Jonghoon Choi

Immuno-hybridization chain reaction for enhancing detection of individual cytokine-secreting human peripheral mononuclear cells.

We present here a new method to enhance the detection of secreted cytokines and chemokines from single human mononuclear cells. The technique uses a hybridization chain reaction (HCR) to amplify signals resulting from sandwich immunoassays. This immuno-HCR employs oligonucleotide-based initiators covalently linked to antibodies to propagate a chain reaction of hybridization events involving a pair of complementary hairpin oligomers bearing fluorescent labels. Integrating this strategy for signal amplification with microengraving (a soft lithographic method for printing arrays of secreted proteins from thousands of single cells) improves both the limits of detection and sensitivity for cytokines and chemokines captured from individual cells by an average of 200-fold relative to methods for direct detection by fluoresence. This approach should enhance the utility of microengraving for defining the immunological signatures of diseases and responses to interventional therapies based on multiplexed single-cell analysis.

Comparison of cytotoxic and inflammatory responses of photoluminescent silicon nanoparticles with silicon micron-sized particles in RAW 264.7 macrophages

Photoluminescent silicon nanoparticles have a bright and stable fluorescence and are promising candidates for bio‐imaging, cell staining and drug delivery. With increasing development of nanotechnology applications for biomedicine, an understanding of the potential toxicity of nanoparticles is needed to assess safety concerns for clinical applications. The objective of this study was to compare biological responses of silicon nanoparticles (SNs, 3 nm diameter) with silicon microparticles (SMs, ∼100–3000 nm diameter) in cultured murine macrophages (RAW 264.7) using standard protocols for assessing cytotoxicity/cell viability and inflammatory responses developed for micron‐sized particles. SNs and SMs were exposed to macrophages with and without addition of endotoxin lipopolysaccharide (LPS), a positive inducer of tumor necrosis factor‐alpha (TNF‐α), interleukin 6 (IL‐6), and nitric oxide (NO). Cytotoxicity was assayed using the dye exclusion and MTT assays. Cell supernatants were assayed for production TNF‐α, IL‐6 and NO. SNs at concentrations ≤20 µg ml−1 exhibited no cytotoxicity or inflammatory responses; however, SNs and SMs >20 and 200 µg ml−1, respectively, increased cytotoxicity compared with controls. SMs induced concentration‐related increases in TNF‐α and IL‐6 production; in contrast, the production of these cytokines was shown to decrease with increasing concentrations of SNs. NO production was not induced by SNs or SMs alone. Fluorescence microscopy demonstrated that SNs were associated with the macrophages, either internalized or attached to cell membranes. In conclusion, evaluating differences in biological responses for nanoparticles compared with microparticles of the same material may help improve tests to assess biological responses of nanoparticles that may be used in biomedical applications. Copyright

Microtools for single-cell analysis in biopharmaceutical development and manufacturing

Biologic drugs are promoting growth in the biopharmaceutical industry. Despite the clinical benefits of these drugs, the time and costs required to bring new biologics to market still are substantial. Three key challenges, among others, persist in the development of biologic drugs: namely, establishing product similarity, product toxicity, and global accessibility. New classes of microtools that facilitate the isolation and interrogation of single cells have the potential to impact each of these challenges. This opinion considers recent examples of microtools with demonstrated or potential utility to address problems in these areas. Integrating these advanced technologies into the development of new biologics could greatly reduce time and costs required to bring alternative products to market, and thus expand their global availability.

Conjugation of the Photoluminescent Silicon Nanoparticles to Streptavidin

We have covalently attached multiple photoluminescent silicon nanoparticles (SNs) to streptavidin molecules. Conjugation of SNs to a target protein is achieved using the multistage photoassisted procedure. In a first step, the terminal hydrogen in the freshly prepared SNs is substituted with an alkane monolayer that serves as a platform for chemical linkage to a heterobifunctional cross-linker: 4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester. A resulting surface coating stabilizes nanoparticles against oxidation and aggregation. Next, an open end of bifunctional cross-linker-diazirine succinimidyl ester is reacted with carboxyl moieties of streptavidin and forms an amide bond. Gel and capillary electrophoresis of the SN-streptavidin complex demonstrated separate elution of the conjugation product and unreacted protein. Then, the number of SNs per protein molecule was determined by measuring complex charge variation by capillary electrophoresis. Conjugate functionality was tested by allowing it to interact with biotinylated polystyrene microbeads. Intense photoluminescence at carefully washed microbeads demonstrated selective binding of silicon nanoparticle bearing streptavidin to biotinylated microbeads. The high quantum yield of streptavidin-SN conjugate in combination with the small size and biocompatibility of silicon nanoparticles presents an attractive platform for the fluorescence labeling in diverse bioassays.

Multimodal imaging of sustained drug release from 3-D poly(propylene fumarate) (PPF) scaffolds.

The potential of poly(propylene fumarate) (PPF) scaffolds as drug carriers was investigated and the kinetics of the drug release quantified using magnetic resonance imaging (MRI) and optical imaging. Three different MR contrast agents were used for coating PPF scaffolds. Initially, iron oxide (IONP) or manganese oxide nanoparticles (MONP) carrying the anti-cancer drug doxorubicin were absorbed or mixed with the scaffold and their release into solution at physiological conditions was measured with MRI and optical imaging. A slow (hours to days) and functional release of the drug molecules into the surrounding solution was observed. In order to examine the release properties of proteins and polypeptides, protamine sulfate, a chemical exchange saturation transfer (CEST) MR contrast agent, was attached to the scaffold. Protamine sulfate showed a steady release rate for the first 24h. Due to its biocompatibility, versatile drug-loading capability and constant release rate, the porous PPF scaffold has potential in various biomedical applications, including MR-guided implantation of drug-dispensing materials, development of drug carrying vehicles, and drug delivery for tumor treatment.

Antibacterial activity and cytotoxicity of multi-walled carbon nanotubes decorated with silver nanoparticles

Recently, various nanoscale materials, including silver (Ag) nanoparticles, have been actively studied for their capacity to effectively prevent bacterial growth. A critical challenge is to enhance the antibacterial properties of nanomaterials while maintaining their biocompatibility. The conjugation of multiple nanomaterials with different dimensions, such as spherical nanoparticles and high-aspect-ratio nanotubes, may increase the target-specific antibacterial capacity of the consequent nanostructure while retaining an optimal biocompatibility. In this study, multi-walled carbon nanotubes (MWCNTs) were treated with a mixture of acids and decorated with Ag nanoparticles via a chemical reduction of Ag cations by ethanol solution. The synthesized Ag-MWCNT complexes were characterized by transmission electron microscopy, X-ray diffractometry, and energy-dispersive X-ray spectroscopy. The antibacterial function of Ag-MWCNTs was evaluated against Methylobacterium spp. and Sphingomonas spp. In addition, the biocompatibility of Ag-MWCNTs was evaluated using both mouse liver hepatocytes (AML 12) and human peripheral blood mononuclear cells. Finally, we determined the minimum amount of Ag-MWCNTs required for a biocompatible yet effective antibacterial treatment modality. We report that 30 μg/mL of Ag-MWCNTs confers antibacterial functionality while maintaining minimal cytotoxicity toward both human and animal cells. The results reported herein would be beneficial for researchers interested in the efficient preparation of hybrid nanostructures and in determining the minimum amount of Ag-MWCNTs necessary to effectively hinder the growth of bacteria.

Electrochemical reduction synthesis of photoluminescent silicon nanocrystals.

An efficient synthesizing procedure of photoluminescent silicon nanocrystals is demonstrated by means of ultrasound assisted electrochemical octyltrichlorosilane reduction that produces octane terminated Si nanocrystals in a single step. The described procedure allows one to make Si nanocrystals with alkyl surface termination and is clean, relatively simple, and potentially scalable to industrial quantities. High resolution transmission electron microscopy, energy dispersive X-ray spectroscopy, UV-vis absorbance, Fourier transform infrared spectroscopy, and photoluminescence spectroscopy are employed to characterize the synthesized photoluminescent Si nanocrystals. Resulting octyl termination provides a stable passivation and could serve as a platform for further particle functionalization. Electrochemical chlorosilane reduction potentially could address the requirement for stable photoluminescent Si nanocrystals in diverse applications.

Biomimetics: forecasting the future of science, engineering, and medicine

Biomimetics is the study of nature and natural phenomena to understand the principles of underlying mechanisms, to obtain ideas from nature, and to apply concepts that may benefit science, engineering, and medicine. Examples of biomimetic studies include fluid-drag reduction swimsuits inspired by the structure of shark’s skin, velcro fasteners modeled on burrs, shape of airplanes developed from the look of birds, and stable building structures copied from the backbone of turban shells. In this article, we focus on the current research topics in biomimetics and discuss the potential of biomimetics in science, engineering, and medicine. Our report proposes to become a blueprint for accomplishments that can stem from biomimetics in the next 5 years as well as providing insight into their unseen limitations.

Facile solvothermal preparation of monodisperse gold nanoparticles and their engineered assembly of ferritin-gold nanoclusters.

Herein, we report a quick and simple synthesis of water-soluble gold nanoparticles using a HAuCl4 and oleylamine mixture. Oleylamine serves as a reduction agent as well as a stabilizer for nanoparticle surfaces. The particle sizes can be adjusted by modulating reaction temperature and time. Solvothermal reduction of HAuCl4 with oleylamine can be confirmed by measuring the product in Fourier transform infrared (FTIR) spectroscopy. The plasmon band shifting from yellow to red confirms a nanosized particle formation. Amide bonds on the surface of the nanoparticles formed hydrogen bonds with one another, resulting in a hydrophobic monolayer. Particles dispersed well in nonpolar organic solvents, such as in hexane or toluene, by brief sonication. Next, we demonstrated the transfer of gold nanoparticles into water by lipid capsulation using 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (MHPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy polyethylene glycol)-2000 (DPPE-PEG2k), and 1,2-dioleoyl-sn-glycero-3-N-{5-amino-1-carboxypentyl}iminodiacetic acid succinyl nickel salt [DGS-NTA(Ni)]. The particle concentration can be obtained using an absorbance in ultraviolet-visible (UV-vis) spectra (at 420 nm). Instrumental analyses using transmission electron microscopy (TEM), energy-dispersive X-ray (EDX) analysis, dynamic light scattering (DLS), and FTIR confirmed successful production of gold nanoparticles and fair solubility in water. Prepared gold particles were selectively clustered via engineered ferritin nanocages that provide multiple conjugation moieties. A total of 5-6 gold nanoparticles were clustered on a single ferritin nanocage confirmed in TEM. Reported solvothermal synthesis and preparation of gold nanoclusters may serve as an efficient, alternate way of preparing water-soluble gold nanoparticles, which can be used in a wide variety of biomedical applications.

Measurement of Nanoparticle Concentration Using Quartz Crystal Microgravimetry

Various nanoscale items (e.g., nanoparticles and nanotubes) have been actively investigated due to their unique physicochemical properties. A common issue encountered in such studies is accurate expression of nanoparticle concentration. Given the critical importance of the dose-response relationship, we present the use of quartz crystal microgravimetry (QCM) to accurately measure nanoparticle concentration in a colloidal suspension. Application of a small drop of the nanoparticle suspension in a volatile solvent to the crystal surface leaves a dry nanoparticle residue after solvent evaporation after which the shift in the crystal resonant frequency is recorded. The instrument was calibrated using a set of serial dilutions of Si and Ag nanopowder in methanol, rhodamine B in methanol, and ferrocene in cyclohexane. Using QCM, a linear response for nanoparticle concentrations up to 1300 μg/mL was determined. The developed method was used to determine the concentrations of size-selected, octyl-terminated Si nanocrystal samples with median diameters in the range 1.1-14.8 nm and also to calculate size-dependent nanocrystal extinction coefficients.