Design of a soluble P-selectin biosensor for detecting platelet activation

Laubscher, Riaan Willem (2019-12)

Thesis (MEng)--Stellenbosch University, 2019.

Masters

ENGLISH ABSTRACT: The main causes of death are shifting from communicable disease, such as Tuberculosis, to non-communicable disease, such as cardiovascular disease. This disease, along with many other diseases, have been associated with chronic in ammation which is also interrelated with activated blood platelets. When platelets activate, they release biomolecules that can be used as biomarkers for diagnosing or monitoring diseases. Currently, there are no highly accurate tests that can measure the level of chronic in ammation and conventional tests ar either costly, time-consuming, or unpractical in remote areas. Therefore, there is room for improvement in the area of diagnostics of chronic in ammation. This project set out to find a potential biomarker that is a good indicator of platelet activation and is upregulated in an in ammatory individual. Soluble P-selectin was identified as a platelet membrane receptor that is shed upon activation into the blood stream. A study was conducted about the different biosensing techniques, and a label-free electrochemical biosensor approach was chosen for this work. Electrochemical biosensors have been shown to be robust and can be manufactured with relatively low-cost materials. Commercial graphene oxide and carbon nanofiber screen printed electrodes were acquired to establish a working proof of concept. The sensors were electrografted with 4-carboxyphenyl diazonium salt to create support groups for the subsequent attachment of human soluble P-selectin antibodies through EDC/NHS crosslinking chemistry. Square wave voltammetry was identified as a very sensitive diagnostic technique that can be used to quantify the concentration of biomolecules in a sample. Ferricyanide/ferrocyanide and hexaammineruthenium(III) chloride was used as the two redox probes for the voltammetry measurements. In parallel, an application specific potentiostat device was developed to apply the square wave potential and measure the sensor response. The portable device had a potential range of ±1:65 V and a current range of ±244 µA. The project used three different approaches for the detection tests over a range of five full scale experiments. Even though none of the approaches were successful in realizing a linear detection range, one approach showed a difference in response between sP-selectin and non-specific CRP. Due to an unfortunate event with the sensors at the closing of the project, the home-built potentiostat could not be used to perform the final detection measurements of the immunosensor. However, it showed excellent performance against a conventional potentiostat device for detecting ferricyanide/ferrocyanide concentrations in the range of 0.5 to 5 mM. The work done in this thesis showed that if the immunosensor was optimized with the necessary equipment and materials, it would have been possible to use the home-built potentiostat to quantify the level of soluble P-selectin in a sample.

AFRIKAANSE OPSOMMING: Die hoof oorsake van dood is besig om te verander van oordraagbare siektes, soos Tuberkulose, na nie-oordraagbare siektes, soos kardiovaskul^ere siektes. Kardiovaskul^ere siektes, tesame met ander siektes, word geassosieer met kroniese in ammasie wat ook interafhanklik is met geaktiveerde bloedplaatjies. Geaktiveerde bloedplaatjies stel biomerkers vry in die bloedstroom wat gebruik kan word om siektes te diagnoseer en monitor. Huidiglik is daar geen hoogs akkurate toetse wat kroniese in ammasie kan meet nie, en konvensionele toeste is soms duur, tydrowend, en onprakties in vèrafgeleë areas. Dit is duidelik dat daar plek is vir verbetering in die diagnosering van in ammasie. Die projek se invalshoek was om 'n biomerker te kry wat 'n goeie aanduiding is van bloedplaatjie aktivering, en wat opgereguleer is in in ammatoriese siektes. Oplosbare P-selectin was geïdentifiseer as 'n plaatjie membraan reseptor wat vrygestel word in die bloedstroom wanneer die plaatjie aktiveer. 'n Studie was gedoen oor die verskillende tipe biosensors, en 'n etiket-vrye elektrochemiese biosensor was gekies. Elektrochemiese biosensors het robuuste kenmerke en dit kan vervaardig word met lae koste materiale. Kommersïele grafiet oksied en koolstof nanovesel gedrukte elektrodes was aangekoop om die sensors te vervaardig. Die elektroplatering van 4-karboksiefeniel diazonium sout was gebruik om ondersteunende groepe te skep op die sensor sodat menslike oplosbare P-selectin teenliggaampies geheg kan word deur middel van EDC/NHS bindings chemie. Vierkantsgolf voltammetrie was geïdentifiseer as 'n baie sensitiewe diagnostiese tegniek wat gebruik kan word om elektrochemiese metings te neem. Ferrisianied/ferrosianied en ruteniumheksamien(III) chloraat was gebruik as die twee redoks probes vir die voltammetriese metings. 'n Potentiostaat toestel was ontwikkel om die vierkantsgolf potensiaal toe te pas op die sensors, en die reaksie te meet. Die draagbare toestel kon spannings toepas in die gebied van ±1:65 V, en strome meet in die gebied van ±244 µA. Die projek het drie verskillende metodes probeer om die proteïen te meet oor 'n reeks van vyf volskaalse toetse. Nie een van die metodes het daarin geslaag om 'n lineére aanwinste toon oor die konsentrasies wat getoets was nie, maar een van die metodes het wel 'n verskil in reaksie getoon tussen sP-selectin en nie-spesifieke CRP. As gevolg van probleme met die sensors aan die einde van die projek, kon die ontwikkelde potentiostaat nie gebruik word om die metings te neem nie. Die toestel het wel uitstekende resultate getoon vir die meting van verskillende konsentrasies (0.5 tot 5 mM) van die ferrisianied/ferrosianied redoks probe, en die werking van die masjien was geverifieer deur 'n konvensionele potentiostaat as 'n maatstaaf te gebruik. Die werk wat in hierdie tesis verrig was het gewys dat, as die P-selectin biosensor geop-timaliseer word met die benodigde toerusting en materiale, dit moontlik sou wees om die ontwikkelde potentiostaat toestel te gebruik om die konsentrasie van oplosbare P-selectin in 'n oplossing te meet.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/107134
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