Boc-Glu-Lys-Lys-AMC, which have low free plasmin efficiency and specificity, cannot measure plasmin’s kringle domain interactions and are insensitive to the effects of plasminogen activator inhibitor-1 (PAI-1). Here, we describe the design and characterization of a (FREQ)-based plasmin sensor (FPS) protein that demonstrated high efficiency for plasmin that is not inhibited by α2-macroglobulin when compared to Boc-Glu-Lys-Lys-AMC and interacts with plasmin’s kringle domain 5. The FPS measures plasmin generation in plasma, where it demonstrated greater sensitivity to tranexamic acid compared to Boc-Glu-Lys-Lys-AMC as well as sensitivity to PAI-1 and the effects of fibrin. Therefore, the FTS and FPS will broaden our understanding of thrombin and plasmin generation in ways that are not attainable with current methods. / Thesis / Doctor of Philosophy (PhD) / Increased blood clot formation leads to strokes and heart attacks while the inability to form blood clots when needed leads to bleeding disorders. In the body, thrombin makes blood clots while plasmin breaks them down. Therefore, clotting disorders occur in the absence of proper thrombin and plasmin function and appropriate diagnostics of these processes help to determine appropriate treatment. Currently available tests of thrombin and plasmin do not just measure their active forms and are restricted to plasma assays that do not reflect physiological settings and are not useful for quick diagnosis. Here, we describe the production of novel sensors for thrombin and plasmin that are specific for thrombin and plasmin’s active forms. These sensors have potential to be developed into portable diagnostic tests of thrombin and plasmin activity and to be valuable research tools for the development of better treatment options of blood clotting diseases.

The development of easily fabricated, highly stable DNA-based microarray and continuous flow concentrating devices is vital for several biomedical and environmental applications. Nucleic acid biosensors can be used for genetic analysis, disease diagnosis, drug discovery, food and water quality control and more, however methods of fabrication are tedious, and the longevity of sensors is compromised by the fragility of the sensing component. In this report, the fabrication and characterization of two biosensing modalities – microarrays and microgels – composed of Rolling Circle Amplification (RCA) product in poly(oligoethylene glycol methacrylate) (POEGMA) hydrogels are investigated. RCA product microarrays were developed by the sequential printing of aldehyde and hydrazide functionalized POEGMA precursors on nitrocellulose paper, exploiting rapid gelling via hydrazone crosslinking to generate thin film hydrogel sensing arrays. POEGMA/RCA product microgels for affinity column applications were synthesized using an inverse emulsion polymerization technique. Inkjet printing evenly deposited RCA product in all wells, with POEGMA effectively stabilizing DNA on the cellulose substrate. Hybridization of complementary probe to the encapsulated RCA product was optimized, yielding a signal to noise ratio of ~4 for a large range of probe concentrations. Microgels were successfully synthesized in the size range of 10-60 μm diameter, and a linear model that can accurately predict size based on initiator and emulsifier concentration was developed. The encapsulation efficiency of RCA product in different sized microgels was explored, with larger microgels entrapping more product and the highest encapsulation efficiency calculated at 56%. These results demonstrate that POEGMA hydrogels can be utilized to encapsulate and stabilize RCA product in two distinct structures, providing a basis for the development of easily fabricated biosensors for more specific applications. / Thesis / Master of Applied Science (MASc)

Synthetic genetic circuits have revolutionized numerous fields, ranging from academic research and point-of-care diagnostics to disease therapeutics and industrial biomanufacturing. These circuits provide a powerful tool for precise spatiotemporal control over biological and biochemical interactions, thereby enhancing our understanding of these complex systems and expanding their applicability. The last few decades have witnessed a surge in research efforts, both in cell-free and cellular systems. These endeavors include those to improve the sensitivity and specificity of diagnostics and optimize the safety, efficacy, and tunability of existing treatments. This dissertation delves into the exploration of Boolean logic gates in the cell-free realm: the development of a 'Cell-Free Recombinase Integrated Boolean Operating System' (CRIBOS) for expanding the capabilities of cell-free sensing applications. Applications of Boolean logic gates have flourished within cellular systems and animal models. However, a persisting gap in the field is in their exploration within the cell-free system. This deficiency has resulted in a constrained toolkit for studying and applying Boolean logic gates in cell-free settings. Recognizing this limitation in the field and aiming to extend the frontiers of genetic circuits beyond traditional boundaries, I introduce CRIBOS, leveraging the advantages of recombinase, known for its high orthogonality, efficiency, and sensitivity. I designed more than 20 multi-input-multi-output recombinase Boolean logic gates in a cell-free context, from which a set of critical rules crucial for building genetic circuits in the cell-free environment was also established. In addition, integrating allosteric transcription factor (aTF)-based sensors with CRIBOS enabled multiplex environmental sensing within the cell-free environment. Moreover, the CRIBOS system showcased its versatility by facilitating the creation of a biological memory storage device, demonstrating robust functionality with high stability over four months. Implementing CRIBOS not only expands the application of multiplex Boolean logic gates from cellular systems to the cell-free environment but also expands their overall versatility, opening new avenues for the design and application of sophisticated genetic circuits.

Biosensors are extremely powerful analytical tools instrumental for detection and quantification of bio-molecules such as DNA, peptides and even metabolites. The recent decade has seen a surge in biosensing applications ranging from molecular diagnostics, environmental monitoring, basic life science research, forensics and biothreat monitoring. The existing biosensor systems of today, however, have several limitations. They are expensive, bulky in size, power hungry, hard to use and with access limited to core facilities. Among other disadvantages, these impediments discourage the availability of point-of-care testing and low cost in-vitro diagnostics (IVD) in locations such as developing and third world countries. The main bottleneck in the development of low-cost and compact biosensors is the effective and efficient integration of several complex components present inside a typical biosensor. These components are the sample preparation, biomolecular recognition, signal transduction and data analysis. With vii the recent advancements in very large scale integration (VLSI) and fabrication technologies, it is now possible to integrate several of these biosensing components into a small form factor. This thesis proposes leveraging the utilization of VLSI technology to develop a low-cost, miniature, portable, fast analysis, high throughput and low power consumption biosensor solution. Apart from the miniaturization bene- fits, employing VLSI technology facilitates low-cost, high yield and low process variation. We present complementary metal-oxide semiconductor (CMOS) integrated microsystem solutions for fluorescence, bioluminescence and electrochemical biosensing. Simulation models are provided for the microsystems and the specifications for the constituent components derived. A common problem in the transducer development of biosensors that we specifically focus on, is the presence of a large non-informative signal called the background signal. This background signal can be several orders of magnitudes higher than the signal of interest and it reduces the overall sensitivity of the biosensor. Existing transducer solutions rely on very high dynamic range, expensive and power hungry solutions to solve the problem of high background signal. To address the problem of overwhelming background signal, this thesis proposes an active background subtraction architecture merged with a Σ∆ modulator. The robust, versatile architecture can be conveniently employed for optical and electrochemical sensing. The proposed architecture attenuates the background signal very early in the signal chain, achieving high dyviii namic range while significantly relaxing the performance requirements of the subsequent circuit blocks in terms of power dissipation, area and bandwidth requirements. To validate the proposed solution, two CMOS IC prototypes were developed for optical and electrochemical sensing respectively. A 12 × 12 array of Σ∆ photodetector with in-pixel background subtraction was developed in 0.18µm standard CMOS technology. The pixel performance has been validated with over 140dB dynamic range and the ability of subtract the background subtraction current validated from 10nA to 10fA. Real time pyrosequencing experiment has also been performed utilizing the photodetector array. A 12 × 12 array of Σ∆ electrochemical sensor with in-pixel background subtraction was developed in 0.18µm standard CMOS technology. Capacitive charge redistribution circuit architecture for bipolar current measurements was employed. The circuit performance was validated over the wide input current range of 100nA to 1pA. / text

Three non-peripherally substituted liquid crystalline bisphthalocyanine (Pc) compounds have been studied to examine the role of central metal ions lutetium (Lu), and gadolinium (Gd) and substituent chain lengths, i.e. octyl (C8H17) and hexyl (C6H13), in determining the physical properties. For the octyl substituted Pc molecules, the head-to-tail or Jaggregates within the as-deposited spun films produced a redshift of the optical absorption Q bands in relation to their 0.01 mgml-1 solutions. Annealing at 80˚C produced a well-ordered discotic liquid crystalline (LC) mesophase causing additional redshifts irrespective of the metal ion in case of C8LuPc2 and C8GdPc2. Formation of face-to-face or H-aggregated monomers led to blueshifts of the Q bands with respect to solution spectra for C6GdPc2, both as-deposited and annealed films. Stretching and bending vibrations of pyrrole, isoindole, and metal-nitrogen bonds in Pc rings showed Raman bands at higher energy for smaller metal ion. However, no change was observed for the difference in chain lengths. As-deposited C8LuPc2 and C6GdPc2 produced comparable Ohmic conductivity, of the value 67.55 Scm-1 and 42.31 Scm-1, respectively. C8GdPc2 exhibited two orders of magnitude less conductivity than the other two due to the size effect of the central ion and side chain length. On annealing, an increase of Ohmic conductivity was noticed in the isostructural octyl substituted phthalocyanines on contrary to a reduced conductivity in hexyl substituted one. An optical band shift of the C8LuPc2 and C8GdPc2 thin films occurred on oxidation by bromine vapour. Oxidations of Pc-coated ITO were also achieved by applying potential at 0.88 V and 0.96 V electrochemically for the C8LuPc2 and C8GdPc2 compounds, respectively. To explore the applications of these compounds in biosensing, in situ interaction studies between bromine oxidised compounds and biological cofactors nicotinamide adenine dinucleotide (NADH) and L-ascorbic acid (vitamin C) were carried out using optical absorption spectroscopy. Thin films of a non-peripherally octyl substituted LC lead phthalocyanine was exposed to 99.9 % pure hydrogen sulfide gas to produce hybrid nanocomposites consisting of lead sulphide quantum dots embedded in the analogous metal free phthalocyanine matrix. Trapping of charge carriers caused hysteresis in the current-voltage characteristics of the film on interdigitated gold electrodes. The charge hopping distance was found to be 9.05 nm, more than the percolation limit and responsible for forming two well-defined conducting states with potential application as a memristor.

Law Wing Cheung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references. / Abstracts in English and Chinese. / Abstract --- p.I / Acknowledgements --- p.V / List of Publications related to this project --- p.VI / Contents --- p.VII / Chapter Chapter 1 --- Introduction --- p.1-1 / Chapter Chapter 2 --- Literature Review / Chapter 2.1 --- Surface Plasmon Waves --- p.2-2 / Chapter 2.2 --- Excitation of Surface Plasmon --- p.2-4 / Chapter 2.2.1 --- Surface Plasmon Coupling Schemes --- p.2-6 / Chapter 2.3 --- Detection Techniques used in SPR sensors --- p.2-13 / Chapter 2.3.1 --- Angular Interrogation --- p.2-14 / Chapter 2.3.2 --- Wavelength Interrogation --- p.2-15 / Chapter 2.3.3 --- Intensity Interrogation --- p.2-16 / Chapter 2.3.4 --- Phase Interrogation --- p.2-16 / Chapter 2.3.5 --- Commercial SPR biosensors --- p.2-18 / Chapter 2.3.6 --- Comparison between Detection Techniques --- p.2-19 / Chapter 2.4 --- Applications of SPR biosensors --- p.2-21 / Chapter Chapter 3 --- Principle of Surface Plasmon Resonance Sensing Technology / Chapter 3.1 --- SPR Phenomenon --- p.3-1 / Chapter 3.2 --- Conditions for Surface Plasmon Resonance --- p.3-5 / Chapter 3.3 --- Wave-vectors --- p.3-7 / Chapter 3.4 --- Surface Plasmon Resonance described by Fresnel's Theory --- p.3-8 / Chapter 3.5 --- Concept of Surface Plasmon Resonance Biosensing --- p.3-10 / Chapter Chapter 4 --- Experiments / Chapter 4.1 --- Highly sensitive differential phase-sensitive surface plasmon resonance biosensor based on Mach-Zehnder configuration --- p.4-1 / Chapter 4.1.1 --- Materials required --- p.4-1 / Chapter 4.1.2 --- Experimental Setup --- p.4-2 / Chapter 4.1.3 --- Principle of Differential Phase Measurement --- p.4-3 / Chapter 4.1.4 --- Photodetector Circuitry --- p.4-6 / Chapter 4.1.5 --- Digital Signal Processing --- p.4-7 / Chapter 4.1.6 --- Polymer based Micro-fluidic System Integrated with SPR Biosensor --- p.4-9 / Chapter 4.2 --- Phase-sensitive Surface Plasmon Resonance Biosensor using the Photoelastic Modulation Technique --- p.4-12 / Chapter 4.2.1 --- Materials required --- p.4-12 / Chapter 4.2.2 --- Experimental Setup --- p.4-13 / Chapter 4.2.3 --- Principle of Photoelastic Modulation Technique and Signal Processing --- p.4-14 / Chapter 4.2.4 --- Operation Principle of Photoelastic Modulator --- p.4-17 / Chapter 4.3 --- Sample Preparations --- p.4-18 / Chapter 4.3.1 --- Glycerin-water Mixtures --- p.4-18 / Chapter 4.3.2 --- "PBS, BSA and BSA antibody" --- p.4-19 / Chapter 4.3.3 --- "RPMI, Trypsin, Cells and SDS" --- p.4-20 / Chapter Chapter5 --- Results amd Discussions / Chapter 5.1 --- Experimental setup I: Highly sensitive differential phase-sensitive surface plasmon resonance biosensor based on Mach-Zehnder configuration --- p.5-1 / Chapter 5.1.1 --- Measuring various glycerin-water concentration mixture with silver-gold sensing layer --- p.5-1 / Chapter 5.1.2 --- Comparison between the sensitivity of our setup and reported setup based on phase detection --- p.5-4 / Chapter 5.1.3 --- Discussion on 0.01° system resolution --- p.5-7 / Chapter 5.1.4 --- Experiment on monitoring BSA-BSA antibody binding reaction --- p.5-9 / Chapter 5.1.5 --- Matching oil and glass slide --- p.5-11 / Chapter 5.1.6 --- Experiments on monitoring BSA-BSA antibody binding reaction with integrated microfluidic system --- p.5-12 / Chapter 5.1.7 --- Experiment on observing cell adhesion properties on gold surface under the influence of trypsin --- p.5-14 / Chapter 5.1.8 --- Discussion on the non-specific binding between trypsin and gold surface --- p.5-16 / Chapter 5.1.9 --- Modifying the gold surface with BSA layer --- p.5-17 / Chapter 5.1.10 --- Experiment on observing cell adhesion properties on the gold surface under the influence Sodium Dodecyl Sulfate (SDS) --- p.5-18 / Chapter 5.2 --- Experimental setup II: Phase-sensitive surface plasmon resonance biosensor using the photoelastic modulation technique --- p.5-21 / Chapter 5.2.1 --- Measurement on difference glycerin-water concentration mixture --- p.5-21 / Chapter 5.2.2 --- Experiment on monitoring BSA-BSA antibody binding reaction --- p.5-23 / Chapter Chapter 6 --- Conclusions and Future Works / Chapter 6.1 --- Conclusions --- p.6-1 / Chapter 6.2 --- Future Works --- p.6-2 / References --- p.R-1 / Appendix / Chapter A. --- Phase Extraction Routine written by Matlab --- p.A-1 / Chapter B. --- Mathematical expressions for calculating the phase angle in the experiment of SPR biosensor using the Photoelastic Modulation Technique --- p.A-6 / Chapter C. --- Relationship between Concentration and Refractive Index of Glycerin-Water Mixture --- p.A-11 / Chapter D. --- Physical Properties of Bovine Serum Albumin --- p.A-12 / Chapter E. --- Simulation Curve written by Matlab --- p.A-13

Detection of oligonucleotide target has been performed with a sandwich assay scheme. We compare the detection performance of strategies using probe oligonucleotide with or without gold nanoparticles (Au-NPs, 20nm) capped on 3'. Our experimental results reveal that while the DNA detection implemented with NIS can provide high sensitivity, both dynamic range and detection limit can be amplified with the aid of Au-NPs on 3' of the probes. The current detection limits of NIS with and without Au-NPs are 0.4 femtomole and 1 nanomole respectively. (Abstract shortened by UMI.) / Finally, this work presents a systematic study of the surface-enhanced Raman-scattering (SERS) properties of nanoparticle island substrates (NIS) and their application for oligonucleotide target detection. To effectively implement SERS on NIS and identify an optimal condition for DNA detection, the relationship between extinction maximum (lambdamax) and SERS enhancement factor (EF) will be explored in detail. This work demonstrates high S/N ratio SERS spectra can be achieved with NIS that has lambdamax located within a spectral window (∼60nm) defined by the excitation wavelength (514nm) and the scattered Raman wavelength. The highest EF measured is about 4x10 8 with a thickness of Ag being 50 A. / In addition, a surface plasmon enhanced ellipsometry (SPEE) biosensor scheme based on the use of a photoelastic modulator (PEM) has been explored. We showed that the polarization parameters of a laser beam, tan psi, cos Delta and ellipse orientation angle &phis;, can be directly measured by detecting the modulation signals at the 1st and 2nd harmonics of the modulation frequency under a certain birefringence geometry. This leads to an accurate measurement of refractive index variations within the evanescent field region close to the gold sensor surface, thereby enabling biosensing applications. Our experimental results confirm that the new scheme offers a decent detection limit of 2x10-7 refractive index unit (RIU) or 5ng/ml of biomolecule solute concentration without any compromise in dynamic range. / We have demonstrated that the sensitivity limit of intensity-based SPR biosensors can be enhanced when we combine the contributions from phase with that of amplitude instead of just detecting the amplitude or phase variation only. Experimental results indicate that an enhancement factor of as much as 20 times is achievable, yet with no compromise in measurement dynamic range. While existing SPR biosensor systems are predominantly based on the angular scheme, which relies on detecting intensity variations associated with amplitude changes only, the proposed scheme may serve as a direct system upgrade approach for these systems. / We have developed a novel design of multi-pass surface plasmon resonance (SPR) biosensor with differential phase interrogation based on multi-pass interferometry. This new configuration provides an intrinsic phase amplification effect of over two-fold by placing the SPR sensor head in a signal arm of the interferometer so that the interrogating optical beam will traverse the sensor surface infinite number of times. Experimental interferometers based on the Michelson and Fabry-Perot configurations have been employed to experimentally verify this amplification effect through the comparison with the Mach-Zehnder configuration. Results obtained from the salt-water mixtures, antibody-antigen, and protein-DNA binding reaction have confirmed the expected phase measurement enhancement. / Yuan, Wu. / Adviser: H. P. Ho. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3582. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 115-132). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

The importance of porous materials has risen substantially in the last few decades due to their ability to reduce the size and cost of bioanalytical devices and fuel cells. First, this work aims to describe the fabrication of nanoporous gold (NPG) electrodes that are resistant to electrode passivation due to fibrinogen biofouling in redox solutions. The effect on potentiometric and voltammetric experiments was seen as a deviation from ideal behavior on planar gold electrodes, whereas NPG electrodes were consistently behaving in a Nernstian fashion at low concentrations of ferri-ferrocyanide (£100 mM). An improvement in electrode behavior on NPG electrodes versus planar gold was seen in solutions containing ascorbic acid as well as blood plasma. Second, cost effective NPG electrodes were fabricated using a glass substrate to test the response in the presence of a variety of redox molecules. The optical transparency of these electrodes allowed for microdroplet measurements to be made using an inverted microscope in several redox solutions for validation and subsequent biological applicability. Nernstian behavior was demonstrated for all one- and two-electron transfer systems in both poised and unpoised solutions. All experiments were conducted using volumes between 280 and 1400 pL producing rapid results in less than one minute. Third, in order to decrease the requirement for complex instrumentation, microdroplet fabrication technique was used to create mini-nanoporous gold (mNPG) electrodes on glass capillary tubes. The cylindrical shape of the electrodes allowed for testing in sample volumes of 100 mL. The response to ferri-ferrocyanide, ascorbic acid, cysteine, and uric acid was then investigated with Nernstian behavior shown. However, the mNPG electrodes were insensitive to glucose and hydrogen peroxide. In order to increase the sensitivity of the electrodes, a minimal amount of platinum was electrodeposited onto the NPG surface using a low concentration of platinum salt (0.75 mM) for a short deposition time (2 seconds) producing a Nernstian response to both glucose and hydrogen peroxide. Lastly, to test the viability of crossover applications, the platinum incorporated NPG electrode was employed as a fuel cell anode material, testing their oxidation capability with methanol, ethanol, and formic acid.

The development of real time, label-free biosensors based on ligand-induced nanomechanical responses of microcantilevers (MCs) allows for sensitive and selective detection. High sensitivity is afforded by the MCs small dimensions. Immobilizing biomolecular recognition phases imparts selectivity from bioaffinity interactions. Biological sensors on a MC platform utilize various proteins, such as antibodies and nuclear receptors, which can be used to detect and screen for potential environmental contaminants. The interaction between contaminants and immobilized receptors induces an apparent surface stress that leads to static bending of the MC, which is monitored by an optical beam bending technique. Biofunctionalized MCs can provide high sensitivity and selectivity on a relatively inexpensive platform that requires small amounts of analyte. The goal of this research is to develop and optimize MCs as biosensors to detect low concentrations of contaminants. Initially, the research utilized specific receptors and antibodies to detect and screen for contaminants that are deemed endocrine disrupting chemicals (EDCs). Immobilizing estrogen receptors and specific antibodies on the MC surface may provide information on the ever expanding list of EDCs, along with fundamental endocrine studies. Then, the MC surface was morphologically and chemically optimized. This optimization included the thickness and metal ratio of the dealloyed surface. The concentration, reaction time, and pH of chemical immobilization reagents, which include aminoethanethiol and glutaraldehyde, were optimized by using an anti-body test system. Antibody and protein functionalization conditions, which are incubation time and concentration, were optimized using the anti-immunoglobulin G (anti-IgG) receptor: IgG and an anti-biotin:biotin test systems. The optimized immobilization conditions were applied to the detection of thyroid disrupting chemicals (TDCs) using MCs functionalized with the transport protein thyroxine-binding globulin. The final project involved developing a nanomechanical transducer to study xenobiotic and EDC interactions with the bioreceptor PXR’s ligand binding domain (LBD). The combination of immobilized LBD PXR with a nanostructured microcantilever (MC) platform allows for the study of ligand interaction with the receptor’s binding domain. PXR shows real-time, reversible responses when exposed to specific pharmaceutical, EDC, and xenobiotic ligands. Three binding interactions that involve EDCs are tested, which include phthalic acid, nonylphenol, and bisphenol A, with PXR.

Adeno-associated virus (AAV) is a 25 nm replication deficient DNA virus most commonly studied for human gene therapy applications. The work detailed in this thesis investigates the controlled delivery of AAV from surfaces for both transduction and biosensing applications. First, AAV was tested for compatibility with substrate-mediated gene delivery approaches. Two reverse transduction applications were investigated: (1) spatial localization of cells and virus vectors for tissue engineering applications and (2) live cell genetic microarrays. To drive the proper differentiation and assembly of cells within tissue engineering constructs, gene expression patterns may need to be tightly regulated. To localize adhesive proteins and AAV, polydimethylsiloxane stamps and protein adhesive alkanethiols were used. By adsorbing AAV onto adhesive proteins, including human fibronectin, laminin, collagen I, elastin and poly-l-lysine, both cell adhesion and gene delivery were localized to a defined pattern. Gene delivery was efficient on all protein surfaces, with higher expression observed on laminin surfaces. AAV was also patterned using a robotic spotter to create live cell genetic microarrays, creating localized cell islands expressing GFP. This potentially high-throughput technique could be extended to study complex genetic interactions within cells, such as stem cells or induced pluripotent stem cells. Additionally, AAV was explored as a biosensor by modifying virus output functionalities. Wild-type AAV2 externalizes an N-terminus region containing a phospholipase A2 (PLA2) domain during intracellular processing, allowing the virus to escape the endosomal pathway and deliver genetic cargo. This externalization can be replicated outside of cells through heat treatment. AAV2-ΔPLA2-His was created by replacing the PLA2 domain with a nickel binding hexahistidine tag. This replacement allows heat-treated mutant virus to bind a nickel affinity column. Finally, directed evolution was used to (1) improve the ability of AAV to deliver genes into target cells or (2) alter AAV biosensor inputs. Virus libraries were created using error-prone polymerase chain reaction (EP-PCR) to introduce random amino acid modifications into the protein capsid. The error rate for these libraries was estimated to be between 5-7 errors per cap gene. Combining surface immobilization with directed evolution could allow for precise control of AAV for gene delivery and biosensing.