Three-dimensional biomolecule patterned hydrogels provide cellular microenvironments that mimic in vivo conditions. We are particularly interested in the fabrication of materials to spatially control stem cell differentiation towards the creation of tissue analogues. To this end, we have designed a 3D protein patterning system where differentiation factors were immobilized within distinct volumes through two-photon chemistry, which provides 3D control since the excitation volume is limited to the focal point of the laser. Agarose hydrogels were modified with 6-bromo-7-hydroxy-coumarin (Bhc) protected amines or thiols, which upon two-photon excitation are deprotected in defined volumes yielding reactive amines or thiols. Fibroblast growth factor-2 (FGF-2) was immobilized onto agarose-thiol-Bhc through either disulfide bond formation with agarose thiols or the physical interaction between human serum albumin (HSA) and the albumin binding domain (ABD). The use of biological binding pairs also provides mild immobilization conditions, minimizing the risk for bioactivity loss. Similarly, two differentiation factors for retinal stem progenitor cells were simultaneously immobilized: 1) ciliary neurotrophic factor (CNTF); and 2) N-terminal sonic hedgehog (SHH). Maleimide modified binding proteins, such as maleimide-streptavidin; react with exposed thiols, yielding 3D patterns of covalently immobilized streptavidin in agarose hydrogels. Growth factors are then introduced as fusion proteins with binding domains, such as biotin-CNTF, for complexation and thus 3D immobilization. By combining multiple binding systems with two-photon patterning, we were able to simultaneously 3D immobilize proteins towards the creation biomimetic hydrogels.

Three-dimensional biomolecule patterned hydrogels provide cellular microenvironments that mimic in vivo conditions. We are particularly interested in the fabrication of materials to spatially control stem cell differentiation towards the creation of tissue analogues. To this end, we have designed a 3D protein patterning system where differentiation factors were immobilized within distinct volumes through two-photon chemistry, which provides 3D control since the excitation volume is limited to the focal point of the laser. Agarose hydrogels were modified with 6-bromo-7-hydroxy-coumarin (Bhc) protected amines or thiols, which upon two-photon excitation are deprotected in defined volumes yielding reactive amines or thiols. Fibroblast growth factor-2 (FGF-2) was immobilized onto agarose-thiol-Bhc through either disulfide bond formation with agarose thiols or the physical interaction between human serum albumin (HSA) and the albumin binding domain (ABD). The use of biological binding pairs also provides mild immobilization conditions, minimizing the risk for bioactivity loss. Similarly, two differentiation factors for retinal stem progenitor cells were simultaneously immobilized: 1) ciliary neurotrophic factor (CNTF); and 2) N-terminal sonic hedgehog (SHH). Maleimide modified binding proteins, such as maleimide-streptavidin; react with exposed thiols, yielding 3D patterns of covalently immobilized streptavidin in agarose hydrogels. Growth factors are then introduced as fusion proteins with binding domains, such as biotin-CNTF, for complexation and thus 3D immobilization. By combining multiple binding systems with two-photon patterning, we were able to simultaneously 3D immobilize proteins towards the creation biomimetic hydrogels.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The aim of this study was to develop a ZnO nanowire-array biosensor that would detect immunoglobulins and record changes in the concentration of an antibody. Early detection of disease-causing agents is essential for an early response. In contrast to conventional methods, biosensors may detect disease-associated agents much faster and more accurate, which holds specific benefits to rural communities. The development of such a biosensor would be favourable for diagnostics in underprivileged communities without infrastructure. The hypothesis was that binding of antibodies to the surface of ZnO nanowires would result in the generation of a piezoelectric potential that, when channelled through a Schottky barrier, would produce a constant voltage reading. Piezoelectricty would be generated due to the bending of the nanowires, or tensile stress applied to the nanowires due to binding of the antibodies. The performance of such a device largely depends on the methods used to construct the ZnO nanowires and methods used to funtionalize the sensor surface. The biggest challenge was thus to chemically modify the self-assembled monolayers (SAMs) and create intermediate monolayers that would react to primary amino groups of lysozyme and form a covalent amide bond. Lysozyme was selected as model antigen, since its structure and reaction with antibodies has been well studied. Alkanethiol and dialkyl disulphides were used to form SAMs. Different SAMs were compared to select the absorbate that would bind the highest concentration of lysozyme. Lysozyme was best immobilized onto Au film layers in the presence of SAM 3-mercaptopropionic acid. Weakest immobilization was in the presence of combined SAM 11-mercaptoundecanoic acid/1-nonanethiol. The sensitivity of the constructed ZnO nanowire biosensor was tested in vitro, in the presence of different concentrations of lysozyme antibodies. An increase in the dimension of the ZnO seed layer led to an increase in the mean diameter of the ZnO seed grains, and subsequently an increase in the mean diameter of the synthesized ZnO nanowires. Deposition of the ZnO seed layer, using the RF cylindrical magnetron sputtering technique, improved the c-axis alignment of the nanowires and produced nanowires with similar dimensions. However, deposition of the ZnO seed layer using the sol-gel spin coating technique, produced nanowires with irregular c-axis alignments and irregular diameters. An increase in the Au film thickness led to a decrease in the mean diameter of the synthesized ZnO nanowires and worsening of the c-axis alignment. In contrast to single crystalline Au (111) film layers, polycrystalline Au layers increased the mean diameter of the synthesized nanowires. The crystal orientation of the Au film layer had no effect on the c-axis alignment. Increased voltage readings were recorded with an increase in antibody binding, indicating that the ZnO nanosensor may be used to record changes in immunoglobulin levels. Antibody concentrations ranging from 10 ng/ml to 20 μg/ml were sensed. This is the first study showing that ZnO nanowires, conformed into piezoelectric transducers, may be used in the detection of antibodies. The current size of the chip with ZnO nanowires is approximately 1 cm², which is too big to incorporate into a compact monitoring device. Apart from the challenge to produce smaller nanowire-arrays, highly sensitive sensors and miniature amplifiers will have to be developed to increase the strength of the signals generated by the nanowires. The biosensor will also have to be optimised to detect a variety of immunoglobulins. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om ‘n ZnO nanodraad biosensor te ontwikkel wat immunoglobuliene kan opspoor en veranderinge in konsentrasies van die teenliggaampies sal reflekteer. Vroë deteksie van siekte veroorsaakende agente is belangrik vir n vroeg tydige respons. In teenstelling tot konvensionele metodes, kan biosensors siekte veroorsaakende agente vining en akkuraat opspoor, wat veral voordele vir gemeenskappe in landelike gebiede inhou. Die hipotese was dat binding van teenliggaampies aan die ZnO nanodrade ‘n piëzo-elektriese potensiaal sal skep, wat dan ‘n konstante leesbare spanningspotensiaal sal lewer nadat dit deur ‘n Schottky versperring gestuur is. Piëzo-elektrisiteit word gegenereer deur die buiging van die nanodrade, of deur spanning wat op die nanodrade geplaas word deur binding van die teenliggaampies. Die sukses van die ontwerp hang grootliks af van die metode wat gebruik word om die ZnO nanodrade te konstrueer en metodes wat gebruik word om die sensor oppervlak te funksionaliseer. Die grootste uitdaging was dus om die monolae wat outomaties saam groepeer (SAMs) chemies so te verander dat intermediêre monolae vorm wat aan primêre aminogroepe van lisosiem bind ten einde kovalente amied-bindings te vorm. Lisosiem is as model antigeen geselekteer omdat die struktuur en reaksie daarvan met teenliggaampies reeds goed bestudeer is. Alkaantiol en di-alkiel disulfied is gebruik om SAMs te vorm. ‘n Verskeidenheid SAMs is vergelyk ten einde die anker te selekteer waaraan die hoogste konsentrasie lisosiem sal bind. Lisosiem is die effektiefste aan Au film lae ge-immobiliseer in die teenwoordigheid van SAM 3-merkapto-propanoësuur. Die swakste immobilisasie is in die teenwoordigheid van kombineerde SAM 11-merkapto-dekanoësuur/1-nanotiol waargeneem. Die sensitiwiteit van die ZnO nanodrade is in vitro getoets, in die teenwoordigheid van verskillende konsentrasies van lisosiem teenliggaampies. ‘n Toename in die dimensie van die ZnO grondlaag het die gemiddelde deursnit van die ZnO grein verhoog en so ook die gemiddelde deursnit van die gesintetiseerde ZnO nanodrade. Toediening van die ZnO grondlaag deur gebruik te maak van die RF silindriese mikrogolf-verstuiwings tegniek het die orientasie van die c-aslyn van die nanodrade verbeter. Toediening met die sol-gel draai-bedekkings tegniek het ‘n onreëlmatige orientasie van die c-aslyn teweeg gebring, asook ‘n variasie in die afmetings van die nanodrade. ‘n Toename in die Au laag het ‘n afname in die gemiddelde afmetings van die nanodrade en ook ‘n onreelmatige oriëntasie van die c-aslyn veroorsaak. In teenstelling met enkel-kristallyne Au (111) het poli-kristallyne Au lagies ‘n toename in die gemiddelde deursnit van die nanodrade veroorsaak. Die kristal-oriëntasie van die Au laag het geen effek op die belyning van die nanodrade gehad nie. Die spanningspotensiaal het verhoog met ‘n toename in teenliggaampie binding. Hiervolgens kan die ZnO nanosensor gebruik word om veranderinge in immunoglobulien vlakke te monitor. Teenliggaampie konsentrasies wat wissel van 10 ng/ml tot 20 μg/ml is opgespoor. Hierdie is die eerste studie wat toon dat ZnO nanodrade, omskep tot piëzo-elektriese transduseerders, gebruik kan word in die opsporing van teenliggaampies. Die grootte van die skyfie met die ZnO nanodrade is tans ongeveer 1 cm² en is te groot om in ‘n kompakte biosensor in te bou. Benewens die uitdaging om kleiner nanodraad skyfies te ontwikkel, sal hoogs sensitiewe sensors en seinversterkers ontwikkel moet word om die sein afkomstig van die nanodrade te versterk. Die biosensor sal ook ge-optimiseer moet word om ‘n verskeidenheid immunoglobuliene op te spoor.

No description available.

Active targeting with controlled delivery of therapeutic agents to bone is an ideal approach for treatment of several bone diseases. Since bisphosphonates (BPs) are known to have high affinity to bone mineral and are being widely used in treatment of osteoporosis, they are well-suited for drug targeting to bone. For this purpose, bifunctional hydrazine-bisphosphonates (HBPs) with spacers of various lengths and lipophilicity were synthesized and studied. Crystal growth inhibition assays demonstrated that the HBPs with shorter spacers bound more strongly to bone mineral, hydroxyapatite (HA), than did alendronate. HBPs were also demonstrated to be non-toxic to MC3T3-E1 pre-osteoblasts. The targeted delivery of the HBP-conjugated model drug, 4-nitrobenzaldehyde, was demonstrated through hydrolysis of the hydrazone linkage at the low pH of bone resorption and wound healing sites. In another series of experiments, a method to orient proteins on HA surfaces was developed to improve protein bioactivity. Enhanced green fluorescent protein (EGFP) and β-lactamase were used as model proteins. These proteins have a Ser or Thr at their N-terminus, which was oxidized to obtain a single aldehyde group that was subsequently used for bonding HBPs of various length and lipophilicity through formation of a hydrazone bond. The amount of protein immobilized through various HBPs was determined and found not to be exclusively dependent on the length of HBPs. The enzymatic activity of HBP-immobilized β-lactamase, measured with cefazolin as substrate, was found to be higher than β-lactamase that was simply adsorbed on HA. In a third set of studies, HBPs were evaluated for delivering parathyroid hormone (PTH) to bone mineral to enhance cell responses for bone formation. PTH was oxidized and conjugated to HBPs, followed by targeting to bone wafers. In vitro bioassays demonstrated that HBP-targeted PTH stimulated greater synthesis of cAMP in pre-osteoblasts compared to surfaces with simply adsorbed PTH. HBPs were also found to have similar pro-apoptotic activity to widely used alendronate. Overall, HBPs can be used for drug delivery to bone and oriented immobilization of proteins and peptides, with or without anti-osteoclastic action, for a variety of applications including bone tissue engineering.

The food safety and human diagnostics markets are in need of faster working, reliable, sensitive, specific, low cost bioassays and biosensors for bacterial detection. This thesis reports the use of P22 bacteriophage tailspike proteins (TSP) immobilized on silanized silicon surfaces, roughened at a nano-scale, for specific capture and detection of Salmonella. Towards developing TSP biosensors, TSP immobilization characteristics were studied, and methods to improve bacterial capture were explored. Atomic force microscopy was used to count TSP immobilized on gold thin-films. Surface density counts are dependent on the immobilization scheme used. TSP immobilized on flat silicon (Si), silanized with 3-aminopropyltriethoxysilane and activated with glutaraldehyde, showed half the bacterial capture of gold thin-films. To improve bacterial capture, roughened mountain-shaped ridge-covered silicon (MSRCS) surfaces were coated with TSP and tested. Measurements of their bacterial surface density show that such MSRCS surfaces can produce bacterial capture close to or better than TSP-coated gold thin-films. / Biomedical Engineering

No description available.

Plasma polymerization techniques were used to synthesize and deposit hydrogel on silicon (Si) substrate. Hydrogel is a network of polymer chains that are water-insoluble and has a high degree of flexibility. The various fields of applications of hydrogel include drug release, biosensors and tissue engineering etc. Hydrogel synthesized from different monomers possess a common property of moisture absorption. In this work two monomers were used namely 1-amino-2-propanol (1A2P) and 2(ethylamino)ethanol (2EAE) to produce polymer films deposited on Si ATR crystal. Their moisture uptake property was tested using FTIR-ATR technique. This was evident by the decrease in -OH band in increasing N2 purging time of the films. Secondly, two monomer compounds namely vinyl acetic acid and glycidyl methacrylate which have both amine and carboxylic groups are used as solid surface for the immobilization of bovine serum albumin (BSA). Pulsed plasma polymerization was used to polymerize these monomers with different duty cycles. Initial works in this field were all about protein surface adsorption. But more recently, the emphasis is on covalent bonding of protein on to the surface. This immobilization of protein on solid surface has a lot of applications in the field of biochemical studies. The polymerization of vinyl acetic acid and glycidyl methacrylate were shown as successful method to attach protein on them. Chemical mechanical polishing (CMP) of Cu is one of the processes in the integrated chips manufacturing industry. Benzotriazole is one of the constituents of this CMP slurry used as corrosion inhibitor for Cu. Benzotriazole (C6H5N3) is a nitrogen heterocyclic derivative having three nitrogen atoms, each with an unshared pair of electrons, forming five-membered ring structure. This molecule coordinates with Cu atoms by loosing a proton from one of its nitrogen atom and thereby forming a film which is polymeric in nature that prevents further oxidation of Cu. In this work, aqueous solution of BTA was used to study the adsorption characteristics on Cu when a Cu electrodeposited Si was immersed in to it. This study was performed using ATR-FTIR technique. The adsorption of BTA on Cu surface was evident by the presence of aryl C-H stretching band at 3061 cm-1.

Las etapas de transferencia electrónica o transferencia de carga involucradas en reacciones electroquímicas juegan un papel muy importante en un gran número de procesos biológicos y bioquímicos. Hoy en día, el interés de la comunidad científica se centra en explorar y entender exhaustivamente la naturaleza biológica y química de los fenómenos bioelectroquímicos que ocurren en los seres vivos, con el objeto de mimetizarlos en el laboratorio. Los procesos bioelectrocatalíticos presentan un amplio abanico de aplicaciones dirigidas al: (i) desarrollo de biorreactores electroquímicos para la mitigación de las emisiones de gases de efecto invernadero, la eliminación de contaminantes presentes en aguas residuales y urbanas, o la síntesis de productos con alto valor añadido para la industria, (ii) el desarrollo de biopilas y biobaterías, y (iii) el desarrollo de (bio)sensores electroquímicos con fines analíticos. Sin embargo, la implantación en el mercado de dispositivos basados en procesos biocatalíticos aún se enfrenta a varios desafíos, como son la robustez, la estabilidad a largo plazo, la reproducibilidad y la rentabilidad de producción en términos de materiales y fabricación de los dispositivos electroquímicos. La motivación de esta tesis doctoral es la de enfrentarse a algunos de los desafíos con los que se encuentra hoy en día la bioelectrocatálisis, para ello esta tesis doctoral se centra, principalmente en el estudio de nuevos materiales y mejora de rutas y estrategias bioelectrocatalíticas, con la finalidad de desarrollar dispositivos electroquímicos con aplicaciones analíticas y en la obtención de productos de valor añadido. En primer lugar esta tesis doctoral recoge el estudio y desarrollo de (bio)sensores electroquímicos para la determinación de lactato, L-cisteína, peróxido de hidrógeno y pH en medios biológicos complejos, y en segundo lugar estudia la bioelectrosíntesis de ácido fórmico a través de la reducción bioelectroquímica de dióxido de carbono.

The demand for economical, efficient protein production, reuse, and recovery has never been greater due to their versatility in a large variety of applications ranging from industrial chemical manufacturing to pharmaceutical drug production. The applications for naturally and artificially produced proteins include protein drugs and other pharmaceutical products, as biocatalysts in environmentally friendly chemical manufacturing, as enzymes for food processing purposes, and as an essential component in many biomedical devices. However, protein production suffers from many challenges, which include the cost of production, protein stability especially under harsh conditions, and recoverability and reusability of the proteins. The combination of two developing technologies, cell-free protein synthesis systems (CFPS) and unnatural amino acid incorporation, provides solutions to these protein production challenges.This dissertation reports on the use of cell-free protein synthesis systems and unnatural amino acid incorporation to develop new proteins and enzyme immobilization techniques that significantly increase activity and stability while simplifying recoverability and reuse.