Engineering tools at the nano-bio interface have enabled transformative advances in molecular diagnostics, therapeutic monitoring, and cellular manipulation. However, challenges remain in achieving continuous real-time sensing, intracellular probing, and controlled actuation within an integrated, multifunctional platform. Nanotechnology, particularly through localized surface plasmons (LSPs), addresses these challenges by leveraging radiative decay for enhanced optical sensing (e.g., SERS) and non-radiative decay for nanoscale actuation (e.g., photothermal effects and vapor nanobubbles). Conventional plasmonic systems, however, are limited in wavelength multiplexing, versatility, and spatial mode overlap. To overcome these shortcomings, this dissertation presents a wavelength-multiplexed multimodal optical nano-bio interfaces enabled by multiresonant plasmonic architectures. These systems combine advanced plasmonic designs with intimate bio-nano interfaces, achieving multifunctionality across a broad spectral range for biochemical sensing and nanoscale actuation. The core platform is built on metal-insulator-metal (MIM) plasmonic nanolaminate nanopillar arrays (NLNPAs), which provide tunable multiresonant responses, nanoscale mode overlap, and an intimate bio-nano interface. For biochemical sensing, the multiband plasmonic resonances enable broadband surface-enhanced Raman scattering (SERS), offering high sensitivity and molecular specificity across a wide spectral range. This capability facilitates high-dimensional molecular fingerprinting, providing insights into spatial-temporal biochemical processes. Additionally, the platform enhances nonlinear optical processes, such as second- and third-harmonic generation (SHG/THG), enabling broadband, label-free sensing and bio-actuation with tunable performance. Beyond sensing, the multiresonant plasmonic interface supports precise nanoscale actuation through femtosecond laser-induced vapor nanobubbles. This approach enables highly localized, minimally invasive membrane permeabilization—optoporation—facilitating intracellular biochemical sensing and molecular delivery with nanoscale precision. Such capabilities hold significant promise for applications in bio-nanophotonics, targeted drug delivery, and cellular biochemical analysis, offering a pathway for advancing molecular diagnostics, minimally invasive therapies, and precise nanosurgery. As a proof-of-concept, a vapor nanobubble-enabled regenerative SERS sensing platform is demonstrated for continuous, wavelength-multiplexed biochemical monitoring. By combining photothermal nanocavitation with plasmonic SERS hotspots, the system achieves ultrasensitive molecular detection in protein-rich biofluids, such as bacterial biofilms associated with chronic wounds. This platform allows real-time monitoring of biochemical evolution in complex biointerfaces, offering a robust tool for continuous molecular fingerprinting in dynamic biological systems. Collectively, these advancements establish the wavelength-multiplexed multimodal optical nano-bio interface as a versatile platform that bridges the gap between nanoscale optical engineering and biological applications. By enabling simultaneous spatial-temporal sensing and actuation with nanoscale precision, this work paves the way for transformative applications in molecular diagnostics, real-time therapeutic monitoring, and cellular biochemical analysis. Future efforts toward portable instrumentation and integration with wearable or implantable technologies will further enhance the platform's potential for non-invasive, real-time monitoring in clinical and healthcare settings, driving forward the future of bio-nanophotonics. / Doctor of Philosophy / The ability to observe, analyze, and control biological processes at the tiniest scales—down to individual cells and molecules— has the potential to transform our understanding of life and revolutionize medicine, diagnostics, and healthcare. Imagine tools that can simultaneously detect disease-related molecules, deliver medicine with pinpoint accuracy, and monitor changes happening inside cells in real time. Achieving this, however, is no small feat. Existing tools often lack the ability to perform multiple tasks at once or adapt to the dynamic nature of living systems. To address this, we developed a new type of nano-bio interface that uses specialized nanostructures to interact with light in unique ways. These tiny structures can trap and amplify light across a wide range of colors, allowing us to achieve multifunctional capabilities at different colors: detecting molecules, probing inside cells, and even triggering specific biological responses using short bursts of laser light. For sensing, the system enhances Raman spectroscopy, a technique that reads the molecular "fingerprints" of chemicals, helping us detect and identify molecules with high precision. For cellular manipulation, we use short laser pulses to generate tiny bubbles that can temporarily open cell membranes—optoporation—enabling drug delivery or accessing the cell's biochemical content without causing harm. Additionally, the system can monitor changes over time, such as the molecular activity within bacterial biofilms, which are responsible for chronic infections. This work opens exciting new possibilities for medicine and biology: detecting diseases earlier, delivering therapies more precisely, and analyzing biological processes in real time. In the future, these nano-tools could be incorporated into portable devices, wearables, or implants, enabling doctors and scientists to monitor health and treat diseases in ways that are faster, safer, and more effective.

Advances in several areas of scientific research is currently hampered by the slow progress in developing a non-viral, high precision technique capable of safely and efficiently injecting targeted single cells with impermeable molecules. To date, one of the most promising techniques employs the laser to temporarily create a pore in the cell membrane to allow the entry of exogenous molecules. This technique has potentially wide applications. In this thesis, I utilised the precision of laser transfection, also known as optoporation, to deliver two histone demethylase inhibitors (8-hydroxyquinoline and FMF1293) of the JmjC-domain protein JMJD3 into vital cells. The enzyme, JMJD3, demethylates histone H3 lysine K27, the methylation state of which has been shown in previous studies to regulate genes in such a way as to play a key role in the formation of tumours and even maintenance of stem cell pluripotency. The research here shows proof of principle that optoporation can be employed to quickly screen and test the efficacy of novel drugs by delivering them into cells at significantly low concentrations while still maintaining inhibition activity. I also used optoporation to deliver relatively large proteins such as bovine serum albumin (BSA), phalloidin and novel synthetic antibodies into living cells without fixatives. This offers the possibility of using reporter systems to monitor living cells over time. Finally, an attempt was made to generate iPS colonies by optoporating plasmid DNA into somatic cells, however, I find that this technique was unable to efficiently transfect and reprogram primary cells. Two automated image-based systems that can be integrated into existing microscopes are presented here. First, an image processing algorithm that can quickly identify stem cell colonies non-invasively was implemented. When tested, the algorithm’s resulting specificity was excellent (95 – 98.5%). Second, because optoporation is a manual and time consuming procedure, an algorithm to automate optoporation by using image processing to locate the position of cells was developed. To my knowledge, this is the first publication of a system which automates optoporation of human fibroblasts in this way.

Abstract This thesis reports on studies of plasmonic nanoparticles and particularly gold nanostars as signal enhancers and contrast agents for biophotonic applications including visualisation, treatment of living cells and chemical sensing. In this thesis, the optical properties of nanoparticles of different size and morphology and their silica composites were compared. Because they are the most suitable plasmonic nanostructures, gold nanostars were utilised for optical imaging modalities such as confocal microscopy and Doppler optical coherence tomography. The ability of gold nanoparticles to enhance the signal in surface-enhanced vibrational spectroscopy, including Raman and Fourier transform infrared spectroscopy was additionally studied. Finally, various gold nanoparticles were applied for cell optoporation to increase the penetration ability of exogeneous substances. In summary, significant advantages of nanostars such as their low-toxicity, high scattering and contrast abilities, in addition to a broad, tunable, plasmon resonance wavelength range, as well as the capability to enhance the signal of analyte molecules in vibrational spectroscopy were demonstrated in this thesis. The results of this study on the effectiveness of nanostars have a broad scope of utility and open a wide perspective for their utilisation in nanobiophotonics and biomedicine. / Tiivistelmä Tämä opinnäytetyö kertoo tutkimuksista, joissa plasmoninanopartikkeleita ja erityisesti kultananotähtiä on käytetty signaalinvahvistimina biofotoniikan sovelluksissa, kuten visualisointi, elävien solujen käsittely ja kemiallinen tunnistus. Tässä työssä verrattiin eri kokoisten ja muotoisten nanopartikkeleiden ja niiden piioksidikomposiittien optisia ominaisuuksia. Sopivimpina plasmoninanorakenteina kultananotähtiä käytettiin optisiin kuvantamismenetelmiin, kuten konfokaalimikroskopiaan ja Doppler-optiseen koherenssitomografiaan. Lisäksi kuvattiin myös kultananopartikkelien kykyä parantaa pinta-aktivoidun värähtelevän spektroskopian signaalia, mukaan lukien Raman- ja Fourier-muunnos-infrapuna-spektroskopia. Lopuksi, eri kultananopartikkeleita käytettiin soluoptoporaatioon eksogeenisten aineiden läpäisevyyden lisäämiseksi. Yhteenvetona, työssä osoitettiin nanotähtien merkittävät edut, kuten matala-myrkyllisyys, suuret sironta- ja kontrastiominaisuudet, laaja plasmoniresonanssin aallonpituusalue ja sen viritettävyys, sekä kyky parantaa analyyttimolekyylien signaalia värähtelyspektroskopiassa. Niinpä tutkimustulokset nanotähtien tehokkuudesta ovat laajasti käyttökelpoisia ja ne avaavat laajan näkökulman niiden hyödyntämiseen nanobiofotoniikassa ja biolääketieteessä.