A Novel Point-of-Care BioNanoSensor for Rapid HIV Detection and Treatment Monitoring.

We report here a new diagnostic approach to the direct detection of HIV in blood or other body fluids that is rapid, sensitive and potentially applicable in a point-of-care setting. The approach follows on the development of a novel BioNanoSensor (BNS) device that utilizes piezoelectric technology to detect the presence of the HIV surface glycoprotein gp120 in a nanoscale format. The detection range of the BNS device for the biomarker gp120 displayed a low-end sensitivity of 6.5×104 HIV viral particles/ml, while using a small fluid sample (5 µl) and with a reaction time of less then 30 seconds. Performance of this device indicated that the BNS has utility for direct detection of HIV particles prior to, and independent from, antibody formation. Accordingly, this device holds utility to monitor the status of HIV infection both early after exposure to virus as well as during chronic HIV infection. The BNS parameters of small sample volume, compact device size, and detection sensitivity indicate that the BNS is potentially useful in the point-of-care and/or home setting for monitoring decisions regarding HIV treatment on a real-time basis.

Development of recombinant Aleuria aurantia lectins with altered binding specificities to fucosylated glycans.

Changes in glycosylation have long been associated with disease. While there are many methods to detect changes in glycosylation, plant derived lectins are often used to determine changes on specific proteins or molecules of interest. One change in glycosylation that has been observed by us and by others is a disease or antigen associated increase in fucosylation on N-linked glycans. To measure this change, the fucose binding Aleuria aurantia lectin (AAL) is often utilized in plate and solution based assays. AAL is a mushroom derived lectin that contains five fucose binding sites that preferentially bind fucose linked (α-1,3, α-1,2, α-,4, and α-1,6) to N-acetyllactosamine related structures. Recently, several reports by us and by others have indicated that specific fucose linkages found on certain serum biomarker glycoprotein's are more associated with disease than others. Taking a site-directed mutagenesis approach, we have created a set of recombinant AAL proteins that display altered binding affinities to different analytes containing various fucose linkages.

Improved protein detection on an AC electrokinetic quartz crystal microbalance (EKQCM).

Microscale electrodes supplied with an AC field can generate rotational fluid patterns known as AC electroosmosis. In the present study, this effect was used to improve antibody binding on a biosensor surface. Antibodies, like many other large, slow moving biomolecules, tend to suffer from transport limitations during a reaction with a surface-bound receptor. Stirring such reactions with AC electroosmosis can alleviate this transport limitation by bringing fresh reagent to the surface. For the first time, the use of this phenomenon was used to improve the capture of protein on a sensor. Directly adsorbed antibodies were bound to the surface of specially modified quartz crystal microbalances, known as electrokinetic QCMs (EKQCMs) and the signal was enhanced by about 5.6 times. Modification of the QCM resulted in little reduction of quality factor (from ∼ 5.3 k to ∼ 4.6k) and an increased sensitivity to viscosity changes (151%). Full immunoassays performed on electrodes fabricated on glass surfaces were used to ensure antibody function was not significantly degraded by the enhancement technique.

Real-time analysis of cell-surface adhesive interactions using thickness shear mode resonator.

The cell adhesion process and the molecular interactions that determine its kinetics were investigated using a thickness shear mode (TSM) sensor. The goal of this study was to correlate sensor readings with the progression of cell adhesion. In particular, the specific effects of receptor-mediated adhesion, the glycocalyx, and surface charge on initial cell-surface attachment and steady-state adhesion of endothelial cells were investigated. We found a strong correlation between resistance changes (DeltaR) and the development of cell adhesion strength by comparing the sensor readings with independently assessed cell adhesion. The result showed that integrin binding determines the kinetics of initial cell attachment while heparan sulfate proteoglycan (HSPG) modulates steady-state adhesion strength. Coating the sensor surface with the positively charged poly-d-lysine (PDL) enhanced the initial interaction with substratum. These data confirm our current understanding of the contribution of these three phenomena to the adhesion process. The real-time monitoring capability of this technique with high temporal resolution provides more detailed information on the kinetics of the different stages of the adhesion process. This technique has the potential to facilitate the evaluation of biomaterials and surface treatments used for implants and tissue-engineering scaffolds for their bioactive effects on the cell adhesion process.

The study of an interaction of solid particles with various surfaces using TSM sensors.

The interaction of solid particles with various surfaces has been experiencing growing interest in many areas of nanotechnology, colloidal science, and biology. In this paper the interactions of solid particles with the surface of piezoelectric thickness shear mode (TSM) sensors have been studied. A mechanical model has been presented to evaluate the effect of particle loading on the behavior of a TSM sensor. The main sources contributing to the interaction, such as Van der Waals force, friction force, and electrostatic force, are discussed. Experiments have been designed for 10-100 microm particles on the 5-MHz and 10-MHz TSM sensors. It has been shown that the resonant frequencies of the TSM sensors might increase or decrease depending on the interaction conditions. The results have shown that the TSM sensor technique could provide the information on the mass/size of a particle and the binding energy between a particle and the sensor surface. This technique may find its applications in characterizing the properties of an interaction between particles and various surfaces.