Author: Tsang, Ming Kiu
Title: Synthesis and characterization of upconversion nanocrystals for biodetection
Advisors: Hao, J. H. (AP)
Degree: Ph.D.
Year: 2018
Subject: Hong Kong Polytechnic University -- Dissertations
Nanostructured materials -- Optical properties
Rare earth metal compounds
Department: Department of Applied Physics
Pages: xxi, 133 pages : color illustrations
Language: English
Abstract: The recent development of lanthanide (Ln³⁺) ions-doped upconversion nanocrystals (UCNCs) had provide new opportunities for them in diverse biological applications, such as bioimaging, biodetection, drug delivery and phototherapeutic systems. The Ln³⁺ ions play important role in these applications because they are able to harvest near infrared (NIR) photons and convert them into visible photons via upconversion (UC). Owing to the transparency of the NIR excitation to the biological samples, the upconversion luminescence (UCL) process poses no damage to the samples with absence of background fluorescence. Among the aforementioned biological applications, the unique UCL property can improve the existing gene-based virus oligonucleotide (oligo) detections. Conventional clinical virus gene detection methods, such as reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assays (ELISA) are limited by some drawbacks. For instance, RT-PCR technique requires trained personnel to execute complicated procedures and the sample turnaround time is long while ELISA presents relatively low sensitivity. Owing to the limitations of these techniques, luminescent assays are developed as alternatives for rapid and sensitive detection. Ultraviolet (UV)-excited semiconductor quantum dots were used in downconversion assays. However, the gene oligo backbones are sensitive to UV and thus, prone to degradation. UV also induces severe background fluorescence that affects the optical readout by resulting in false positive or negative detections. As a result, these factors pave the way for the emergence of UCL in biodetection assays.
Luminescent assays are divided into homogeneous and heterogeneous assays. In general, the heterogeneous assays present superior sensitivity to homogeneous assays owing to the high binding affinity between the nanoprobes and analytes. Therefore, a hybrid nanoprobe/nanoporous membrane heterogeneous platform was designed for ultrasensitive detection of Ebola virus oligos. The detection principle was manifested by the luminescence resonance energy transfer (LRET) between the BaGdF₅:Yb/Er UCNCs and gold nanoparticles (AuNPs). The amine-functionalized UCNCs were anchored onto the nanochannels of the amine modified anodized alumina (NAAO) membrane by using glutaraldehyde. The probe oligo was conjugated onto the surface of the anchored UCNCs by the same method. The proof-of-concept system was firstly tested with simulated Ebola virus oligo sequence and yielded a limit of detection (LOD) at 300 femtomolar (fM). Furthermore, an improvement in LOD was found at around 20 folds compared to that of the homogeneous system. The improvement may be attributed to the increased surface area for hybridization in the NAAO membrane. Finally, the assay was tested with extracted oligos from inactive Ebola viron samples. The results indicated that the NAAO-based heterogeneous assay was able to achieve the detection in the fM range. The novel sensor also featured low-cost, rapid and high sensitivity. Although the heterogeneous sensor showed high sensitivity, the system has high potential for further improvements, such as specificity, fabrication cost and ease of handling. Therefore, the sandwich detection scheme consisting of two sections of oligo probes on the nanoprobes was devised to enhance the specificity of the nanosensor. The oleate-capped NaGdF₄:Yb/Er@NaGdF₄:Yb/Nd core-shell upconversion nanocrystals (csUCNCs) were synthesized by the coprecipitation method. The UCNCs were treated with acid and conjugated with polyacrylic acid for probe conjugation using covalent coupling. 15 nm AuNPs were selected as the quencher because of its high quenching efficiency and overlapping with the emission profile of the csUCNCs. On the other hand, the instantaneous oligo probe conjugation via acid-assisted method increased the fabrication efficiency. In order to retain the high LOD of heterogeneous assay, the csUCNCs were coupled with the nanoporous polystyrene spheres (nPS) to form a composite. Although the system is not validated with simulated or clinical samples at the moment, it is expected that the cs/nPS composite can increased the LOD and ease of handling for future on-site virus rapid screening application.
Rights: All rights reserved
Access: open access

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