Zhen Gu

Chrominance to Dimension: A Real-Time Method for Measuring the Size of Single Gold Nanoparticles

Noble metal nanoparticles have excellent optical and chemical properties and are widely used in optics, sensors, and biomedicines. The inherent characteristics of metal nanoparticles, particularly their size, play important roles in their applications. The ability to readily measure the size of single nanomaterials on-site is crucial to the rapid development of single-particle sensors. In this study, we developed a facile and real-time method for estimating the diameter of single gold nanoparticles (GNPs) that range from 35 to 110 nm in diameter; this technique uses the chrominance of the GNPs plasmon resonance scattering light that is captured by a dark-field microscope (DFM). The RGB (three primary colors, red, green, and blue) chrominance information from the dark-field image can be directly converted into the diameters of the GNPs using the relationship between the particle size and the scattering light peak wavelength; this conversion was carried out using Matlab program based on an RGB-To-Wavelength (RTW) process. This approach is more convenient, less time-consuming, and enables observation under arbitrary conditions compared to the scanning electron microscopy (SEM) technique. The differences between the diameters of the GNPs that were calculated using this method and those that were measured using SEM were less than 5 nm. The RTW method has also been applied in the monitoring of the refractive index of the media surrounding the GNPs, and their dynamic acting within cells in real-time.

Ubiquinone-quantum dot bioconjugates for in vitro and intracellular complex I sensing

Quantum dots (QDs) have attracted increasing interest in bioimaging and sensing. Here, we report a biosensor of complex I using ubiquinone-terminated disulphides with different alkyl spacers (QnNS, n = 2, 5 and 10) as surface-capping ligands to functionalise CdSe/ZnS QDs. The enhancement or quenching of the QD bioconjugates fluorescence changes as a function of the redox state of QnNS, since QDs are highly sensitive to the electron-transfer processes. The bioconjugated QnNS-QDs emission could be modulated by complex I in the presence of NADH, which simulates an electron-transfer system part of the mitochondrial respiratory chain, providing an in vitro and intracellular complex I sensor. Epidemiological studies suggest that Parkinsons patients have the impaired activity of complex I in the electron-transfer chain of mitochondria. We have demonstrated that the QnNS-QDs system could aid in early stage Parkinsons disease diagnosis and progression monitoring by following different complex I levels in SH-SY5Y cells.

Capturing intercellular sugar-mediated ligand-receptor recognitions via a simple yet highly biospecific interfacial system

Intercellular ligand-receptor recognitions are crucial natural interactions that initiate a number of biological and pathological events. We present here the simple construction of a unique class of biomimetic interfaces based on a graphene-mediated self-assembly of glycosyl anthraquinones to a screen-printed electrode for the detection of transmembrane glycoprotein receptors expressed on a hepatoma cell line. We show that an electroactive interface confined with densely clustered galactosyl ligands is able to ingeniously recognize the asialoglycoprotein receptors on live Hep-G2 cells employing simple electrochemical techniques. The only facility used is a personal laptop in connection with a cheap and portable electrochemical workstation.

Investigating electron-transfer processes using a biomimetic hybrid bilayer membrane system

Here we report a protocol to investigate the electron-transfer processes of redox-active biomolecules in biological membranes by electrochemistry using biomimetic hybrid bilayer membranes (HBMs) assembled on gold electrodes. Redox-active head groups, such as the ubiquinone moiety, are embedded in HBMs that contain target molecules, e.g., nicotinamide adenine dinucleotide (NADH). By using this approach, the electron-transfer processes between redox molecules and target biomolecules are mediated by mimicking the redox cycling processes in a natural membrane. Also included is a procedure for in situ surface-enhanced Raman scattering (SERS) to confirm the electrochemically induced conformational changes of the target biomolecules in the HBMs. In addition, each step in constructing the HBMs is characterized by electrochemical impedance spectroscopy (EIS), high-resolution X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The time required for the entire protocol is ∼12 h, whereas the electrochemical measurement of electron-transfer processes takes less than 1 h to complete.

Analysis of a Single α-Synuclein Fibrillation by the Interaction with a Protein Nanopore

The formation of an α-synuclein fibril is critical in the pathogenesis of Parkinsons disease. The native unfolded α-synuclein monomer will translocate through an α-hemolysin nanopore by applied potential at physiological conditions in vitro. Applying a potential transformed α-synuclein into a partially folded intermediate, which was monitored by capture inside the vestibule of an α-hemolysin nanopore with a capture current of 20 ± 1.0 pA. The procedure involves the critical early stage of α-synuclein structural transformation. Further elongation of the intermediate produces a block current to 5 ± 0.5 pA. It is revealed that the early stage fibril of α-synuclein inside the nanopore is affected by intrapeptide electrostatic interaction. In addition, trehalose cleared the fibrillation by changing the surface hydrophobic interaction of A53T α-synuclein, which did not show any inhibition effect from WT α-synuclein. The results proved that the interpeptide hydrophobic interactions in the elongation of A53T α-synuclein protofilaments can be greatly weakened by trehalose. This suggests that trehalose inhibits the interpeptide interaction involved in protein secondary structure. The hydrophobic and electrostatic interactions are associated with an increase in α-synuclein fibrillation propensity. This work provides unique insights into the earliest steps of the α-synuclein aggregation pathway and provides the potential basis for the development of drugs that can prevent α-synuclein aggregation at the initial stage.

Accurate Data Process for Nanopore Analysis

Data analysis for nanopore experiments remains a fundamental and technological challenge because of the large data volume, the presence of unavoidable noise, and the filtering effect. Here, we present an accurate and robust data process that recognizes the current blockades and enables evaluation of the dwell time and current amplitude through a novel second-order-differential-based calibration method and an integration method, respectively. We applied the developed data process to analyze both generated blockages and experimental data. Compared to the results obtained using the conventional method, those obtained using the new method provided a significant increase in the accuracy of nanopore measurements.

In situ high throughput scattering light analysis of single plasmonic nanoparticles in living cells.

Plasmonic nanoparticles have been widely applied in cell imaging, disease diagnosis, and photothermal therapy owing to their unique scattering and absorption spectra based on localized surface plasmon resonance (LSPR) property. Recently, it is still a big challenge to study the detailed scattering properties of single plasmonic nanoparticles in living cells and tissues, which have dynamic and complicated environment. The conventional approach for measuring the scattering light is based on a spectrograph coupled to dark-field microscopy (DFM), which is time-consuming and limited by the small sample capacity. Alternatively, RGB-based method is promising in high-throughput analysis of single plasmonic nanoparticles in dark-field images, but the limitation in recognition of nanoparticles hinders its application for intracellular analysis. In this paper, we developed an automatic and robust method for recognizing the plasmonic nanoparticles in dark-field image for RGB-based analysis. The method involves a bias-modified fuzzy C-means algorithm, through which biased illumination in the image could be eliminated. Thus, nearly all of the gold nanoparticles in the recorded image were recognized both on glass slide and in living cells. As confirmed, the distribution of peak wavelength obtained by our method is well agreed to the result measured by conventional method. Furthermore, we demonstrated that our method is profound in cell imaging studies, where its advantages in fast and high-throughput analysis of the plasmonic nanoparticles could be applied to confirm the presence and location of important biological molecules and provide efficiency information for cancer drug selection.

Brightening Gold Nanoparticles: New Sensing Approach Based on Plasmon Resonance Energy Transfer

Scattering recovered plasmonic resonance energy transfer (SR-PRET) was reported by blocking the plasmon resonance energy transfer (PRET) from gold nanoparticle (GNP) to the adsorbed molecules (RdBS). Due to the selective cleavage of the Si-O bond by F− ions, the quenching is switched off causing an increase in the brightness of the GNPs,detected using dark-field microscopy (DFM) were brightened. This method was successfully applied to the determination of fluoride ions in water. The SR-PRET provides a potential approach for a vitro/vivo sensing with high sensitivity and selectivity.

Analyzing Carbohydrate–Protein Interaction Based on Single Plasmonic Nanoparticle by Conventional Dark Field Microscopy

We demonstrated a practical method to analyze carbohydrate-protein interaction based on single plasmonic nanoparticles by conventional dark field microscopy (DFM). Protein concanavalin A (ConA) was modified on large sized gold nanoparticles (AuNPs), and dextran was conjugated on small sized AuNPs. As the interaction between ConA and dextran resulted in two kinds of gold nanoparticles coupled together, which caused coupling of plasmonic oscillations, apparent color changes (from green to yellow) of the single AuNPs were observed through DFM. Then, the color information was instantly transformed into a statistic peak wavelength distribution in less than 1 min by a self-developed statistical program (nanoparticleAnalysis). In addition, the interaction between ConA and dextran was proved with biospecific recognition. This approach is high-throughput and real-time, and is a convenient method to analyze carbohydrate-protein interaction at the single nanoparticle level efficiently.

Enhanced resolution of low molecular weight poly(ethylene glycol) in nanopore analysis.

A design with conjugation of DNA hairpin structure to the poly(ethylene glycol) molecule was presented to enhance the temporal resolution of low molecular weight poly(ethylene glycol) in nanopore studies. By the virtue of this design, detection of an individual PEG with molecular weight as low as 140 Da was achieved at the single-molecule level in solution, which provides a novel strategy for characterization of an individual small molecule within a nanopore. Furthermore, we found that the current duration time of poly(ethylene glycol) was scaled with the relative molecular weight, which has a potential application in single-molecule detection.