BT 11: principle, apparatus, applications and limitations of various biophysical techniques

Biophysics is an interdisciplinary science using methods of, and theories from, physics to study biological systems. Biophysics spans all scales of biological organization, from the molecular scale to whole organisms and ecosystems.Biophysical research shares significant overlap with biochemistry, nanotechnology, bioengineering, computational biology and (complex) systems biology. It has been suggested as a bridge between biology and physics.

Biophysical techniques:Biophysical methods are techniques to study the structure, properties, dynamics or function of biomolecules at an atomic or molecular level. They encompass a range of techniques including microscopy, spectroscopy, electrophysiology, single-molecule methods and molecular modelling.There include:Microscopy, gel electrophoresis, chromatography, cell disruption methods, centrifugation, NMR, crystallography etc.

Principle, apparatus, applications and limitations of various biophysical techniques

I) Centrifugation:Centrifugation is a process which involves the use of the centrifugal force for the sedimentation of heterogeneous mixtures with a centrifuge, used in industry and in laboratory settings. This process is used to separate two immiscible liquids. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture migrate towards the axis. Chemists and biologists may increase the effective gravitational force on a test tube so as to more rapidly and completely cause the precipitate (pellet) to gather on the bottom of the tube. The remaining solution is properly called the "supernate" or "supernatant liquid". The supernatant liquid is then either quickly decanted from the tube without disturbing the precipitate, or withdrawn with a Pasteur pipette.

The rate of centrifugation is specified by the angular velocity measured in revolutions per minute (RPM), or acceleration expressed as g. The conversion factor between RPM and g depends on the radius of the centrifuge rotor. The particles' settling velocity in centrifugation is a function of their size and shape, centrifugal acceleration, the volume fraction of solids present, the density difference between the particle and the liquid, and the viscosity. The most common application is the separation of solid from highly concentrated suspensions, which is use in the treatment of sewage sludges for dewatering where less consistent sediment is produced. Centrifuges are further classified into various subtypes depending on the speed, rotor types and intended applications.

Apparatus:

A typical bench top centrifuge separate or concentrate substances suspended in a liquid medium by density. Space-saving fixed- and variable-speed benchtop or tabletop centrifuges are used for applications including tissue culture, protein work, DNA/RNA research, and cell harvesting. Although spinning is used to achieve separation in all centrifuges, the rotor’s rpm only indicates the power of the motor. The best indication of separation power is its RCF, or relative centrifugal force. Versatile multipurpose centrifuges are the most common type, with an RCF up to about 24,000 × g, a variety of volume ranges, and the ability to spin plates. They can accommodate different types of rotors, including fixed angle, swinging bucket, and continuous flow. Ultraspeed centrifuges offer g-forces up to 1,000,000 × g, useful in nanotechnology. Microcentrifuges spin small sample volumes, such as 0.2-mL PCR tubes, at very high speeds. Other factors to consider include noise level, easy bowl access, refrigeration capabilities, and rotor material, which can be metal, plastic, or composite.

Applications of centrifugationClinical and blood bankingMicrobiologyTissue cultureMolecular biology and genomicsDrug discovery and proteomics

Limitations of centrifugation• Limitations of the centrifuge include high capital and operational costs as they are

energy-intensive. In addition, the maintenance costs and number of incidents of breakdown are higher than with static separators due to the moving parts.

• Another limitation is the narrow range for optimum performance with variable conditions like feed acceleration, positioning of the interface and solid discharge method. In addition, sealing materials, especially the dynamic sealing materials, must be carefully chosen to be chemically and thermally resistant.; therefore, a

much more extensive design and optimization program is required than with the static separators; therefore, a much more extensive design and optimization program is required than with the static separators.

The actual achieved separative power of gas centrifuge will be always lower than its theoretical separative power reflecting additional inefficiencies in the centrifuges when running as individual machines and in cascades. For example, The IR-1 centrifuge achieves an average separative power in cascades of less than one SWU/year, significantly less than its theoretical maximum separative power of 4.9 SWU/year. Although its value when run individually is greater, it is still far below the theoretical value. It also experiences a relatively high breakage rate, which accounts for much of the additional reduction of its separative power when run in production cascades.

II) Gel electrophoresis:Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge and/or size (IEF agarose, essentially size independent) and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.

Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through a matrix of agarose or other substances. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving.

Proteins are separated by charge in agarose because the pores of the gel are too large to sieve proteins.

Gel electrophoresis can also be used for separation of nanoparticles.

Gel electrophoresis uses a gel as an anticonvective medium and/or sieving medium during electrophoresis, the movement of a charged particle in an electrical field. Gels suppress the thermal convection caused by application of the electric field, and can also act as a sieving medium, retarding the passage of molecules; gels can also simply serve to maintain the finished separation, so that a post electrophoresis stain can be applied.[3] DNA Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via PCR, but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further characterization.

Apparatus:

In simple terms, electrophoresis is a process which enables the sorting of molecules based on size. Using an electric field, molecules (such as DNA) can be made to move through a gel made of agar or polyacrylamide. The electric field consists of a negative charge at one end, which pushes the molecules through the gel, and a positive charge at the other end that pulls the molecules through the gel. The molecules being sorted are dispensed into a well in the gel material. The gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric current is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster. The different sized molecules form distinct bands on the gel.

The term "gel" in this instance refers to the matrix used to contain, then separate the target molecules. In most cases, the gel is a cross-linked polymer whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed. When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually composed of different concentrations of acrylamide and a cross-linker, producing different sized mesh networks of polyacrylamide. When separating larger nucleic acids (greater than a few hundred bases), the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet porous matrix. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and must be handled using appropriate safety precautions to avoid poisoning. Agarose is composed of long unbranched chains of uncharged carbohydrate without cross links resulting in a gel with large pores allowing for the separation of macromolecules and macromolecular complexes.

"Electrophoresis" refers to the electromotive force (EMF) that is used to move the molecules through the gel matrix. By placing the molecules in wells in the gel and applying an electric field, the molecules will move through the matrix at different rates, determined largely by their mass when the charge to mass ratio (Z) of all species is uniform. However, when charges are not all uniform then, the electrical field generated by the electrophoresis procedure will affect the species that have different charges and therefore will attract the species according to their charges being the opposite. Species that are positively charged will migrate towards the cathode, which is negatively charged (because this is an electrolytic rather than galvanic cell). If the species are negatively charged they will migrate towards the positively charged anode.

If several samples have been loaded into adjacent wells in the gel, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows

separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved component. Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are molecular weight size markers available that contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule.

Applications of electrophoresis:

• Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in restriction mapping of cloned DNA.

• Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting• Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to

Northern transfer.Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and biochemistry. The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device. The image is recorded with a computer operated camera, and the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel. The measurement and analysis are mostly done with specialized software.

Limitations of electrophoresis:

1. Electrophoresis Measurements Are Not Precise

Gel electrophoresis can effectively separate similar proteins with different weights (this is a technique called Western blotting). It can separate them more precisely through a technique known as 2d electrophoresis; this is common in proteomics.Unfortunately, all of the measurements made from this technique are semi-quantitative at best. In order to obtain the precise mass (weight) of proteins, mass spectroscopy must be employed after the protein has been purified by electrophoresis. Furthermore, comparing the relative amounts of different molecules relies on the band density (darkness) of different spots on the gel. This method has some degree of error, and samples are usually run multiple times to get clean results.

2. Substantial Starting Sample is Required

Electrophoresis is a technique of isolating and visually identifying different biomolecules. This is done by passing an electrical current through the gel to separate charged molecules of different weights. If the molecule you're interested in isn't common enough, its band will be virtually invisible and difficult to measure.DNA and RNA can be amplified somewhat before running electrophoresis, but it isn't practical to do this with proteins. Therefore, a large

tissue sample is needed to run these assays. This can limit the usefulness of the technique, especially in medical analysis. It's virtually impossible to run electrophoresis on samples from a single cell; flow cytometry and immunohistochemistry are more commonly used to assess cell-by-cell expression of proteins. A technique called PCR is excellent at precisely measuring tiny amounts of RNA.

3. Gel electrophoresis requires a large amount of DNA or protein to start with. Only Certain Molecules Can Be Visualized

Electrophoresis is excellent at separating and identifying medium- to large-sized biomolecules. However, many of the molecules that researchers wish to look at are smaller; small hormones, neurotransmitters, and ions cannot be measured by electrophoresis. This is for two reasons: they don't properly react with the electrophoresis preparation (usually a technique called SDS PAGE) and, even if they did, they are too small to separate properly and would rush out the bottom of the gel. These molecules are instead measured by techniques such as RIAAs (radio immunoassays) and ELISAs (enzyme-linked immunosorbant assay).

4. Electrophoresis is Low Throughput

Gel electrophoresis is generally low throughput, meaning it doesn't produce data especially rapidly. Contrast electrophoresis, where you can look at a small handful of RNA molecules at a time, with PCR (polymerase chain reaction), which can simultaneously assess thousands of samples. Similarly, flow cytometry can take measurements from thousands of individual cells and make complex correlations, while electrophoresis looks at cells en masse and cannot make such fine discriminations. PCR and flow cytometry represent massively parallel and serial processes respectively, and both far outstrip the abilities of electrophoresis to generate research data.

III) X-ray crystallography

X-ray crystallography can locate every atom in a zeolite, an aluminosilicate.X-ray crystallography is a tool used for identifying the atomic and molecular structure of a crystal, in which the crystalline atoms cause a beam of incident X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their disorder and various other information.

Since many materials can form crystals—such as salts, metals, minerals, semiconductors, as well as various inorganic, organic and biological molecules—X-ray crystallography has been fundamental in the development of many scientific fields. In its first decades of

use, this method determined the size of atoms, the lengths and types of chemical bonds, and the atomic-scale differences among various materials, especially minerals and alloys. The method also revealed the structure and function of many biological molecules, including vitamins, drugs, proteins and nucleic acids such as DNA. X-ray crystallography is still the chief method for characterizing the atomic structure of new materials and in discerning materials that appear similar by other experiments. X-ray crystal structures can also account for unusual electronic or elastic properties of a material, shed light on chemical interactions and processes, or serve as the basis for designing pharmaceuticals against diseases.

In a single-crystal X-ray diffraction measurement, a crystal is mounted on a goniometer. The goniometer is used to position the crystal at selected orientations. The crystal is bombarded with a finely focused monochromatic beam of X-rays, producing a diffraction pattern of regularly spaced spots known as reflections. The two-dimensional images taken at different rotations are converted into a three-dimensional model of the density of electrons within the crystal using the mathematical method of Fourier transforms, combined with chemical data known for the sample. Poor resolution (fuzziness) or even errors may result if the crystals are too small, or not uniform enough in their internal makeup.

Apparatus:

x-ray tubes provides a means for generating x-ray radiation in most analytical

instruments. An evacuated tube houses a tungsten filament which acts as a cathode opposite to a much larger, water cooled anode made of copper with a metal plate on it. The metal plate can be made of any of the following metals: chromium, tungsten, copper, rhodium, silver, cobalt, and iron. A high voltage is passed through the filament and high energy electrons are produced. The machine needs some way of controlling the intensity and wavelength of the resulting light. The intensity of the light can be controlled by adjusting the amount of current passing through the filament; essentially acting as a temperature control. The wavelength of the light is controlled by setting the proper accelerating voltage of the electrons. The voltage placed across the system will determine the energy of the electrons traveling towards the anode. X-rays are produced when the electrons hit the target metal.

Applications:

• Chemistry:    characterising new products and materials, investigating their properties

• Physics:   determining fundamental properties

• Engineering: identifying properties of materials; stress analysis; tomography    

• Biology, medicine:   structures of macromolecules such as proteins and DNA

• Surface science: studying the structures of surfaces and interfaces under various environmental conditions

• Geology:    identification of minerals and understanding their transformations

• Planetary science: studying the behaviour of atmospheric components under extreme conditions

• Pharmaceutical:         structure-based drug design, physical properties, polymorph screening, patent applications, quality control

Nanotechnology: investigation of structures, properties and their interdependence

Limitations

The uncertainties introduced during the derivation of an atomic model from the experimentally observed electron density data are not always appreciated.

Uncertainties in the atomic model can have significant consequences when this model is subsequently used as the basis of manual design, docking, scoring, and virtual screening efforts.

Docking and scoring algorithms are currently imperfect. A good correlation between observed and calculated binding affinities is usually

only observed only when very large ranges of affinity are considered. Errors in the correlation often exceed the range of affinities commonly

encountered during lead optimization.