Environmental Stability of Plasmonic Biosensors Based on Natural versus Artificial Antibody.

Plasmonic biosensors based on the refractive index sensitivity of localized surface plasmon resonance (LSPR) are considered to be highly promising for on-chip and point-of-care biodiagnostics. However, most of the current plasmonic biosensors employ natural antibodies as biorecognition elements, which can easily lose their biorecognition ability upon exposure to environmental stressors (e.g., temperature and humidity). Plasmonic biosensors relying on molecular imprints as recognition elements (artificial antibodies) are hypothesized to be an attractive alternative for applications in resource-limited settings due to their excellent thermal, chemical, and environmental stability. In this work, we provide a comprehensive comparison of the stability of plasmonic biosensors based on natural and artificial antibodies. Although the natural antibody-based plasmonic biosensors exhibit superior sensitivity, their stability (temporal, thermal, and chemical) was found to be vastly inferior to those based on artificial antibodies. Our results convincingly demonstrate that these novel classes of artificial antibody-based plasmonic biosensors are highly attractive for point-of-care and resource-limited conditions where tight control over transport, storage, and handling conditions is not possible.

Single Molecule Force Spectroscopy to Compare Natural versus Artificial Antibody-Antigen Interaction.

Biorecognition is central to various biological processes and finds numerous applications in virtually all areas of chemistry, biology, and medicine. Artificial antibodies, produced by imprinting synthetic polymers, are designed to mimic the biological recognition capability of natural antibodies, while exhibiting superior thermal, chemical, and environmental stability compared to their natural counterparts. The binding affinity of the artificial antibodies to their antigens characterizes the biorecognition ability of these synthetic nanoconstructs and their ability to replace natural recognition elements. However, a quantitative study of the binding affinity of an artificial antibody to an antigen, especially at the molecular level, is still lacking. In this study, using atomic force microscopy-based force spectroscopy, the authors show that the binding affinity of an artificial antibody to an antigen (hemoglobin) is weaker than that of natural antibody. The fine difference in the molecular interactions manifests into a significant difference in the bioanalytical parameters of biosensors based on these recognition elements.

Polydopamine-Mesoporous Silica Core-Shell Nanoparticles for Combined Photothermal Immunotherapy.

Cancer immunotherapy involves a cascade of events that ultimately leads to cytotoxic immune cells effectively identifying and destroying cancer cells. Responsive nanomaterials, which enable spatiotemporal orchestration of various immunological events for mounting a highly potent and long-lasting antitumor immune response, are an attractive platform to overcome challenges associated with existing cancer immunotherapies. Here, we report a multifunctional near-infrared (NIR)-responsive core-shell nanoparticle, which enables (i) photothermal ablation of cancer cells for generating tumor-associated antigen (TAA) and (ii) triggered release of an immunomodulatory drug (gardiquimod) for starting a series of immunological events. The core of these nanostructures is composed of a polydopamine nanoparticle, which serves as a photothermal agent, and the shell is made of mesoporous silica, which serves as a drug carrier. We employed a phase-change material as a gatekeeper to achieve concurrent release of both TAA and adjuvant, thus efficiently activating the antigen-presenting cells. Photothermal immunotherapy enabled by these nanostructures resulted in regression of primary tumor and significantly improved inhibition of secondary tumor in a mouse melanoma model. These biocompatible, biodegradable, and NIR-responsive core-shell nanostructures simultaneously deliver payload and cause photothermal ablation of the cancer cells. Our results demonstrate potential of responsive nanomaterials in generating highly synergistic photothermal immunotherapeutic response.

Aromatic Functionality of Target Proteins Influences Monomer Selection for Creating Artificial Antibodies on Plasmonic Biosensors.

Natural antibodies used as biorecognition elements suffer from numerous shortcomings, such as limited chemical and environmental stability and cost. Artificial antibodies based on molecular imprinting are an attractive alternative to natural antibodies. We investigated the role of aromatic interactions in target recognition capabilities of artificial antibodies. Three proteins with different aromatic amino acid content were employed as model targets. Artificial antibodies were formed on nanostructures using combinations of silane monomers of varying aromatic functionality. We employed refractive index sensitivity of plasmonic nanostructures as a transduction platform for monitoring various steps in the imprinting process and to quantify the target recognition capabilities of the artificial antibodies. The sensitivity of the artificial antibodies with aromatic interactions exhibited a protein-dependent enhancement. Selectivity and sensitivity enhancement due to the presence of aromatic groups in imprinted polymer matrix was found to be higher for target proteins with higher aromatic amino acid content. Our results indicate that tailoring the monomer composition based on the amino acid content of the target protein can improve the sensitivity of plasmonic biosensors based on artificial antibodies without affecting the selectivity.

PEGylated Artificial Antibodies: Plasmonic Biosensors with Improved Selectivity.

Molecular imprinting, which involves the formation of artificial recognition elements or cavities with complementary shape and chemical functionality to the target species, is a powerful method to overcome a number of limitations associated with natural antibodies. An important but often overlooked consideration in the design of artificial biorecognition elements based on molecular imprinting is the nonspecific binding of interfering species to noncavity regions of the imprinted polymer. Here, we demonstrate a universal method, namely, PEGylation of the noncavity regions of the imprinted polymer, to minimize the nonspecific binding and significantly enhance the selectivity of the molecular imprinted polymer for the target biomolecules. The nonspecific binding, as quantified by the localized surface plasmon resonance shift of imprinted plasmonic nanorattles upon exposure to common interfering proteins, was found to be more than 10 times lower compared to the non-PEGylated counterparts. The method demonstrated here can be broadly applied to a wide variety of functional monomers employed for molecular imprinting. The significantly higher selectivity of PEGylated molecular imprints takes biosensors based on these artificial biorecognition elements closer to real-world applications.

Bio-Enabled Gold Superstructures with Built-In and Accessible Electromagnetic Hotspots.

The bio-enabled synthesis of a novel class of surface enhanced Raman scattering probes is presented for functional imaging with built-in and accessible electromagnetic hotspots formed between densely packed satellites grown on a plasmonic core. The superstructures serve as nanoscale sensors to spatiotemporally map intravesicular pH changes along endocytic pathways inside live cells.