Applications of Photothermal Conversion of Gold Nanoparticles in Diagnostics and Cryopreservation

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Applications of Photothermal Conversion of Gold Nanoparticles in Diagnostics and Cryopreservation

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2022-06

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Lateral flow assays (LFAs) are paper-based point-of-care (POC) diagnostic tools that are widely used because of their low cost, ease of use, and rapid format. Within LFA, the presence of disease analytes was visualized by capturing and accumulating gold nanoparticles (GNPs) labels at the test line. Unfortunately, traditional commercial LFAs have significantly poorer sensitivities (µM) than laboratory tests (enzyme-linked immunosorbent assay, ELISA: pM – fM; polymerase chain reaction, PCR: aM), thus limiting their impact in disease control. To boost the sensitivity of LFAs, thermal contrast amplification (TCA) was used in this dissertation as a simple add-on reading step after assay completion. Specifically, TCA used a laser to excite the GNPs from test line, whose photothermal signals were then recorded by a sensitive IR camera. As a result, subvisual positive (i.e., visually false negative) LFAs were identified.To demonstrate the advantage of TCA for influenza rapid diagnosis, a prospective cohort study was conducted on 345 clinical specimens collected for influenza A and B testing during the 2017-2018 influenza season. Through the TCA detecting sub-visual weak positives, TCA reading improved the overall influenza sensitivity by 53% for influenza A and 33% for influenza B over the visual LFA readings. It was also observed that the specificity was compromised slightly by the TCA protocol (i.e., slightly increased false positives), which was mainly due to amplified noise from nonspecific binding (NSB) of GNPs at the test line. Despite this, the overall performance was still better than that achieved by visual readout based on comparison of their plots in receiver operating characteristic space and F1 scores. To further boost sensitivity of LFAs, assay optimization was also needed to substantially increase specific binding (SB) while reducing NSB. To accomplish this, we created an advanced LFA with comprehensive assay redesign for enhanced SB/NSB ratio and TCA as signal amplification, which enabled fM – aM detection sensitivity for SARS-CoV-2 spike receptor-binding domain (RBD) proteins within 30 min. The advanced LFA can visually detect RBD proteins down to 3.6 aM and 28.6 aM in buffer and human nasopharyngeal wash, respectively. This is the first reported LFA achieving sensitivity comparable to that of the PCR (aM-zM) by visual reading, which was much more sensitive than traditional LFAs. A fast (< 1 min) TCA reading algorithm was also reported, with results showing that this TCA could distinguish 26%~32% visual false negatives for clinical commercial LFAs. When our advanced LFAs were applied with this TCA, the sensitivities were further improved by 8-fold to 0.45 aM (in buffer) and 3.6 aM (in human nasopharyngeal wash) with semi-quantitative readout. In addition to assay improvement, this dissertation also explored the limit TCA reading of gold nanoparticles (GNPs/mm2) at test regions in immunoassays. More specifically, we built and compared fast (minute scale) and ultrafast (seconds scale) TCA setups using continuous-wave (CW) and ms pulsed lasers, respectively. TCA improved the limit of detection (LoD) for silica-core gold nanoshells (GNSs) in lateral flow immunoassays (LFAs) by10~20-fold over visual reading. While the ultrafast TCA led to higher thermal signals, this came with a 2-fold loss in LoD vs. fast TCA primarily due to noise within the infrared sensor and a necessity to limit power to avoid burning. To allow higher laser power, and therefore amplification fold, we also explored transparent glass coverslip substrate as a model microfluidic immunoassay (MIA). It was found the ultrafast TCA reading of GNS-coated coverslips achieved a maximal signal amplification (57-fold) over visual reading of LFAs. Therefore, ultrafast TCA-MIA is promising for ultrasensitive and ultrafast diagnostics. Further advantages of using TCA in MIA vs. LFA could include lower sample volume, multiplexed tests, higher throughput, and fast reading. In summary, TCA technology is able to enhance the sensitivity and speed of reading GNPs (GNPs/mm2) within both LFAs and MIAs. To further explore the use of photothermal GNPs, a fundamental study was carried out in this dissertation to characterize the photothermal properties of various GNPs. Different gold nanorods (GNRs) and GNSs were synthesized and characterized by using a modified laser-cuvette calorimetry method with complementary Monte Carlo modeling. Results showed that GNSs had much larger scattering-to-absorption ratios (γ) than GNRs. GNSs with larger γ could have ~30% over-estimation of photothermal conversion efficiency if scattering and reabsorption inside their solutions was not considered, while GNRs with lower ratios were less impacted. As an example application in laser warming of vitrified droplets where biospecies can be loaded, simulations (i.e., Monte Carlo and COMSOL) were used to predict the heating behavior of GNP-loaded hemispherical droplets. Results demonstrated that the GNS case with larger γ achieved more uniform heating than the GNR case. Within the model, uniform heating could be potentially achieved by tuning the γ of droplets via loading designed GNPs. In summary, this dissertation mainly focused on ultrasensitive point-of-care diagnostics based on photothermal GNPs. It demonstrated improved sensitivity by TCA reading of commercial LFAs in a cohort study and explored ultrasensitive rapid testing by both assay optimization for LFA and upgraded TCA systems. In addition, a fundamental study was conducted to understand photothermal properties of various GNPs and their possible application in laser warming of vitrified droplets.

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University of Minnesota Ph.D. dissertation. June 2022. Major: Mechanical Engineering. Advisor: John Bischof. 1 computer file (PDF); xxvi, 243 pages.

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Liu, Yilin. (2022). Applications of Photothermal Conversion of Gold Nanoparticles in Diagnostics and Cryopreservation. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/241602.

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