Photostable Single-Molecule Nanoparticle Optical Biosensors for Real-Time Sensing of Single Cytokine Molecules and Their Binding Reactions

Abstract

We synthesized tiny stable silver nanoparticles (2.6 ± 1.1 nm) and used its small surface area and functional groups to control single molecule detection (SMD) volumes on single nanoparticles. These new approaches allowed us to develop intrinsic single molecule nanoparticle optical biosensors (SMNOBS) for sensing and imaging of single human cytokine molecules, recombinant human tumor necrosis factor-α (TNFα), and probing its binding reaction with single monoclonal antibody (MAB) molecules in real-time. We found that SMNOBS retained their biological activity over months and showed exceptionally high photostability. Our study illustrated that smaller nanoparticles exhibited higher dependence of optical properties on surface functional groups, making it a much more sensitive biosensor. Localized surface plasmon resonance spectra (LSPRS) of SMNOBS showed a large red shift of peak wavelength of 29 ± 11 nm, as single TNFα molecules bound with single MAB molecules on single nanoparticles. Utilizing its LSPRS, we quantitatively measured its binding reaction in real time at SM level, showing stochastic binding kinetics of SM reactions with binding equilibrium times ranging from 30 to 120 min. SMNOBS exhibited extraordinarily high sensitivity and selectivity, and a notably wide dynamic range of 0-200 ng/mL (0-11.4 nM). Thus, SMNOBS is well suited for the fundamental study of biological functions of single protein molecules and SM interactions of chemical functional groups with the surface of nanoparticles, as well as development of effective disease diagnosis and therapy.

Bioanalytical chemistry of cytokines – A review

“…Noble metals do not suffer some of the problems of photobleaching associated with fluorophores and blinking associated with quantum dots. The Xu research group at Old Dominion has described their major progress towards creating stable noble metal nanoparticles that have been called single molecule nanoparticle optical nanobiosensors (SMNOBS) that have been used for sensing TNF-α [243]. This work demonstrated the ability to track the on-off kinetics of TNF-α onto the Ag nanoparticles.…”

Silver and gold nanoparticles in silica matrices: synthesis, properties, and application

“…The toxicity of the nanoparticles is discussed in [118]. In large amounts, however, their toxicity is high, and the biocompatibility is substantially lower than for gold nanoparticles [119][120][121][122].…”

Toxicity of Silver Nanoparticles as Assessed by Global Gene Expression Analysis

“…5 In addition, Ag-NPs have been studied for industrial use in optics, sensors, imaging, and catalysis. [6][7][8][9][10] However, some studies have shown that Ag-NPs have adverse effects on mammalian cells. [11][12][13][14][15] It has been reported that the generation of reactive oxygen species (ROS) is one of causes of Ag-NP toxicity, because cells exposed to Ag-NPs show elevated ROS levels, lipid peroxidation, expression of oxidative stress responsive genes, and reduction of glutathione.…”