A Systematic Approach toward Enabling Maximal Targeting Efficiency of Cell Surface Proteins with Actively Targeted SERS Nanoparticles.

With their intricate design, nanoparticles (NPs) have become indispensable tools in the quest for precise cellular targeting. Among various NPs, gold NPs stand out with unique features such as chemical stability, biocompatibility, adjustable shape, and size-dependent optical properties, making them particularly promising for molecular detection by leveraging the surface-enhanced Raman scattering (SERS) effect. Their multiplexing abilities for the simultaneous identification of multiple biomarkers are important in the rapidly evolving landscape of diverse cellular phenotypes and biomolecular profiling. However, the challenge is ensuring that SERS NPs can effectively target specific cells and biomarkers among intricate cell types and biomolecules with high specificity. In this study, we improve the functionalization of SERS NPs, optimizing their targeting efficiency in cellular applications for ca. 160 nm NP-based probes. Spherical SERS NPs, conjugated with antibodies targeting epidermal growth factor receptor and human epidermal growth factor receptor 2, were incubated with cells overexpressing these proteins, and their specific binding potential was quantified at each stage by using flow cytometry to achieve optimal targeting efficiency. We determined that maintaining an average of 3.5 × 105 thiols per NP, 300 antibodies per NP, 18,000 NPs per cell, conducting a 15 min staining incubation at 4 °C in a shaker, and using SM(PEG)12 as a cross-linker for the NP conjugation were crucial to achieve the highest targeting efficiency. Fluorescence and Raman imaging were used with these parameters to observe the maximum ability of these NPs to efficiently target suspended cells. These highly sensitive contrast agents demonstrate their pivotal role in effective active targeting, making them invaluable for multiplexing applications across diverse biological environments.

Nanomaterial-based contrast agents.

Medical imaging, which empowers the detection of physiological and pathological processes within living subjects, has a vital role in both preclinical and clinical diagnostics. Contrast agents are often needed to accompany anatomical data with functional information or to provide phenotyping of the disease in question. Many newly emerging contrast agents are based on nanomaterials as their high payloads, unique physicochemical properties, improved sensitivity and multimodality capacity are highly desired for many advanced forms of bioimaging techniques and applications. Here, we review the developments in the field of nanomaterial-based contrast agents. We outline important nanomaterial design considerations and discuss the effect on their physicochemical attributes, contrast properties and biological behaviour. We also describe commonly used approaches for formulating, functionalizing and characterizing these nanomaterials. Key applications are highlighted by categorizing nanomaterials on the basis of their X-ray, magnetic, nuclear, optical and/or photoacoustic contrast properties. Finally, we offer our perspectives on current challenges and emerging research topics as well as expectations for future advancements in the field.

Multiplexing potential of NIR resonant and non-resonant Raman reporters for bio-imaging applications.

Multiplexed imaging, which allows for the interrogation of multiple molecular features simultaneously, is vital for addressing numerous challenges across biomedicine. Optically unique surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have the potential to serve as a vehicle to achieve highly multiplexed imaging in a single acquisition, which is non-destructive, quantitative, and simple to execute. When using laser excitation at 785 nm, which allows for a lower background from biological tissues, near infrared (NIR) dyes can be used as Raman reporters to provide high Raman signal intensity due to the resonance effect. This class of imaging agents are known as surface-enhanced resonance Raman scattering (SERRS) NPs. Investigators have predominantly utilized two classes of Raman reporters in their nanoparticle constructs for use in biomedical applications: NIR-resonant and non-resonant Raman reporters. Herein, we investigate the multiplexing potential of five non-resonant SERS: BPE, 44DP, PTT, PODT, and BMMBP, and five NIR resonant SERRS NP flavors with heptamethine cyanine dyes: DTTC, IR-770, IR-780, IR-792, and IR-797, which have been extensively used for biomedical imaging applications. Although SERRS NPs display high Raman intensities, due to their resonance properties, we observed that non-resonant SERS NP concentrations can be quantitated by the intensity of their unique emissions with higher accuracy. Spectral unmixing of five-plex mixtures revealed that the studied non-resonant SERS NPs maintain their detection limits more robustly as compared to the NIR resonant SERRS NP flavors when introducing more components into a mixture.

Plier Ligands for Trapping Neurotransmitters into Complexes for Sensitive Analysis by SERS Spectroscopy.

Catecholamines-dopamine, noradrenaline and adrenaline are important biomarkers of neurotransmitter metabolism, indicating neuroendocrine tumors and neurodegenerative diseases. Surface-enhanced Raman spectroscopy (SERS) is a promising analytical technique with unprecedented multiplexing capabilities. However, not all important analytes exhibit strong SERS signals on stable and robust nanostructured substrates. In this work, we propose a novel indicator system based on the formation of mixed ligand complexes with bispidine-based bis-azole ligands which can serve as pliers to trap Cu(II) ions and stabilize its complexes with catecholamines. Four synthesized ligands with different functional groups: carboxyl, amino, benzyl, and methoxybenzyl, were applied for forming stable complexes to shift maximum absorbance of catecholamines from the ultraviolet region to 570-600 nm. A new absorbance band in the visible range resonates with the local surface plasmon resonance (LSPR) band of metal nanoparticles and most used laser wavelengths. This match allowed use of Molecular Immobilization and Resonant Raman Amplification by Complex-Loaded Enhancers (MIRRACLE) methodology to measure intense Raman signals on a nanostructured silver-based SERS-active substrate. The synthesized plier-like ligands fixed and stabilized catecholamine complexes with Cu(II) on the SERS sensor surface, which facilitated the determination of dopamine in a 3.2 × 10-12-1 × 10-8 M concentration range.

Expanding the Multiplexing Capabilities of Raman Imaging to Reveal Highly Specific Molecular Expression and Enable Spatial Profiling.

Profiling the heterogeneous landscape of cell types and biomolecules is rapidly being adopted to address current imperative research questions. Precision medicine seeks advancements in molecular spatial profiling techniques with highly multiplexed imaging capabilities and subcellular resolution, which remains an extremely complex task. Surface-enhanced Raman spectroscopy (SERS) imaging offers promise through the utilization of nanoparticle-based contrast agents that exhibit narrow spectral features and molecular specificity. The current renaissance of gold nanoparticle technology makes Raman scattering intensities competitive with traditional fluorescence methods while offering the added benefit of unsurpassed multiplexing capabilities. Here, we present an expanded library of individually distinct SERS nanoparticles to arm researchers and clinicians. Our nanoparticles consist of a ∼60 nm gold core, a Raman reporter molecule, and a final inert silica coating. Using density functional theory, we have selected Raman reporters that meet the key criterion of high spectral uniqueness to facilitate unmixing of up to 26 components in a single imaging pixel in vitro and in vivo. We also demonstrated the utility of our SERS nanoparticles for targeting cultured cells and profiling cancerous human tissue sections for highly multiplexed optical imaging. This study showcases the far-reaching capabilities of SERS-based Raman imaging in molecular profiling to improve personalized medicine and overcome the major challenges of functional and structural diversity in proteomic imaging.

Plasmonic features of free-standing chitosan nanocomposite film with silver and graphene oxide for SERS applications.

A scalable procedure of SERS substrates design was developed using a novel plasmonic structure based on a freestanding chitosan film, silver nanoparticles, and graphene oxide. Chitosan provides a uniform distribution of silver nanoparticles from a colloidal suspension and, therefore, a reproducible Raman signal from local areas of measurements of several tens of microns. The addition of graphene oxide (GO) to the colloidal solution of silver nanoparticles suppresses the tortuous background fluorescence signal from the analyte and leads to an increase in the signal-to-fluorescence background intensity ratio by up to 6 times as compared to structures without GO. The manufactured plasmonic polymer nanocomposite provides a detection limit of down to 100 pM for R6G using a laser wavelength of 532 nm through a portable ×10 objective. The high colloidal stability of GO in water and the use of an aqueous colloid of silver nanoparticles simplify the procedure for creating a substrate by applying the GO-silver composite on the surface of a chitosan film without a need to form a GO film. Therefore, our approach paves a promising avenue to provide more sensitive detection even for the fluorescent analytes with short-wavelength lasers (532, 633 nm) instead of IR (785, 1024 nm) and foster the practical application of the developed plasmonic composites on portable Raman spectrometers.

Molecular Immobilization and Resonant Raman Amplification by Complex-Loaded Enhancers (MIRRACLE) on copper (II)-chitosan-modified SERS-active metallic nanostructured substrates for multiplex determination of dopamine, norepinephrine, and epinephrine.

A unique approach based on Molecular Immobilization and Resonant Raman Amplification by Complex-Loaded Enhancers (MIRRACLE) on copper (II)-chitosan-modified SERS-active metallic nanostructured substrates is proposed for sensitive and rapid determination of the catecholamines (CA) dopamine, norepinephrine, and epinephrine. The ternary (CA)2Cu(4AAP)2 complexes were characterized by the appearance of new absorbance bands at 555, 600, and 500 nm for dopamine, norepinephrine, and epinephrine, respectively. The new absorbance band matched with a broad surface plasmon resonance band of utilized silver nanoparticles: 450-600 nm, and 633 excitation wavelength. We observed enhancement factors up to 3.6·106 due to the additional resonant enhancement. The multiplexing capabilities of quantitative spectral unmixing for Raman spectra of a group of CAs, which differ by only either hydroxy or methyl group, at the fingerprint region were successfully demonstrated with the direct classic least squares model. The achieved nM limits of detection with only 1.5 mW laser power and analysis of spiked human blood plasma samples proved the possibility of the multiplex determination of the catecholamines at the level of reference concentrations in the blood of healthy people as well as promise for the future facilitation in the precision diagnosis of neuroendocrine tumors and neurodegenerative diseases.

Selecting Surface-Enhanced Raman Spectroscopy Flavors for Multiplexed Imaging Applications: Beyond the Experiment.

Multiplexing capabilities and sensitivity of surface-enhanced Raman spectroscopy (SERS) nanoparticles (NPs) are strongly dependent on the selected Raman reporter. These Raman-active molecules are responsible for giving each batch of SERS NPs its unique spectral fingerprint. Herein, we studied four types of SERS NPs, namely, AuNPs labeled with trans-1,2-bis(4-pyridyl)ethylene (BPE), 4,4'-bis(mercaptomethyl)biphenyl (BMMBP), 5-(4-pyridyl)-1,3,4-oxadiazole-2-thiol (PODT), and 5-(4-pyridyl)-1H-1,2,4-triazole-3-thiol (PTT), and demonstrated that the best level of theory could be chosen based on inner products of DFT-calculated and experimental Raman spectra. We also calculated the theoretical spectra of these Raman reporters bound to Au20 clusters to interrogate how SERS enhancement would affect their spectral fingerprint. Importantly, we found a correlation between B3LYP-D3 calculated and experimental enhancement factors, which opens up an avenue toward predicting which Raman reporters could offer improved sensitivity. We observed 0.5 and 3 fM limits of detection for BMMBP- and PTT-labeled 60 nm AuNPs, respectively.

Dual-Purpose SERS Sensor for Selective Determination of Polycyclic Aromatic Compounds via Electron Donor-Acceptor Traps.

Toxic, carcinogenic, and mutagenic properties of polycyclic aromatic hydrocarbons (PAHs) and environmental pollution caused by polycyclic aromatic sulfur heterocycles (PASHs) postulate the importance of their selective and sensitive determination in environmental and oil fuel samples. Surface-enhanced Raman spectroscopy (SERS) opens up an avenue toward multiplex analysis of complex mixtures, however not every molecule gives high enhancement factors and, thus, cannot be reliably detected via SERS. However, the sensitivity can be drastically increased by additional resonant enhancement as a result of the analyte absorption band overlapping with the surface plasmon band of nanoparticles (NPs) and the laser excitation wavelength. Using this idea, we developed a dual-purpose SERS sensor based on trapping the target PAHs and PASHs into colored charge-transfer complexes (CTCs) with selected organic π-acceptor molecules on the surface of AgNPs. Studying, computing, and then comparing stability constants of the formed CTC served as a powerful explanation and prediction tool for a wise choice of π-acceptor indicator systems for the further silver surface modification. Moreover, we show that CTC formation can be effectively utilized for increasing both selectivity and sensitivity by simple liquid-liquid extraction prior to SERS measurements. For the first time, the dual-purpose SERS sensor allowed determination of two different classes of polycyclic aromatic fuel components down to 10 nM concentration, lower than that restricted by the ASTM regulation, and demonstrated multi-purpose capabilities of the developed approach.

A colorful approach towards developing new nano-based imaging contrast agents for improved cancer detection.

Providing physicians with new imaging agents to help detect cancer with better sensitivity and specificity has the potential to significantly improve patient outcomes. Development of new imaging agents could offer improved early cancer detection during routine screening or help surgeons identify tumor margins for surgical resection. In this study, we evaluate the optical properties of a colorful class of dyes and pigments that humans routinely encounter. The pigments are often used in tattoo inks and the dyes are FDA approved for the coloring of foods, drugs, and cosmetics. We characterized their absorption, fluorescence and Raman scattering properties in the hopes of identifying a new panel of dyes that offer exceptional imaging contrast. We found that some of these coloring agents, coined as "optical inks", exhibit a multitude of useful optical properties, outperforming some of the clinically approved imaging dyes on the market. The best performing optical inks (Green 8 and Orange 16) were further incorporated into liposomal nanoparticles to assess their tumor targeting and optical imaging potential. Mouse xenograft models of colorectal, cervical and lymphoma tumors were used to evaluate the newly developed nano-based imaging contrast agents. After intravenous injection, fluorescence imaging revealed significant localization of the new "optical ink" liposomal nanoparticles in all three tumor models as opposed to their neighboring healthy tissues (p < 0.05). If further developed, these coloring agents could play important roles in the clinical setting. A more sensitive imaging contrast agent could enable earlier cancer detection or help guide surgical resection of tumors, both of which have been shown to significantly improve patient survival.

Silver-chitosan nanocomposite as a plasmonic platform for SERS sensing of polyaromatic sulfur heterocycles in oil fuel.

Herein, a silver-chitosan nanocomposite for application in surface enhanced Raman spectroscopy (SERS) sensing was proposed. It was shown that optically transparent chitosan coatings with 0.8 µm thickness allow penetration of target analytes to silver nanoparticles and the analysis in both polar and nonpolar solvents. Under the chosen conditions, chitosan formed continuously smooth films and coatings stabilizing rough nanostructured metallic surfaces and served as a suitable matrix for immobilization, uniform spreading, and preconcentration of the analytes. Polycyclic aromatic sulfur heterocycles were chosen as target analytes being one of the most important fuel quality markers, hazardous components, and the hardest-to-remove impurities. For the most effective immobilization and even distribution of the analytes onto a nanostructured metallic surface, an additional polymer layer of chitosan was found to be needed. The presence of thin films of chitosan resulted in higher reproducibility of SERS spectra as compared to bare nanostructured silver substrates. Additionally, the developed nanocomposite SERS sensors provided the rapid determination of dibenzothiophene and its derivatives in isooctane with the threshold of detection better than 0.1 µM. This approach was successfully applied in the analysis of real fuel samples and the results agreed well with independently measured FTIR and GC-MS data.

Optically transparent chitosan hydrogels for selective sorption and fluorometric determination of dibenzothiophenes.

Solid-phase extraction of polycyclic aromatic sulfur heterocycles (PASHs) and their rapid determination in oil fuel without tedious sample pretreatment are of high interest. We propose porous and optically transparent hydrogels prepared from the covalently crosslinked chitosan (CS) as the basis for a sensor system for the rapid and robust monitoring of PASHs. We efficiently combined the ability of the crosslinked CS to sorb PASHs, the capacity of microcavities in a molecularly imprinted polymer to selectively recognize and trap analytes, and the optical transparency of CS materials for selective sorption and solid-phase fluorometric determination of dibenzothiophenes. For the screening of PASHs in organic nonpolar media, ortho-phtalic dialdehyde appeared to be the most appropriate crosslinker. Synthetic and analytical procedures performed in microplate mode allowed obtaining CS hydrogels with suitable reproducible properties and their further time- and labor-efficient applying in analysis (particularly, as little as 2 µM dibenzothiophene oxide can be determined).

Novel Multilayer Nanostructured Materials for Recognition of Polycyclic Aromatic Sulfur Pollutants and Express Analysis of Fuel Quality and Environmental Health by Surface Enhanced Raman Spectroscopy.

A novel concept of advanced SERS (surface enhanced Raman spectroscopy) planar sensors is suggested for fast analysis of sulfur-containing hazardous oil components and persistent pollutants. The main advantage of the proposed sensors is the utilization of an additional preconcentrating layer of optically transparent chitosan gel, which is chemically modified with appropriate π-acceptor compounds to selectively form charge-transfer complexes (CTCs) at the interface with nanostructured silver coatings. The CTCs shift absorption bands of polycyclic aromatic sulfur heterocycles (PASHs) and other important analytes in a controllable way and thus provide a surplus enhancement of vibration modes due to resonant Raman scattering. This novel indicator system provides multiplex determination of PASHs in different forms in a small volume of oil without any tedious sample pretreatment steps. This approach opens new possibilities of increasing either spectral and concentration sensitivity or specificity of SERS-based sensors, allowing for new developments in the fields of ecology, advanced fuel analysis, and other prospective applications.