- A Nano-Plasmonics-Based Technique for Tracking the Viral Membrane Fluidity on a Single-Virus Level (2014-2016)
The viral membrane fluidity and liquid order has been shown to influence the infectivity of human immunodeficiency virus (HIV). Liquid order of membranes is a function of lipid composition and especially the cholesterol content. Increasing cholesterol content enhances the membrane liquid orders and decreases its fluidity. Therefore, being able to measure the cholesterol content and membrane fluidity in a single-virus level would make us able to study the infection mechanisms with unprecedented accuracy. This has not been possible, so far, through fluorescence microscopy techniques (due to their intrinsic limitations and the nano-scale size of virus particles) that are commonly used for membrane fluidity measurements such as General Polarization of Laurdan and fluorescence anisotropy method. We have developed a new technique which takes advantage of the high scattering cross section of plasmonic nanoparticles by temporally tracking them on the membrane of a virus while they are linked to the membrane lipids. The idea was first developed in-silico through predicting the kinetic behavior of the plasmonic nanoparticles through finite-difference time-domain simulations. The translational motions of nanoparticles attached to the virus membrane, and therefore their scattering polarization fluctuations over time, is correlated with the membrane fluidity. We have shown (through this technique and independently through a Laurdan assay) that changes in the cholesterol content of liposomes and virus-like particles, which result in changes in membrane fluidity as we calculated by molecular dynamics (MD) simulations, affect the polarization fluctuation trajectories that we experimentally recorded, in a quantifiable manner. The basics and initial results of this technique are submitted to be published soon.
- Quantification of Viral Surface Lipids Using Plasmon-Coupling-Based UV-Vis Spectrophotometry (2014-2015)
Specific lipids on the surface of viruses have been found in recent years to be playing important roles in their infection process. In the case of HIV-1, phosphatidylserine and gangliosides such as GM1 have been shown to contribute to the attachment and entry of the virus particles to various host cells. Therefore, the ability to quantify these surface lipids seems to be crucial in understanding the underlying mechanisms and finding therapeutic strategies against such viral diseases. Conventionally, mass spectrometry has been used as a precise method for lipid quantification. However, it suffers from the complicated sample preparation and relatively large sample sizes which cannot easily be provided by patient samples. We had previously developed a plasmon coupling microscopy-based technique which takes advantage of DNA-conjugated metal nanoparticles as probes and allows for clinically-relevant sample sizes, yet provides a precise quantification. Since the microscope sample preparation, measurement and data processing in this method requires some sophistication, we have simplified this technique by replacing the microscopy with UV-Vis spectrophotometry. This has given us the opportunity for a simpler and more time-efficient quantification procedure which only slightly compromises in precision. The assay works based on the reaction of biotinylated metal nanoparticles and liposomes or virus-like particles surface-functionalized with biotinylated cholera toxin B and neutravidin in colloid. The basics of this techniques are submitted to be published soon.
- Quantifying Lipid Contents in Enveloped Virus Particles with Plasmonic Nanoparticles (2012-2014)
Phosphatidylserine (PS) and monosialotetrahexosylganglioside (GM1) are examples of two host-derived lipids in the membrane of enveloped virus particles that are known to contribute to virus attachment, uptake, and ultimately dissemination. A quantitative characterization of their contribution to the functionality of the virus requires information about their relative concentrations in the viral membrane. We introduce herein a gold nanoparticle (NP) binding assay for probing relative PS and GM1 lipid concentrations in the outer leaflet of different virus-like particles (VLPs) using sample sizes of less than 3×106 particles. The assay evaluates both scattering intensity and resonance wavelength and determines relative NP densities through plasmon coupling as a measure for the target lipid concentrations in the NP-labeled VLP membrane. A correlation of the optical observables with absolute lipid contents was achieved by calibration of the plasmon coupling-based methodology with unilamellar liposomes of known PS or GM1 concentration. The performed studies reveal significant differences in the membrane of VLPs that assemble at different intracellular sites and pave the way to an optical quantification of lipid concentration in virus particles at physiological titers. The basics of this technique and its initial findings are published in Small, and it has been utilized in a few other studies by the Boston Medical Center and the National Cancer Institute researchers in collaboration with us, whose results are published in Virology and mBio.
Optimizing the performance of electrochemical biosensors using a continuous system of fabrication and qualifying the best combination of different strip variables (2009-2011)
Abstract: The possibility of making some improvements in the overall structure of the electrochemical portable biosensors to reduce the amount of their error and obtain a higher performance was investigated. Glucose biosensor was chosen as a case study, because it is now commercially produced and widely used, so the results of this study can be compared with a reliable source. This optimizing process was performed by designing and manufacturing a new complex of systems for producing the biosensor test strips, using novel methods such as inkjet printing of conductive electrodes and enzyme solution. Also, the design of the electrical circuits and digital monitoring system was developed.
This project resulted in registering some patents on a digital monitor for showing the concentration of different analytes in blood, capable of working with all available commercial test strips and an optimized semi-industrial production line for biosensor test strips.
Producing a carbon monoxide sensor based on PEDOT-PSS as a conductive polymer (2007-2009)
Abstract: Poly (3,4-ethylenedioxy) thiophene–poly (styrenesulfonate) has been used as a base to produce a carbon monoxide chemiresistive detector. Poly (3,4-ethylenedioxy) thiophene/poly (styrenesulfonate) (PEDOT/PSS) thin films were prepared by spin coating method. The polymer was tested by some common gases in the atmosphere to determine its discrimination ability to detect CO. Particular impositions of Fe, Al and morpholine were also added to the polymer solution to improve thin films sensitivity (percent of resistance variations with respect to its initial value) to recognize carbon monoxide from other gases like water vapor. Room air contains a partial pressure of water vapor which has the main effect on pure polymer. As doping agents were added to the polymer, prepared thin films showed a satisfying sensitivity to CO which was now comparable with the effects of room air. The sensor behavior in approach to room air and carbon monoxide and also the effect of dopants on the polymer has been described. (Results were published in Sensors and Actuators B as shown here.)