Coupling of surface plasmon resonance (SPR) detection to asymmetric flow field-flow fractionation (AF4) offers the possibility to study active fractions of bio-separations on real samples, such as serum and saliva, including the assessment of activity of possibly aggregated species. The coupling of SPR with AF4 requires the possibility to select fractions from a fractogram and redirect them to the SPR. The combination of SPR with chromatography-like methods also requires a mechanism for regeneration of the receptor immobilised onto the SPR sensor surface. In recent work, the combination of size exclusion chromatography (SEC) with SPR was pioneered as a successful methodology for identification, characterisation and quantification of active biocomponents in biological samples. In this study, the approach using AF4 is evaluated for the antibody trastuzumab in buffer and serum. The particular object of this study was to test the feasibility of using AF4 in combination with SPR to detect and quantify proteins and aggregates in complex samples such as blood serum. Also, in the investigation, three different immobilisation methods for the receptor HER-2 were compared, which involved (1) direct binding via EDC/NHS, the standard approach; (2) immobilisation via NTA-Ni-Histag complexation; and (3) biotin/avidin-linked chemistry using a regenerable form of avidin. The highest specific activity was obtained for the biotin-avidin method, while the lowest specific activity was observed for the NTA-Ni-Histag linkage. The data show that AF4 can separate trastuzumab monomers and aggregates in blood serum and that SPR has the ability to selectively monitor the elution. This is an encouraging result for automated analysis of complex biological samples using AF4-SPR.
A variable-wavelength Kretschmann configuration surface plasmon resonance (SPR) apparatus with angle scanning is presented. The setup provides the possibility of selecting the optimum wavelength with respect to the properties of the metal layer of the sensorchip, sample matrix, and biomolecular interaction of interest. Monitoring SPR curves over a wide angular range (39 •) permits simultaneous determination of the total internal reflection angle (TIR), the resonance angle, and the intensity and width of the SPR dip, which are essential parameters for measuring binding events and achieving optimum sensitivity. The new apparatus was evaluated by recording full SPR curves at different wavelengths ranging from 600 to 890 nm using sensor surfaces of silver, gold and gold with deposited silicon oxide, aluminum oxide, titanium oxide and indium tin oxide which were exposed to air and an aqueous solution of sodium chloride. Clear wavelength dependencies of sensor-material resonance angles and SPR-dip widths were demonstrated. In order to investigate the capability of the system to probe molecular binding to different sensor surfaces, the layer-by-layer adsorption of charged polyelectrolytes was monitored in angular scanning mode at 600, 670, 785, and 890 nm. Although at longer wavelengths lower angular shifts were observed as result of layer deposition, the sharper dip, wider detection window and better signal-to-noise ratios at these wavelengths can be beneficial for binding studies. The applicability for biosensing was tested by immobilizing human serum albumin (HSA) on an aluminum-oxide-coated gold sensor using a new procedure and measuring the binding of anti-HSA antibodies at the optimal wavelength (890 nm) in angular-scanning and fixed-angle mode. The HSA biosensor showed negligible non-specific interaction and yielded almost ten times better sensitivity than obtained with a conventional gold-dextran-based sensor operated at 670/785 nm. Analysis of anti-HSA samples pre-incubated with different concentrations of HSA allowed measurement of the IC50 value. The reported data demonstrate the usefulness of the presented variable-wavelength angle-scanning SPR instrument, permitting continuous recording of full SPR curves in time at any selected wavelength in the 600-890 nm range using a sensor material of choice.
Merging biological and non-biological matter to fabricate nanoscale assemblies with controllable motion and function is of great interest due to its potential application, for example, in diagnostics and biosensing. Here, we have constructed a DNA-based bionanoactuator that interfaces with biological and non-biological matter via an electric field in a reversibly controllable fashion. The read-out of the actuator is based on motion-induced changes in the plasmon resonance of a gold nanoparticle immobilized to a gold surface by single stranded DNA. The motion of the gold nanoparticle and thus the conformational changes of the DNA under varying electric field were analyzed by dark field spectroscopy. After this basic characterization, another actuator was built utilizing hairpin-DNA coated gold nanoparticles, where the hairpin-DNA induced discrete transitions between two specific open-loop and folded-loop states. These two states and the transition dynamics between them were clearly visible in the actuator behavior. The demonstrated nanoactuator concept could be readily extended to inspection of conformational changes of other biomolecules as well. Besides, this concept enables other possibilities in applications like surface-enhanced Raman spectroscopy and fluorescence enhancement, since the specific wavelength of the plasmon resonance of the actuator can be tuned by the external voltage.
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