Biophysical Methods

SPR is a powerful technique that measures biomolecular interactions in real time. It is based on optical technology where mass concentration of biomolecules in close proximity to a sensor chip surface is measured (Figure 2.1). The typical sensor chip is a glass slide coated with a gold layer which has a carboxymethylated dextran matrix where the ligand can be immobilised. When biomolecules bind to the immobilised ligand at the sensor chip, they cause a mass increase and a corresponding increase in the refractive index. This will alter the angle of the reflected light. The SPR angle is measured by the detector which monitors the changes in the reflected light intensity (Figure 2.1 A, from I to II). This change is recorded as a function of time in a sensorgram (Figure 2.1 B). The response increases when the analyte associates with the ligand. When the injection is stopped and the analyte dissociates from the ligand due to displacement by added buffer (regeneration buffer or recovery solution), the response falls. The response is measured in resonance units (RU) and is proportional to the mass at the surface.

Photo:

Schematic illustration of the SPR detection system. A) Change of the reflected light angle due to changes of the adsorbed mass on the surface. B) This change is recorded as a function of time in a sensorgram. Figure is redrawn from www.rci.rutgers.edu

DLS is a well-established method to characterise nanoparticles and biological macromolecules including proteins and polymers in the submicron region. This technique is mainly used to determine the size or size distribution, the molecular weight or the zeta potential of a particle. DLS analyses the Brownian motion of particles which have been hit by a monochromatic laser light, and measures the intensity fluctuation in the scattered light. Small particles diffuse quickly in fluid giving large pattern changes, while larger particles give smaller pattern variations (Figure 2.3). Thus, intensity changes result in an autocorrelation function which gives the rate of the particle diffusion. The size of the particle can then be determined by the use of the Stokes-Einstein relationship. 

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Schematic representation of a DLS instrument and data analysis. Diffusion of particles of different sizes changes the intensity pattern, which outcomes in an autocorrelation function from which the size of the particle can be determined based on Stokes-Ei

DLS is a good method to investigate conformational stability of a protein and their tendency to oligomerize and aggregate over time or depending on temperature. 

CD is a type of absorption spectroscopy that can examine various aspects of protein structure in solution including secondary structure composition, tertiary structure fingerprint and conformational changes, protein folding and denaturation. The principle is as follows: the difference between the absorption of left and right handed circularly-polarised light is measured as a function of the wavelength. Circularly polarised light consists of two polarised wavecomponents with the same amplitude and a phase difference of 90°. If the combined vector of the wave is rotated clockwise, right handed-circular polarised light is produced; vice versa for the left handed-circular polarisation. After passing through an optically active sample, the right and left handed polarised light have different amplitudes. The combination of the two unequal beams gives elliptically polarised light. Thus, CD measures the light that is not absorbed, as well as the change in optical rotation relative to the incident light (Figure 2.2 A). For the purpose of CD on protein and peptides, CD spectra are divided into two different regions, far-UV spectral region, which lies between 190 nm and 250 nm, and the nearUV range (250–350 nm). In far-UV, changes in the secondary structure can be studied (Figure 2.2 B), while CD in near-UV provides information about the tertiary structure. 

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The principle of CD spectroscopy. A) The light passes through an optically active sample which causes an amplitude change of the right and left handed polarised light. Figure taken from www.nmsu.edu B) Three typical far-UV CD spectra with the associated s

CD can be used to gain structural information on proteins and peptides. Furthermore, it can be used for examining the effect of solvent conditions, pH and changing temperature on protein conformation and stability. It should be noted, however, that CD cannot provide information at residue level and it is thus low in structural detail. 

AFM is a high resolution imaging method that can be used to study the surface topography in atomic resolution and the adhesion of e.g. nanoparticles, macromolecules and cells to a surface. Furthermore it can provide information on the mechanical, electrical and chemical properties of the material. The principle is based on a laser beam deflection system. Briefly, a laser beam is reflected from the back side of the cantilever onto a position-sensitive photodetector (Figure 2.5). In this arrangement, a small deflection of the cantilever will tilt the reflected beam and change the position of the beam on the photodetector. As the tip moves along the surface of the sample it goes up and down the contours of the surface and causing the laser beam to be deflected by the cantilever into a dual photodiode. A photodetector measures the difference in light intensity between the upper and lower photodiode and this difference is converted to a voltage. 

Photo:

The principle of AFM. Beam deflection system, using a laser and photodector to measure the beam position. When the cantilever moves up and down the beam is moved across the surface of the detector. Figure is redrawn from www.iept.tu-clausthal.de

Imaging can take place in different modes, where the most common are alternating current (AC) mode and contact mode. The AC mode allows high resolution topographic imaging of sample surfaces that are easily damaged and loosely hold to their substrate. Imaging is implemented by alternately placing the tip in contact with the surface and then lifting the tip off the surface. Thus, the cantilever oscillates near its resonant frequency using a piezoelectric crystal. During AC mode operation, the cantilever oscillation amplitude is maintained constant by a feedback loop. In contact mode the tip scans in close contact across the surface while the tip is constantly adjusted to maintain a constant deflection due to the feedback amplifier. The force on the tip is repulsive and set by pushing the cantilever against the sample surface with a piezoelectric positioning element. In addition to topographical measurements, AFM can also be used to measure forces on the cantilever as the tip comes close to the sample surface and is pulled off again. This technique can be used to measure a series of attractive and repulsive forces between the tip and the sample surface to map the local chemical and mechanical properties such as adhesion, elasticity and thickness of the adsorbed molecule layer.