Plasmonics in Biology and Medicine XXII

We used a digital approach for quantification in surface-enhanced Raman scattering (SERS). SERS present large temporal or spatial variations in intensities, particularly at low concentrations. The digital approach can then be implemented when the fluctuations involve only a small (preferably just one) number of species. In that case, the quantification can be done by producing analytical calibration curves that involve SERS digital event counts rather than absolute SERS intensity values. The procedure improve the linearity of the calibration range to lower concentrations and it can be applied in several applications, such as environmental analysis and immunoassays.

The target of this work is the detection of miRNA-183, that is highly expressed in chronic obstructive pulmonary disease (COPD). This microRNA was detected with a labelled molecular beacon (MB) immobilized on silver nanowires (AgNWs) used as SERS active surface. Sub-femtomolar detection limits were obtained with this approach after optimization of the sensing surface. Good specificity and the possibility of performing multiple cycles of measurements after regeneration were also demonstrated.

Plasmonic nanostructures have a wide application in biomedical monitoring via Surface-Enhanced Raman Spectroscopy (SERS). SERS is a promising approach for bacterial identification however reproducibility has been a long-standing challenge. Electrostatics holds promise towards a controllable bacterial SERS by enabling specific coverage of target cells. Here, we perform systematic study of electrostatic interactions between 785nm resonating plasmonic gold nanorods with surface charges of 29, 16 and -9 mV and representative Gram-negative Escherichia coli and Gram-positive Staphylococcus epidermidis bacteria with surface charge densities of 0.01 and 1.02 r/e (-) / 10-6 mm2 respectively in water. We illuminate the cell-nanorod interaction using cryo-electron microscopy and zeta potential measurements revealing charge densities and the local binding affinities and Raman spectroscopy testing effective signal enhancements. We also simulated the interaction energies revealing systematic understanding at the single particle level. Our work contributes to the reliability of plasmonic nanomaterials for precision diagnostics towards clinical translation.

The use of circulating miRNA biomarkers offers a rapid and non-invasive way to diagnose multiple cancers and other diseases at an early stage. We developed a platform combining the Inverse Molecular Sentinel (iMS) miRNA probe technology with a reproducible SERS substrate foundation to facilitate rapid and direct miRNA sensing. To address current challenges in plasmonics-based detection, we discuss the automation of bimetallic nanostar synthesis. This platform has been successfully used to detect various miRNA panels from patient samples with different diseases in less than hour 1. Additionally, we will discuss augmenting this biosensing platform with innovative sample processing techniques to enhance its diagnostic capabilities. This integration of sample processing, purification, and amplification-free sensing represents a significant step forward in creating a rapid point-of-care miRNA profiling system.

Surface-enhanced Raman scattering (SERS) tags, combining nanoparticles with Raman reporter molecules, offer high sensitivity and quantification for detecting target molecules. However, analyzing SERS spectra, especially in mixtures, is challenging due to interference and noise. This study developed a Convolutional Neural Network-based model trained on 10,000 augmented SERS spectra to identify components in mixtures. Baseline correction using airPLS and the Nonnegative Elastic Net algorithm enabled accurate ratio estimation of each reporter. The system used antibody-functionalized SERS tags for multiplex labeling and AI spectral analysis for component classification and concentration calculation in single cancer cells, showing high detection specificity. This method significantly enhances multispectral recognition, promising advances in tumor cell analysis.

In this work, we demonstrate a textured SiO2 substrate with Au plasmonic layer that can be fabricated on a standard 4” wafer, providing a path towards commercially viable SERS substrates. The resulting SERS substrate is characterized at the wafer scale using automated tools to measure uniformity, paving the way for consistent SERS chips.

In this study, we propose a simple and cost-effective approach to create a flexible SERS patch by integrating multi-branched gold nanostars (GNS) onto commercially available adhesive Scotch Tape, resulting in exceptionally high SERS sensitivity, reproducibility, and stability. Our SERS measurements show that GNS with a 2.5 nm branch tip diameter (GNS-4) delivers the most significant SERS enhancement. Using rhodamine 6G (R6G) as a benchmark analyte, we demonstrated that the flexible SERS patch achieved a limit of detection as low as 1 pM for R6G. The enhancement factor of the GNS-4 SERS patch was calculated to be 6.2×10^8, highlighting its potential for ultra-high sensitivity. The reliability of the substrate was confirmed through tests on 30 different spots, showing a relative standard deviation (RSD) in SERS intensity of about 5.4%, indicating consistent reproducibility.

Surface-enhanced Raman scattering (SERS) allows for detection of analytes using plasmonics-active noble metal nanoparticles, but there remains a push to optimize the morphologies of these structures to obtain higher sensitivity. Gold nanostars have shown exceptional plasmonic and SERS sensing capabilities, but with the implementation of magnetic properties, these characteristics can be further enhanced. The nanostar morphology is represented through sharp spikes that hold high localized surface plasmon resonance (LSPR), and through a method of “controlled aggregation”, the particles become clustered, allowing for the overlapping of the LSPR “hot spots”, leading to higher plasmonic activity. Here, we utilize surfactant-free magneto-plasmonic gold nanostars (mpGNS) for SERS sensing of pollutants. Our results have shown that mpGNS significantly improves SERS sensitivity, making it an optimal candidate for point of care (POC) detection and monitoring that can be used for a wide variety of analytes.

The present culture-based bacterial detection technique is slow and demands an alternative fast, sensitive and accurate bacterial detection methodology. We propose a porous Si microcavity-based surface-enhanced Raman spectroscopy substrate for sensitive and accurate detection of bacteria. The 3D finite difference time domain (FDTD) simulation is utilised to investigate the local field enhancement due to the microcavity structure. Further, the porous Si is strategically designed to increase the SERS signal reproducibility by surface functionalisation. The optimum enhancement was investigated by considering various layers of closely placed Au and Ag nanoparticles of 60 nm diameter to explore the local field enhancement. The advantage of cavity structure is that it allows the bacteria to passively trap inside the nanocavity due to the high surface topology of the cavity along the XY- direction and laser along the -Z direction. Hence, the study emphasises the strategy to remove reproducibility issues in bacteria detection using the SERS technique, which enables rapid and accurate identification of bacterial pathogens.

Detecting miRNAs is crucial for early disease diagnosis and prognosis. Traditional methods face significant challenges due to the intrinsic properties of miRNAs, such as their small size, short sequence length, low concentration levels, and high sequence similarity in complex real samples. To address these issues, we developed novel plasmonic biosensors using MXene to enhance performance for real-time, label-free miRNA detection, achieving a 10 fM detection limit and the capability of distinguishing single-base mismatches. Additionally, our dual-mode phase imaging plasmonic sensor improved sensitivity in complex environments and reduced nonspecific adsorption by 20%, enhancing the detection limit by 548 times, enabling simultaneous detection of multiple miRNA markers in clinical serum samples.

This study leverages the unique optical response and biocompatibility of plasmonic particles for biosensing. By probing sub-wavelength particles of varying dimensions synthesized using seed-mediated colloidal growth, we tune the resonance wavelength in the mid-infrared (mid-IR) range for multiplexed detection. Integrated with a quantum cascade laser (QCL)-based mid-IR spectral imaging system, this biosensor offers advantages such as superior plasmonic enhancement, high-throughput production, and ease of use in biological sample preparation. Numerical investigations were performed including both Finite Difference Time Domain (FDTD) simulation and multipole expansion computation. The platform was also benchmarked experimentally through the detection of Si-C absorption band of polydimethylsiloxane (PDMS) and amide I and II bands of bovine serum albumin (BSA). The method is simple, powerful, and versatile for future molecular detection and analysis in medical diagnostics and therapeutics.

Plasmonic sensors are known for their high sensitivities due to electromagnetic field enhancements at sub-wavelength scales, providing a powerful platform for early disease diagnosis. We present an innovative plasmonic biosensor on a compact photonic integrated circuit (PIC) enhanced by exceptional points (EPs). EPs are singularities in non-Hermitian systems, offer extreme sensitivity to external perturbations. We demonstrated EP in a single coupled nanoantenna particle within a novel silicon nitride junction-waveguide, by laterally shifting two different nanobars. This setup allows high-efficiency coupling and broadband measurements. Integrated with a four-port Mach-Zehnder interferometer, it enables accurate real-time eigenmode extraction. Encapsulated in a microchannel, our EP sensor outperforms linear diabolic point (DP) systems even at large perturbations. The integrated EP biosensor has shown single molecule sensitivity for both protein- and exosome-like particles. Our sensor, with its extreme sensitivity and compact size, offers a novel approach for detecting and quantifying biomarkers, facilitating early disease diagnosis.

A novel non-toxic method for diagnosing biofluid biomarkers using magneto-plasmonic nanoparticles is proposed. This method employs small magnetic nanoparticles with fluorescent surface-bound biomarker traps and larger magnetic nanoparticles (covered by a thin plasmonic shell) that capture the smaller ones in a strong magnetic field gradient. The interaction between an external permanent gradient magnetic field and the magnetic field of the large nanoparticles enables the capturing of small nanoparticles from fluid trajectories. The dependence of the fluorescent signal intensity of the dye molecule on the surface of a small magnetic nanoparticle on the distance and location relative to the plasmonic shell of a large plasmonic magnetic nanoparticle is studied. Simulations have shown that the intensity can be increased by coating large magnetic nanoparticles with a thin metal layer to enhance the plasmonic field intensity at the dye molecule area. It is shown that plasmonic nanoparticles significantly influence fluorescence intensity.

We have carried out the design, modeling, and fabrication of novel plasmonic nanoantennas and other plasmonic nanostructures as surface-enhanced Raman scattering (SERS) substrates. The main objective of this work is to achieve the highest possible enhancement factor of SERS, both theoretically and experimentally. Numerical modeling of the SERS substrates was carried out for different kinds of plasmonic nanoantennas and other nanostructures to determine the SERS electromagnetic enhancement factors. We have employed different nanolithography methods to fabricate the SERS substrates in a repeatable and reliable manner. These SERS substrates were employed for detecting biological molecules which are markers of infectious diseases.

We have developed a new nanoplasmonic platform called caged gold nanostars (C-GNS), which integrates the adjustable optical properties of nanostar-based particles with hollow core-shell loadable structures. The described synthesis does not require galvanic replacement and produces surfactant-free C-GNS particles to ensure high biocompatibility. Through finite element method (FEM) modeling, strong areas of electric field enhancement have been identified in the toroidal zone surrounding the nanostar branches within the hollow shell region of the C-GNS. These unique particles can be loaded with small-molecule cargo, enabling the detection of nanoparticle accumulation in living organisms before the photothermal treatment of solid tumors. Our team has also successfully carried out in vivo hyperspectral imaging of these particles and observed a notable rise in the temperature of the tumor area during photothermal therapy using several different modalities. Additionally, these particles may be coated in silver and incorporated into biosensing applications.

Gold nanostars (GNS) have garnered significant attention due to their remarkable optical properties, specifically localized surface plasmon resonance (LSPR), and their practical real-world sensing applications. Core-shell GNS, incorporating various core materials such as iron-oxide cores and silver shells, can enhance the LSPR properties of GNS. This enhancement depends on factors like size, shape, and core characteristics of the nanostars, including magnetic core GNS (mGNS) and silver-coated GNS (SGNS). LSPR combines the strong surface-enhanced Raman scattering (SERS) enhancement from gold nanostar spikes with the magnetic concentration effect to generate multiple hotspots for mGNS and the robust scattering properties of silver-gold nanostructures for SGNS. In this presentation, we introduce a novel seed-mediated synthesis approach for mGNS and SGNS that allows precise control over the morphology of gold nanostars.

Optothermal tweezers represent a novel approach in nanoparticle manipulation, offering a versatile and biocompatible method for trapping, sensing, and assembling a wide range of nanomaterials. These tools have been developed to address the limitations of traditional optical tweezers, such as high laser power requirements and the diffraction limit constraint. In this study, we introduce optothermal nanotweezers as a highly adaptable, low-power alternative for manipulating and sensing bionanoparticles in a solid medium. By leveraging the principles of thermophoresis and solid-liquid interface interactions, these nanotweezers can effectively trap and sort nanoparticles of various sizes, charges, and materials, including functional mesoporous silica for controlled cargo delivery, nanostructure assembling, and biosensing.

MicroRNAs (miRNAs) are crucial biomarkers for various diseases, including multiple cancers and neurodegenerative diseases like Alzheimer's, due to their regulatory roles in gene expression. Traditional detection methods often lack the sensitivity, speed, and simplicity required for accurate miRNA detection at low concentrations in point-of-care settings. We present an advanced genetic algorithm approach to optimize the design of DNA probes for our point-of-care inverse molecular sentinel (iMS) technology. This technology utilizes Surface-Enhanced Raman Scattering (SERS) for miRNA detection in blood and saliva samples. The novel genetic algorithm works in conjunction with the UNAFold algorithm to refine DNA probe parameters such as length, structure, melting temperature, and off-target binding. By iterating through multiple generations, the algorithm refines the probe design, leading to improved performance in detecting miRNA at low concentrations. This optimized iMS technology demonstrates enhanced sensitivity and specificity, which is vital for early diagnosis and monitoring of various diseases.

The rapid identification of different plastics and textiles is essential on the way towards efficient recycling pathways. Raman spectroscopy is a well-established method providing substance-specific information, but laser-induced fluorescence and strong absorption in case of carbon black colored materials are challenges. Shifted excitation Raman difference spectroscopy (SERDS) is a physical approach to overcome these issues. Pilot SERDS laboratory investigations are exemplarily conducted on 21 dyed and undyed textiles and 8 black plastics. An in-house developed 785 nm dual-wavelength diode laser serves as excitation light source. An excitation spot size of ca. 100 μm diameter and optical powers of 20-30 mW at the sample were selected. SERDS enables an efficient separation of material-specific target Raman signals from backgrounds and thus permits for rapid material identification of textiles and plastics with measurement times in the order of seconds. Results highlight the capability of SERDS as potential inspection tool for the recycling industry.

The colorimetric lateral flow immunoassay (LFIA) demonstrates remarkable potential for point-of-care (POC) diagnostics due to its portability, cost-effectiveness, and rapid visual biomarker detection capabilities, which eliminate the need for analytical instruments for quantitative detection. The sensitivity of colorimetric LFIA relies on the color visualization of antibody-labeled nanoparticles bound to target analytes at the test line. Traditional gold nanospheres demonstrate good optical brightness, however, the nanostar morphology significantly improves this characteristic. Through the implementation of a magnetic core, these properties can be even further enhanced, through “hot spot” overlap, leading to better visualization. To improve LFIA sensitivity, we have employed magneto-plasmonic gold nanostars (mpGNS) to enhance sensitivity through magnetic concentration of target analytes. We selected the SARS-CoV-2 spike protein-S1 as a model analyte system and investigated LFIA efficiency using visual colorimetric measurements. Our results demonstrated that maximum LFIA sensitivity and that our nanoplatform has the potential to significantly impact at-home diagnostics.

Diabetic retinopathy (DR) is a primary cause of vision impairment globally, yet current treatments are inadequate in preventing its progression. This study introduces a novel imaging approach utilizing multi-modal photoacoustic microscopy (PAM), optical coherence tomography (OCT), and fluorescent imaging to monitor retinal neovascularization (RNV) in DR. The imaging system leverages bipyramids gold nanoparticles (BPNPs) to enhance visualization of RNV. Animal related DR models were induced in Dutch belted rabbits using DLAAA, resulting in stable RNV development by 3 days post-injection and sustained over 1 year. At 1 month, BPNPs at a final concentration of 2.5 mg/mL (400 uL) were intravenously administered via the marginal ear vein. PAM images were acquired using two distinct wavelengths: 578 nm and 650 nm. The results indicate a significant 17-fold enhancement in PAM signal intensity post-injection, which gradually decreased after 14 days. This integrated imaging approach holds promise for advancing understanding of RNV dynamics in diabetic retinopathy and potentially guiding more effective therapeutic strategies.

Serial dilution of Balb/c rat DNA in the visible broadband plasmonic spectroscopy was explored as a promising technique to reduce this genomic substance concentration in the nanoscale. The physical DNA of a living creature, after extraction from the resources (such as blood, nail, hair, tissue, and other possible organs) assumes amorphous pile of strands in the microscale or larger. The serial dilution technique was exploited to exponentially decrease the DNA concentration of BALB/c rat – the target living mammal – in the nanoscale, and the diluted DNA was deposited over a thin film of gold (for fixturing and removing moisture). Plasmonic surface waves of different frequencies were found to promote wave propagation more efficiently when lower thickness of unstructured-nanoscale-DNA layer was deposited (over gold thin film). The results manifest that the most diluted DNA sample reveals distinguishable plasmonic conditions. The sensitivity of the configuration was enhanced in the presence of a DNA sample (as compared to the case of non-existing measurand), which was further increased for larger incidence angles.

Periodic nanostructured thin films of plasmonic materials like gold show great potential for biological and chemical sensors due to their sensitivity and specificity. However, market adoption is hindered by the lack of a scalable fabrication process. This talk demonstrates a scalable wafer-based approach using nanoimprint lithography to create periodic gold nanostructures. After optimizing materials and processes, a mini fabrication run of 24 x 6” wafers produced nearly 10,000 active devices with excellent control of nanofeature dimensions and no degradation in quality.