Design and Simulation of the Microcantilever Biosensor for MITF Antigen and D5 Monoclonal Antibody Interaction Finite Element Analysis, and Experimental.

BACKGROUND: Biosensors and MEMS have witnessed rapid development and enormous interest over the past decades. Constant advancement in diagnostic, medical, and chemical applications has been demonstrated in several platforms and tools. In this study, the analytical and FEA of the microcantilever used in biomolecular analyses were compared with the experimental analysis results. METHODS: In this study, MITF antigen, which is a melanoma biomarker, and anti-MITF antibody (D5) were selected as biomolecules. A MEMS-type microcantilever biosensor was designed by functionalizing the AFM cantilever by utilizing the specific interaction dynamics and intermolecular binding ability between both molecules. Surface functionalization of cantilever micro biosensors was performed by using FEA. The stress that will occur as a result of the interactions between the MITF-D5 has been determined from the deviation in the resonant frequency of the cantilever. RESULTS: It has been found that the simulation results are supported by analytical calculations and experimental results. CONCLUSION: The fact that the results of the simulation study overlap with the experimental and mathematical results allows us to get much cheaper and faster answers compared to expensive and time-consuming experimental approaches.

Discontinuation of lamivudine treatment in HBeAg-negative chronic hepatitis B: a pilot study with long-term follow-up.

BACKGROUND: Finite treatment of hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB) with nucleoside/nucleotide analogues (NAs) is important in resource-limited countries. Outcome of treatment discontinuation in patients on long-term lamivudine (LVD) was assessed in a single centre observational pilot study in the current study. METHODS: Non-cirrhotic patients on LVD for at least 5 years with undetectable HBV DNA on at least two consecutive assessments were offered to stop treatment. Biochemical, serological and virological measures were determined at 3-6 month intervals after treatment discontinuation. Serum quantitative hepatitis B surface antigen (HBsAg) was determined at treatment discontinuation and 5-6 years thereafter. NA treatment was re-instituted in patients with confirmed viral rebound defined as HBV DNA >20,000 IU/ml. Relapser patients were no longer followed but were re-assessed 6 years after treatment cessation. RESULTS: LVD was discontinued in 23 patients. 8 patients relapsed within 1 year and NA treatment was restarted; 15 patients (65%) were non-relapsers. Thirteen of them were followed for at least 5 years. Two patients had undetectable HBV DNA throughout the follow-up period. In the rest, HBV DNA fluctuated at low levels. Two patients cleared HBsAg 24 and 36 months after stopping treatment. Quantitative HBsAg levels 5-7 years after treatment discontinuation were lower in non-relapser compared to relapser patients (1.21 IU/ml ±0.98 versus 2.71 ±0.76; P=0.002). Of 8 relapser patients 1 patient had HBsAg levels less than 100 IU/ml compared to 11 out of 13 non-relapser patients (P=0.0022). CONCLUSIONS: These data suggest that cessation of NA treatment is a viable option after a reasonable treatment duration in patients with HBeAg-negative CHB and that HBsAg clearance may become an achievable target in these patients.

Nanomechanics on FGF-2 and Heparin Reveal Slip Bond Characteristics with pH Dependency.

Fibroblast growth factor 2 (FGF-2), an important paracrine growth factor, binds electrostatically with low micromolar affinity to heparan sulfates present on extracellular matrix proteins. A single molecular analysis served as a basis to decipher the nanomechanical mechanism of the interaction between FGF-2 and the heparan sulfate surrogate, heparin, with a modular atomic force microscope (AFM) design combining magnetic actuators with force measurements at the low force regime (1 × 101 to 1 × 104 pN/s). Unbinding events between FGF-2-heparin complexes were specific and short-lived. Binding between FGF-2 and heparin had strong slip bond characteristics as demonstrated by a decrease of lifetime with tensile force on the complex. Unbinding forces between FGF-2 and heparin were further detailed at different pH as relevant for (patho-) physiological conditions. An acidic pH environment (5.5) modulated FGF-2-heparin binding as demonstrated by enhanced rupture forces needed to release FGF-2 from the heparin-FGF-2 complex as compared to physiological conditions. This study provides a mechanistic and hypothesis driven model on how molecular forces may impact FGF-2 release and storage during tissue remodeling and repair.

An Atomic Force Microscope with Dual Actuation Capability for Biomolecular Experiments.

We report a modular atomic force microscope (AFM) design for biomolecular experiments. The AFM head uses readily available components and incorporates deflection-based optics and a piezotube-based cantilever actuator. Jetted-polymers have been used in the mechanical assembly, which allows rapid manufacturing. In addition, a FeCo-tipped electromagnet provides high-force cantilever actuation with vertical magnetic fields up to 0.55 T. Magnetic field calibration has been performed with a micro-hall sensor, which corresponds well with results from finite element magnetostatics simulations. An integrated force resolution of 1.82 and 2.98 pN, in air and in DI water, respectively was achieved in 1 kHz bandwidth with commercially available cantilevers made of Silicon Nitride. The controller and user interface are implemented on modular hardware to ensure scalability. The AFM can be operated in different modes, such as molecular pulling or force-clamp, by actuating the cantilever with the available actuators. The electromagnetic and piezoelectric actuation capabilities have been demonstrated in unbinding experiments of the biotin-streptavidin complex.

Dually actuated atomic force microscope with miniaturized magnetic bead-actuators for single-molecule force measurements.

We report a novel atomic force microscopy (AFM) technique with dual actuation capabilities using both piezo and magnetic bead actuation for advanced single-molecule force spectroscopy experiments. The experiments are performed by manipulating magnetic microbeads using an electromagnet against a stationary cantilever. Magnetic actuation has been demonstrated before to actuate cantilevers, but here we keep the cantilever stationary and accomplish actuation via free-manipulated microstructures. The developed method benefits from significant reduction of drift, since the experiments are performed without a substrate contact and the measured force is inherently differential. In addition, shrinking the size of the actuator can minimize hydrodynamic forces affecting the cantilever. The new method reported herein allows for the application of constant force to perform force-clamp experiments without any active feedback, profiled for a deeper understanding of biomolecular interactions.