Plasmonics in Biology and Medicine XXI

Surface-enhanced Raman spectroscopy (SERS) has made significant progress in recent decades, primarily driven by the principles of plasmon on metal surfaces. In contrast, SERS on non-metal substrates is based on the chemical mechanism involving charge transfer (CT) processes within irradiated molecules and the resonance Raman effect. This plasmon-free SERS mechanism proves highly suitable for detecting biomedical samples, as it suppresses the photo-thermal conversion associated with plasmon. In this study, we developed non-metal SERS substrates using conducting polymer nanofibers through electropolymerization. We evaluated the CT process and performance of the conducting polymer SERS substrates.

Circulating miRNA biomarkers have been shown to have diagnostic potential for several types of cancer and other diseases. Here, we present the integration and optimization of the inverse Molecular Sentinel (iMS) miRNA probe technology with a reproducible SERS substrate, resulting in a sensing platform capable of amplification-free multiplexed detection of miRNAs of interest. In this work, we showcase the versatility of this SERS-based liquid biopsy by detecting different miRNA panels from patient samples with various types of disease. Further, we highlight the integration of this sensing platform with novel upstream sample processing techniques capable of improving diagnostic utility.

We investigate the effect of proximity of a Surface Enhanced Raman Scattering (SERS) substrate on the Raman signal intensity. We deposited Rhodamine monolayers onto the concave side of a glass lens which was "sandwiched" with a SERS substrate. An airgap of variable thickness was formed between the lens and SERS substrate, which exhibited interference fringes, consisting of concentric "Newton's Rings" around the point of contact. We observed pronounced Raman signals in these structures. The Raman signal varied by position within the rings, with the intensity being higher closer to the contact point, compared to the intensity measured outside the rings.

We will discuss here a new seed-mediated synthesis of BGNS-Ag in which the morphology of the BGNS-Ag can be tuned precisely. Thus their plasmon resonances can be tuned by changing the concentration of the chemicals required in this synthesis. Herein we show how the concentration of AgNO3 plays a significant role in the spike length and spike sharpness. To illustrate the usefulness of the BGNS-Ag morphology in practical real-world in-field applications, we will discuss that the BGNS-Ag can be utilized as a solution-based SERS detection of a variety of analytes including illegal drugs (heroin, cocaine, and fentanyl), biomarkers (pyocyanin, methimazole), pesticides (thiram, ziram), and many more.

Extracellular vesicles (EVs) are readily being explored for disease prediction and diagnostics. We demonstrated the utility of a nanobowl SERS substrate for reproducicble, and label-free characterization of EVs.

In this work, we have employed several novel large-area nanofabrication methods to fabricate different surface-enhanced Raman scattering (SERS) sensor chips. The SERS sensor chips can be uniformly developed over a large-area with high reliability and reproducibility. These SERS sensor chips are employed for the detection of chemical molecules (such as pesticides and dyes) and biological molecules. Furthermore, numerical modeling of the proposed substrates was carried out using Finite Difference Time Domain (FDTD) modeling to study the effect of structural parameters of the plasmonic nanostructures on the resonance wavelengths and the electromagnetic (EM) enhancement factors.

We develop highly sensitive graphene-based surface-enhanced Raman spectroscopy (SERS) substrates where monolayer graphene is coated on the Ag/mica substrates. SERS has attracted attention in biosensor applications that allow label-free detection with high specificity. Recently two-dimensional (2D) materials such as graphene, h-BN, and MoS2 have been explored as a new platform for SERS substrates. These materials have the advantage of being non-metal, with excellent biological compatibility, endurance, and uniformity characteristics. Then, we study the stacked SERS substrates where monolayer graphene is transferred on the mica substrates with single-crystal Ag thin films deposited on the back to improve SERS efficiency. The fabricated SERS substrates show stronger Raman signals than the mica substrates with Ag thin films. Then sandwich enzyme-linked immunosorbent assay (ELISA) is fabricated on the SERS substrates to detect bovine interleukin-6 (IL-6), inciting bovine mastitis.

To improve the LFIA sensitivity, we have utilized high aspect-ratio plasmonic gold nanostars (GNS), which possess higher optical brightness than traditional gold nanospheres. We will discuss here the surfactant-free GNS synthesis in which the aspect ratio of the GNS can be tuned by changing the concentration of three reagents, ascorbic acid, silver nitrate (AgNO3), and hydrochloric acid (HCl). Herein, we selected the bacterium Yersinia pestis as a model analyte system, and the LFIA efficiency was investigated with the optical density measurements of the test line by utilizing different aspect-ratio GNSs. Our results showed that the maximum LFIA sensitivity was achieved with the GNS morphology having the maximum aspect-ratio spikes. Compared to other nanoparticle-based LFIA systems, high aspect-ratio GNS exhibits high analytical sensitivity, indicating it to be a promising candidate to become a much more versatile and tunable LFIA sensor.

Optical sensors offer a wide spectrum of subtance detection capabilities, from biological sample diagnostics to hazardous material identification. As a valuble alternative to complex analytical techniques, these sensors ensure rapid results with minimal or no sample preparation, though lacking reusability. By exponsing metal nanoantennas to light at resonant wavelengths, collective oscilations termed localized surface plasmon resonances (LSPR) are induced through the excitation of conduction electrons. Inthis study, we present an innovative optical sensor featuring achieved using electron beam lithography (EBL) to create a 10x10 micron array, with each triangular gold nanoantenna measuring 285.3 nm in height. Analyzing the sensor´s optical properties through Fourier Transformation Infrared Spectroscopy (FTIR), we examined reflectance as a fuction of wavelength to determine resonance wavelengths.

Here we showcase caged gold nanostars (C-GNS), a novel nanoplasmonic platform developed by our group. This platform technology merges the tunable optical properties of nanostar-based particles with loadable hollow core-shell structures. A galvanic replacement-free synthesis was deployed for the synthesis of the surfactant-free C-GNS particles to improve biocompatibility. FEM modeling of the C-GNS structure reveals areas of intense electric field enhancement around the nanostar branches within the hollow shell region. These particles can be effectively loaded with small-molecule cargo to enable in vivo detection of nanoparticle accumulation prior to photothermal treatment of solid tumors.

The quantification of human immunodeficiency virus at point of care remains a challenge in resource limited settings. Optical biosensors offer a rapid and sensitive optical method for various biological materials by monitoring the dielectric permittivity changes at the interface of a transducer substrate and the analyte. This work focuses on exploring photonic crystal biosensor efficiency and sensitivity for viral load measurement. Photonic crystals were functionalized with anti-HIV-gp120 antibody before the addition of various concentrations of HIV pseudovirus. The samples were analyzed on photonic crystal coupled to a custom-built transmission spectroscopy setup that used white light as a light source. The results showed a red shift at different virus concentrations. These outcomes demonstrate that photonic crystal biosensors are sensitive enough to detect differences in virus concentrations. Therefore, photonic crystals have a potential in the development of photonic crystal-based biosensors for viral load quantification.

Unlocking the potential of colloidal metamaterials—artificial materials mirroring molecular structures—holds promise for diverse applications, from optical engineering to catalytic chemistry. Yet, orchestrating precise self-assembly of colloidal metamaterials remains challenging due to the lack of regioselective surface chemistry. Addressing this, we introduce a novel strategy employing DNA-patched nanoparticles to drive the self-assembly of colloidal metamolecules. By utilizing magnetic bead-assisted DNA cluster transfer, we overcome geometrical constraints, enabling regioselective DNA patches. This approach is highly scalable and versatile, affording diverse configurations. We showcase the creation of gold and silver nanoparticle-based colloidal metamolecules, demonstrating the strategy's broad applicability. Notably, we employ this method to position fluorescent nanodiamonds within silver nanocube dimers, enabling precise control over photophysical properties. Our approach revolutionizes colloidal metamaterial synthesis, paving the way for tailored nanoscale functionalities in fields such as biological sensing and optical physics.

Choroidal neovascularization (CNV) is a leading cause of vision loss in people with macular degeneration. Early detection and precise localization of CNV remain challenging using current imaging techniques. In this study, we present a triple imaging modality comprising photoacoustic microscopy (PAM), optical coherence tomography (OCT), and fluorescence microscopy (FM) imaging to enhance the diagnosis of CNV by leveraging the strengths of each modality. However, the long-term toxicity of traditional contrast agents limits their clinical application. To address this issue, we developed a novel contrast agent based on ultrasmall gold nanospheres (GNS), which improved renal excretion and enhanced PAM, OCT, and FM contrast. The ultrasmall GNS were fabricated using pulsed laser ablation (PLA) techniques and subsequently surface-modified with CALNN peptides and cysteamine molecules to form chain-like GNS clusters (CGC). The surface of the CGC was further conjugated with RGD peptides and indocyanine green (ICG) fluorescent dye to create ICG-CGC-RGD. The synthesized ICG-CGC-RGD contrast agent was intravenously injected into three New Zealand White rabbits with CNV models at a dose of 400 µ

We present numerical modeling results of plasmonic sensor chips employed for highly sensitive bulk sensing and localized sensing, with high tunability of the wavelengths of operation from the visible wavelengths to the infrared wavelengths. We investigate periodic arrays of plasmonic nanostructures present on a continuous thin film such that these plasmonic nanostructures have a combination of surface plasmon polaritons (SPPs) propagating on the surface of the nanostructures and localized surface plasmons (LSPs) in the vicinity of the nanostructures. In order to measure the sensitivity of these plasmonic sensors, we determine the shifts in the reflectivity minima are determined to obtain information on perturbations in localized (local binding analyte layer sensing) and bulk refractive index (bulk medium sensing) on the sensor surface.

We propose and experimentally demonstrate a surface plasmon resonance sensor based on D-shaped single mode silica optical fiber and Indium Tin Oxide (ITO) that operate in the infrared wavelength region. The experimental result revealed that, a sensor fabricated with 0.6dB insertion loss and coated with 120nm thickness ITO layer presented a resonance in the wavelength range of 1200nm - 1400nm, and a refractive index sensitivity of 1409.34 nm/RIU with R-squared 0.99 for the range 1.33 - 1.42 RIU. We investigated also the effects of the polishing depth of D-shaped fiber and the thickness of ITO film on the performance of the sensor sensitivity. Both the experimental result and the COMSOL numerical simulation result indicate that when the thickness of ITO film increases, the resonance wavelength shift to the longer wavelength. However, in order to ensure that the sensor performance is not affected, the polishing depth should be appropriately reduced when the thickness of ITO film is increases; conversely, when the thickness of ITO film decreases, the polishing depth should be increased.

This paper aims to identify the presence of early-stage cancer in individual living cells through the utilization of a surface plasmon resonance (SPR) prism-based biosensor device. The proposed investigation employs SPR phenomena to differentiate between healthy and cancerous cells, employing a multilayer sensing structure. A BK7 glass prism is used as the sensing platform, coated with a nanocomposite layer consisting of gold (Au), titanium dioxide (TiO2), and graphene. The refractive index (RI) range of cancerous adrenal gland (PC12) cells is found to be between 1.381 and 1.395. The numerical results demonstrate that the proposed biosensor, equipped with single and multilayer nanocomposite structures, exhibits high sensitivity, figure of merit (FoM), detection accuracy (DA), and signal-to-noise ratio (SNR) for both healthy and cancerous PC12 cells. As the concentration of cancerous PC12 biomolecules increases in healthy cells, the SPR angle shifts, indicating variations in the refractive index due to the presence of cancerous cell biomolecules. The measurement of refractive index modifications in cancerous PC12 cells of the adrenal gland is achieved through an angle interrogation

We fabricated and characterized sensitivity enhanced surface plasmon resonance sensor based on cladding etched multimode polymer optical fiber coated with gold/Indium Tin Oxide (ITO) bilayer. The fibre use for experiment has a core and cladding of 486μm and 500 μm, respectively. The cladding is completely etched out from 1cm long section of a piece of fibre using Dimethyl Sulfoxide. The etched section of the fiber is coated with 40nm gold layer followed by 30nm ITO layer. The sensor was tested by immersing it in a glucose solution of different refractive index ranging from 1.33 to 1.40. The experimental result shows that, the sensor exhibited a refractive index sensitivity of 2053 nm/RIU for the range 1.33-1.37 RIU and 3081 nm/RIU for the range 1.37-1.40 RIU, both with R-squared of 0.99. These sensitivity values are higher than other sensitivity enhancement approaches reported in the literatures. We belive that, the proposed sensor has the advantages of simple structure and easy fabrication process (which does not require tedious side polishing), cost effective and high sensitivity, which has a potential prospect in the field of biosensing and chemical sensing.

We present a Surface Plasmon Resonance Imaging (SPRI) biochip system to quantitatively detect micro-RNAs involved in the cytokine storm during an inflammatory response. The thiol composition of the self-assembled monolayer on the biochip gold surface was tuned to maximize the capture of RNAs at low concentrations. To further amplify this signal, we have developed a sandwich-like assay using oligonucleotides functionalized gold nanoparticles (AuNPs), synthesized at ambient temperature and optimized to have a high solubility in saline solutions. Sub-picomolar detection limit of those small RNAs was achieved with all these combined improvements.

In this work, we show a highly sensitive grating-based SPR sensor with wavelength interrogation enabled by the tunable laser from 1528 nm to 1565 nm. The SPR sensor was designed in COMSOL Multiphysics and fabricated using UV nanoimprinting lithography. The realized SPR sensor shows a compelling sensitivity of nearly 1200 nm/RIU. The figure of merit (FOM) as a ratio between the sensitivity and the full width at half maximum of the SPR dip is above 400. To our knowledge, such high sensitivity and FOM put our sensor on the forefront in the field.

Super-resolution imaging techniques enable the detailed examination of living organisms at the nanoscale, offering valuable insights into the interactions and collective movements within living soft matter. Previously, deterministic super-resolution methods like STED, MINSTED, and MINFLUX deliberately induce photobleaching to achieve sub-10nm resolution. Conversely, stochastic techniques such as STORM, dSTORM, PALM, and FPALM attain super-resolution by capturing signals from complex temporal behavior. However, increasing optical resolution often leads to more pronounced photobleaching. Even the advanced fluorescence microscopy method, lattice light sheet (LLS), is not exempt from this limitation. In this study, we present a non-photobleaching, long-term investigation technique called PINE. Using PINE, we identified emergent dynamics based on SYNC as a model for exploring group-level movements and shape changes at the macroscale, driven by nanoscopic rearrangements. PINE will pave the way for new long-term investigations of previously unexplored cellular dynamics.

CRISPR-Cas9 is significantly potential and versatile gene-editing treatment for neurodegenerative disorders. The CRISPR-Cas9 system incorporates a single guide RNA (sgRNA) and Cas9 nuclease, which helps system to bind to the target sequence, and makes a double-strand break respectively. Viral vectors, as traditional delivery system of CRISPR-Cas9, present potential safety issues such as immunogenic complications. In this work, directly observe in real time the dynamic CRISPR-Cas9 process in biological cells. Optical manipulation and imaging of nanocarriers of CRISPR-Cas with nanometer precision is desirable for noninvasiveness. We demonstrate precise trapping and rapid rotation of nanocarriers of CRISPR-Cas. With noninvasive nanoimaging and nanomanipulation, it is anticipated this work will make contributions to biological sciences and biotechnology fields.

Nanospeckle Illumination Microscopy (NanoSIM) utilizes plasmonic nanoisland structures to enable super-resolution surface imaging of live cells. By analyzing the intensity fluctuations of plasmonic nanospeckles, we achieved three-fold improved spatial resolution and the ability to identify multiple cellular structures. Experimental results demonstrate the potential of NanoSIM as an effective and versatile tool for investigating dynamic cellular processes within live cell membranes of HeLa cells, providing crucial insights into complex cellular interactions.

Phase Intensity Nanoscope (PINE) is a new super-resolution microscopy method to further improve the resolution of existing techniques. PINE utilizes gold nanorods as non-photobleaching probes and modulates phase differences between electric field components to distinguish nanorods within a diffraction-limited region. This phase-intensity separation enables continuous imaging without photobleaching. PINE achieved sub-10nm resolution of cellular structures through precise localization of populations of randomly distributed nanorod probes. The integrated phase-intensity device isolates nanorods within a diffraction-limited spot by modulating phase differences. The distribution of localized nanorods forms patterns of underlying structures. By defining features from probe distribution patterns and minimizing distances of each probe to its feature projection, PINE extracts sub-10nm structural information. This statistical analysis of precision localizations and probe distributions provides super-resolution below 10nm. PINE revealed 8nm actin filament widths in cells, a resolution not achievable by other techniques.

Surface plasmon resonance is a label free optical detection technique, which responds to refractive index variations that are induced by molecular binding incidents or binding affinities. The angle of incidence that triggers surface plasmon resonance is linked to the refractive index of the material. Because of its sensitivity, it is used as a real-time analytical approach that can be used for many different applications. Here, it was investigated for the measurement of human immunodeficiency virus concentrations. This was achieved by functionalizing gold coated slides by an antibody. To the functionalized gold coated surface, different viral concentrations were added. The samples were analyzed by a surface plasmon resonance biosensing system. The system detected differences in viral concentrations as demonstrated by the angular resonance dip shifts. These findings are useful towards the development of an optical biosensor to be used at point of care for the detection of viral load.

Surface-enhanced Raman scattering (SERS) generally occurs on a planar base plate. However, when the concentration of the detected object is reduced to a certain extent, the presence of the object cannot be detected even by using surface-enhanced Raman scattering. In this paper, we utilized micro-shaped capillary tubes with an inner diameter of 20 μm as SERS substrates to measure the high-intensity Raman signals that could not be detected using planar SERS substrates. According to our calculations, the micro-shaped capillary tube should be able to obtain approximately 350,000 times the scattered light intensity compared to traditional SERS substrates. Flat planar substrates can only measure a portion of the substrate, whereas substrates using capillary tubes can capture the entire substrate at once. Additionally, by creating thinner and longer glass tubes, we hope to achieve even higher scattered-light intensity.