Surface Functionalization of Nanowires Using Silane-PEG Compounds

Abstract

Introduction: GaP nanowires were functionalized using silane-PEG-NH2 and compared with cellulose acetate, PAcrAm™-g-(PMOXA, amine, silane) and a non-functionalized reference sample. Functionalization would allow strong, covalent anchoring of antibodies instead of adsorption.

Aim: The thesis work focused on deciding on, and optimizing, a protocol for nanowire surface modification of the three mentioned functional compounds.

Methods: The different surface treatments were qualitatively compared using optical microscopy. The fluorescence of the different samples was analyzed using a full-factorial DOE of four concentrations of each functionalization incubated in four different concentrated antibody solutions. The functionalizations' success and the protocol alterations' impact were validated using SEM and TEM imaging.

Results: The functionalized samples had considerable drying effects following the initial treatments, visible in the optical microscope and SEM. All protocols were altered to allow slow drying, by covering the Petri dishes with a lid, resulting in significantly decreased drying effects. The detected fluorescent signal was higher for the silanized samples compared to the other functionalities and the reference. However, all functionalized samples without antibodies did reveal an exceedingly high signal, indicating cross-contamination. Further TEM examination of the different samples revealed that none of the functionalized nanowires had any additional compound on their surfaces. This indicates unsuccessful surface treatments for all functional compounds or functionalization of a small fraction of the sample, thus requiring a more extensive TEM analysis.

Conclusion: The surface treatment protocols were improved by implementing slow drying. However, a more extensive TEM examination is required to quantify the extent of the different surface modifications. Furthermore, repeats of the final protocol are required to validate the possible signal-enhancement observed for the silanized nanowires.

Popular Abstract

There are 104,000 people in the UK, aged 0-40, who are living with a life-limiting or life-threatening disease.[1] There are 300-400 million people in the world living with a rare disease, with 50% of them being untreated for decades. These diseases may be life-limiting or life-threatening, and almost always reduce the quality of life.[2, 3] Imagine a medical test that could detect these conditions at early stages, potentially saving many lives[4] and increasing the quality of life because the right treatment is provided. One newly developed immunological test, a.k.a. immunoassay, can according to Aligned Bio AB detect biomolecule concentrations 20 times lower than medical tests (e.g. ELISA) used today. These tests bind fluorescently labelled antibodies, antigens or biomarkers to signal-enhancing nanowires. The aim of my thesis was to come up with a protocol for surface functionalization of nanowires and investigate the potential signal effects.

Samples containing GaP nanowires grown in parallel rows and columns had already shown astonishing signal-enhancing properties when bound to fluorescently labelled biomolecules. All GaP nanowire samples were covered with a thin SiO2 layer, as this compound is commonly used for immunoassays because they readily bind to biomolecules such as antibodies.[5] The thesis work investigated the effect of surface modification of GaP/SiO2 nanowires. The hope was for these modifications, or functionalizations would create a strong covalent linkage between the investigated antibody and the nanowire surfaces. Theoretically, this would allow harsher washing procedures and utilize a stable bond in order to prolong the shelf life. A harsher washing procedure may decrease the background noise which would result in a decreased limit of detection (LOD). A lower LOD allows the detection of smaller biomolecule concentrations. A longer shelf life stabilizes the nanowire samples, preventing the nanowire-biomolecule complex from breaking down before analysis. [5, 6, 7]

The compounds used to modify the nanowire surfaces were silane-PEG-NH2 and compared with cellulose acetate, PAcrAm™-g-(PMOXA, amine, silane). These were chosen because of their ability to covalently bind to SiO2 and the fluorescently-labelled antibody used. These compounds also have antifouling properties, meaning that their chemical composition prevents unwanted molecules from binding.[6, 7] A nonfunctionalized sample was also used, where antibodies were adsorbed directly onto the nanowires. The protocol for functionalizing nanowire surfaces was based on literature done on flat surfaces. The protocol for PAcrAm™ was based on recommendations from the manufacturer because it is relatively new on the market and without many relevant references in the literature.

The main issue with all functionalization processes was the drying effects that appeared on all samples in optical microscope and scanning electron microscope (SEM). Drying effects indicate that uneven evaporation of the solvent occurred, thus creating an uneven film thickness formed around the nanowires. The protocol, therefore, had to be altered in order to increase the diffusion and allow slow drying. This was done by incubating the samples on see-saw shakers and drying the samples inside a Petri dish with lid on. This decreased the drying effects significantly. Furthermore, the detected fluorescent signal was higher for the silanized samples compared to the other functionalities and the reference. However, the control sample for all functionalized samples, which did not contain any fluorescing antibodies, did reveal a considerably high signal. The most likely reason behind this high signal is cross-contamination, invalidating all fluorescent results for the functionalized samples. Further transmission electron microscope (TEM) examination of the different samples revealed that none of the functionalized nanowires had any additional compound on their surfaces. These results reveal unsuccessful surface treatments for all functional compounds. The lack of functionalization compounds on the nanowire surfaces could also be a result of functionalization of a small fraction of the nanowires on each sample. A more extensive TEM analysis is therefore required to analyze a larger number of nanowires.

References
1 How many children and young people are affected by a life-limiting or life-threatening condition? [homepage on the Internet]. Together for Short Lives; [updated 2018 Jan; cited 2022 Sep 1]. Available from: https://www.togetherforshortlives.org.uk/app/uploads/2018/01/ProRes-How-
Many-Children-Young-People-Affected-By-A-Life-Limiting-or-Life-Threatening-Condition-Factsheet.pdf.
2 Horizon - the EU Research and Innovation Magazine [homepage on the Internet]. European Commission;[updated 2022 Feb 2; cited 2022 Apr 26]. Expanding research into rare diseases. Available from: https://ec.europa.eu/research-and-innovation/en/horizon-magazine/expandingresearch-rare-diseases.
3 Marwaha S, Knowles JW, Ashley EA. A guide for the diagnosis of rare and undiagnosed disease: beyond the exome. Genome Medicine. 2022;14:2–23. Available from: https://doi.org/10.1186/s13073-022-01026-w.
4 Harvard Health Publishing [homepage on the Internet]. Robert H. Shmerling; [updated 2021 Jan 28; cited 2022 Apr 26]. Are early detection and treatment always best?. Available from: https://www.health.harvard.edu/blog/are-early-detection-and-treatmentalways-best-2021012821816.
5 Verardo D, Lindberg FW, Anttu N, Niman CS, Lard M, Dabkowska AP, et al. Nanowires for Biosensing: Lightguiding of Fluorescence as a Function of Diameter and Wavelength. Nano Lett. 2018;18:47964802. Available from: http://dx.doi.org/10.1021/acs.nanolett.8b01360.
6 Migliorini E, Weidenhaupt M, Picart C. Practical guide to characterize biomolecule adsorption on solid surfaces (Review). Biointerphases. 2018;13:06D303. Available from: https://doi.org/10.1116/1.5045122.
7 Suwatthanarak T, Thiodorus IA, Tanaka M, Shimada T, Takeshita D, Yasui T, et al. Microfluidic-based capture and release of cancer- derived exosomes via peptide–nanowire hybrid interface. Lab Chip. 2021;21:597–607. Available from: http://dx.doi.org/10.1039/d0lc00899k.

• author: Ekfors Elvin, Hedvig
• supervisor: Martin Ek; Karin Blume; Niklas Mårtensson; Søren Echwald
• organization: Centre for Analysis and Synthesis
• alternative title: Optimization of Cutting-Edge Nanowire Immunoassay
• course: KASM10 20221
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Functionalization, Nanowires, Silanization, Surface Chemistry, Materials Chemistry
• language: English
• id: 9101146
• date added to LUP: 2022-10-11 14:54:12
• date last changed: 2022-10-11 14:54:12