RESUMO
Magneto-elastic (ME) sensors have a great advantage in microbiology due to their ability to be queried wirelessly. Staphylococcus aureus is one of the most common bacteria widespread in the environment and a major human pathogen related to numerous illnesses. Immunosensors are affinity-based assays where the analyte is highly selective. The immobilization of antibodies (Ab) is an important step in the development of such devices. This study compared the effects of two antibody immobilization strategies on the analytical performance of a magneto-elastic immunosensor: (1) random antibody covalent immobilization (CysAb) and (2) specific-oriented antibody covalent immobilization (PrGAb). Immunosensors were exposed to solutions containing S. aureus at different concentrations (104 to 108CFU/ml) and sensor resonant frequencies were measured. In order to confirm that the frequency shifts were mainly caused by the binding of S. aureus to the sensor's surface, scanning electron microscope (SEM) and indirect immunofluorescence (IIF) images were taken after bacteria exposure at 108CFU/ml. Sensor surfaces were further monitored by non-contact topographic atomic force microscopy (AFM) images. In the covalent-oriented strategy, PrG was first bound covalently to the surface, which in turn, then binds the anti-S. aureus antibody in an oriented manner. Topographic AFM images showed different surface patterns between the two antibody immobilization strategies. Specific-oriented antibody covalent immobilization (PrGAb) strategy gave the highest anti-S. aureus antibody immobilization density. Therefore, the covalent-oriented strategy presented the best performance for S. aureus capture, detecting 104CFU/ml.
Assuntos
Staphylococcus aureus , Anticorpos , Técnicas Biossensoriais , Microscopia de Força AtômicaRESUMO
Magneto-elastic materials (ME) have important advantages when applied as biosensors due to the possibility of wireless monitoring. Commercial Metglas 2826MB3™ (FeNiMoB) is widely used, however sensor stabilization is an important factor for biosensor performance. This study compared the effects of biocompatibility and degradation of the Metglas 2826MB3™ alloy, covered or not with a gold layer, when in contact with cell culture medium. Strips of amorphous Metglas 2826MB3™ were cut and coated with thin layers of Cr and Au, as verified by Rutherford Backscattering Spectroscopy (RBS). Using Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES), the presence of metals in the culture medium was quantitatively determined for up to seven days after alloy exposure. Biocompatibility of fibroblast Chinese Hamster Ovary (CHO) cultures was tested and cytotoxicity parameters were investigated by indirect means of reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) at 1, 2 and 7 days. Cell death was further evaluated through in situ analysis using Acridine Orange/Ethidium Bromide (AO/EB) staining and images were processed with ImageJ software. Ions from Metglas(®) 2826MB3™ induced a degradation process in living organisms. The cytotoxicity assay showed a decrease in the percentage of live cells compared to control for the ME strip not coated with gold. AO/EB in situ staining revealed that most of the cells grown on top of the gold-covered sensor presented a normal morphology (85.46%). Covering ME sensors with a gold coating improved their effectiveness by generating protection of the transducer by reducing the release of ions and promoting a significant cell survival.
Assuntos
Ligas/farmacologia , Técnicas Biossensoriais , Materiais Revestidos Biocompatíveis/farmacologia , Ouro/farmacologia , Ligas/química , Animais , Células CHO , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromo/química , Cromo/farmacologia , Materiais Revestidos Biocompatíveis/química , Cricetulus , Ouro/química , Espectrofotometria AtômicaRESUMO
Escherichia coli are bacteria that must be controlled in the food industry and the hospital sector. Magnetoelastic biosensors offer the promise of rapid identification of these and other harmful antigens. In this work, strips of amorphous Metglas 2826MB3 were cut to size (5 mm × 1 mm) with a microdicing saw and were then coated with thin layers of Cr and Au, as verified by Rutherford backscattering spectroscopy (RBS). Several sensor surfaces were studied: 1) as-cast strip, wheel side; 2) as-cast strip, free surface; and 3) thinned and polished surface. A layer of cystamine was applied to the Au-covered magnetoelastic substrate, forming a self-assembledmonolayer (SAM), followed by antibodies, using a modified Hermanson protocol. The cystamine layer growth was verified by Fourier transform infrared spectroscopy (FTIR) and scanning electronmicroscopy (SEM). The biosensors were exposed to solutions of bacteria and the resonant frequency of the sensors was measured with an impedance analyzer for times up to 100 min. Reductions in the resonant frequency, corresponding to bacteria capture, were measured after optimizing the signal amplitude. For times up to 40 min, high capture rates were observed and thereafter saturation occurred. Saturation values of the frequency shifts were compared with the number of bacteria observed on the sensor using fluorescence microscopy. Parameters associated with capture kinetics were studied for different sensor surfaces. The rough surfaces were found to show a faster response, while the thinned and polished sensors showed the largest frequency shift.