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.

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

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Three protein targets are used in malaria rapid diagnostic tests (RDTs). These are Plasmodium falciparum histidine rich protein 2, Plasmodium lactate dehydrogenase and aldolase. A thrust of research in RDTs is to improve on their specificity and sensitivity. In this study the current diagnostic target, P. falciparum lactate dehydrogenase (PƒLDH) was compared to a new target glyceraldehyde-3-phosphate dehydrogenase (PƒGAPDH) that was identified based on transcriptional data. These proteins are conserved amongst all Plasmodium species, with minor amino acid sequence variations which were evaluated as possible species-specific peptide epitopes for PƒLDH: LISDAELEAIFDRC and PƒGAPDH: CADGFLLIGEKKVSVFA; CAEKDPSQIPWGKCQV, where common peptides were identified as pan-malarial epitopes for pLDH: APGKSDKEWNRDDLC and pGAPDH: CKDDTPIYVMGINH. The chosen peptides were located on the surface of their predicted 3D crystal structure models. Antibodies were raised against these peptides in chickens (IgY) and affinity purified. PƒLDH and PƒGAPDH were recombinantly expressed in E. coli BL21(DE3) cells and their coding inserts confirmed by sequencing. The recombinant proteins were detected in Western blots with specific anti-His₆ tag antibodies at approximately 35 kD (PƒLDH ~ 36 kD and PƒGAPDH ~ 39 kD) which compared with their expected values. Both recombinant proteins were found to form tetramers in solution and were used to raise IgY antibodies for comparison of Pheroids™ and Freund’s adjuvants. Pheroids™, like Freund’s appeared to exhibit a depot effect, however Freund’s adjuvant gave higher affinity purified IgY yields. The anti-recombinant and anti-peptide IgY specifically detected their respective recombinant and native antigens and did not cross-react with other human blood proteins. Immunoprecipitation detected higher levels of PƒGAPDH to PƒLDH in P. falciparum culture lysates. A double antibody sandwich ELISA detected 17.3 ng/ml PƒLDH and 138.5 ng/ml PƒGAPDH at 1% parasitemia in in vitro cultures, however this needs to be further evaluated. These findings suggest PƒGAPDH to be at least as good a protein target as PƒLDH for malaria diagnosis and further trials using it as a target in an RDT format should be considered. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Infectious bursal disease virus (IBDV) has a tropism for the lymphoid tissue of poultry and infects actively dividing and differentiating B-lymphocytes in the bursa of Fabricius. This results in a high mortality rate and severe immunosuppression. These immunodepressed chickens are highly susceptible to secondary infections and have a reduced capacity to respond to vaccination. The principal method to control IBDV is through extensive vaccination using either attenuated live or inactivated IBDV vaccines. However, in recent years due to the emergence of new virulent strains, risk of reversion to pathogenicity, cost considerations and intervention by maternal antibodies, the effectiveness of these vaccines in the veterinary field is being reduced. An alternative approach to prevent infection is by interfering with the binding of IBDV to its receptor protein on the surface of bursal cells. Hence this study was undertaken on the characterisation of a possible IBDV receptor on bursal membranes. Infectious bursal disease virus was isolated from infected bursal tissue using CsCl density gradient centrifugation and visualised with Tris-Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy. Following purification of double stranded RNA from infected bursal tissue and commercially available live IBDV vaccines, a polymerase chain reaction (PCR)-based diagnostic assay based on sequences from the highly conserved viral protein (VP2) region was performed. The presence of the virus was demonstrated by the amplification of a 150 bp band in 2% agarose and 15% nondenaturing PAGE gels. The correctness of this product was confirmed byrestriction digestion with a specific restriction endonuclease (BamHI) that resulted in the predicted digestion fragments of 93 and 57 bp. Following preparation of bursal membrane proteins from uninfected bursal tissue, using sucrose density gradient centrifugation, isolation of IBDV receptor protein was carried out by immobilising IBDV on a Sepharose 4B chromatography matrix. After affinity purification, two prominent protein bands around 40 kDa were visualised using a silver stained Tris-Tricine SDS-PAGE gel. Previous work in this laboratory identified two possible IBDV receptor proteins on bursal membranes of 32 and 40 kDa. Antibodies against peptide sequences derived from the 32 kDa receptor protein were raised in rabbits in the present study. These anti-IBDV receptor peptide antibodies recognised the affinity purified native 40 kDa IBDV receptor proteins in an enzyme-linked immunosorbent assay (ELISA). However, due to the possible epitope denaturation by the reducing treatment buffer prior to Tris-Tricine SDS-PAGE such as SDS and 2-mercapthethanol or detergent (Na-deoxycholate) used during the affinity purification of the IBDV receptor protein, the anti-IBDV receptor peptide antibody did not recognise the receptor protein on a western blot. An inhibition assay was performed in an ELISA format by coating the 40 kDa IBDV receptor protein to see if the anti-IBDV receptor peptide antibody could inhibit IBDV binding to the receptor. The result showed that the anti-IBDV receptor peptide antibody effectively inhibited the binding of IBDV to the receptor. This result could pave the way for reducing IBDV infection by interfering at the viral attachment stage prior to crossing the bursal cell membrane barrier. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

No description available.