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Role of Protein in Stem Cell Research

1.1 Background/ Stem Cells and Developments

The human body is a highly complicated bio-system with each organ made of several tissues playing a dedicated and highly specific role in the overall functioning of the human body. The body itself has an innate ability to repair itself when subjected to many stresses or injury to a certain extent over time. This regenerative capability has captured the interest of scientists for a very long time in order to assist them to take advantage of the regenerative properties of cells using synthetic devices, implants and laboratory cultured tissue for regenerative medicine (Tabar & Studer 2014). This is where stem cells come into the picture. Stem cells are undifferentiated cells that have the potential of replicating themselves via mitosis and differentiating into any kind of specialised cell.
The recognition and earliest study of stem cells dates back to 1868, when Ernst Haeckel, a German biologist, first used the word stem cell that is derived from the German word “Stammzelle”. He described a stem cell as a unicellular ancestor of multicellular organisms and the fertilised egg giving rise to a whole organism comprised of innumerable different cell types arising from the development of the hematopoietic (system of blood making organs and tissue; chiefly the bone marrow, thymus, lymph nodes and spleen) system. The touchstone of stem cells being referred to as undifferentiated embryonic cells occurred later in the 19th century (Ramalho-Santos & Willenbring).
Fast forwarding to the present day, the development of stem cell technologies and applications have revolutionised the medical approach to wound healing, aesthetic implants, tissue repair, cosmetic surgery along with other health and body related modifications and reinforcements (Maguire & Friedman 2014). Easy culturing of in vitro stem cells will also promote an individual-specific treatment with a high acceptance rate from the individual’s body with rapid recovery over a short period of time.
Laboratory culture induced pluripotent stem cells (iPSC) are identified and characterised by the expression of a cluster of four prominent proteins or cytokines (interferon, interleukin, and growth factors) OSKM [Oct 3/4, Sox2, Klf4 and c-Myc] factors (Takahashi et al. 2007) or OSNL [Oct4, Sox2, Nanog, and Lin28t] factors cultivated on specialised gelatin-coated, Matrigel®-coated culture plates (Yu et al. 2007).
In order for us to understand the underlying communications and interactions between the cells, the culture medium or substrates they grew on, we must look at a nucleic stage where proteins perform all the tedious workshop activities (Pawson & Nash 2000). Proteins are essential in defining the structure, functions and regulation of a cell and tissue.

1.2 The position of proteins in cell cultures

Proteins have very important roles within a cell as they can act as messengers, activation agents, binding agents and catalysts for many mechanisms that take place. They also play a prominent role in the motility and adhesion of cells when they are placed in an unfamiliar environment or subjected to unfavourable conditions. The adhesion or retraction of cells in accordance with the external environment is a result of the transmembrane proteins called cell adhesion molecules (CAMs). These are located on the surface of cells and play a fundamentally imperative role in cell developmental and pathological transformational processes as they mediate the attachment of cells to substrates through the extracellular matrix (ECM) and neighbouring cells (Mecham 2011). CAMs are mainly comprised of three prominent domains or regions of a protein complex which include:

  1. an intracellular domain cooperating with the cytoskeleton within a cell
  2. a transmembrane domain spanning across the width of the cell membrane functioning as a gateway for molecular transport
  3. an extracellular domain protruding out of the membrane involved in interactions with compatible CAMs on other cells [Homophilic binding] or with protein complexes in the extracellular matrix [Heterophilic binding] (Freemont & Hoyland 1996).

Figure 1 is a representation of the integration and receptivity of the four major
families of CAMs. Source: (Goodman 2008).
Illustrated in the figure above are the four most important CAMs devoted to the modulation of cellular adhesion and proliferation on substrates. The greater majority of all the known CAMs can be classified into four families of proteins.

  1. Immunoglobulin (Ig) or antibody superfamily: Involved in recognition, binding and adhesion process.
  2. Integrins: Involved in the ligand-based binding of a cell to the ECM.
  3. Cadherins: Calcium ion (Ca2+) dependent transmembrane proteins occupied with cell-cell binding within a tissue structure.
  4. Selectins: Single chain glycoproteins associated with binding with sugar complexes. Are also dependent on calcium ions for functioning (Goodman 2008).

1.2.1 Vitronectin

A member of the hemopexin family (evolutionary based plasma proteins), vitronectin (VN) is a multifunctional glycoprotein found mostly in the extracellular matrix material, cell surface and bone tissue of the human body. It is found to be synthesised prominently in the liver of adults but found in other tissue areas during foetal development, especially the stomach, intestine and adrenal areas. Having a molecular weight of 75000 Daltons, it is composed of 478 amino acids (AA). The first 19 units (Sequence no: 1-19) made up of a signalling peptide (Sigurdardottir & Wiman 1994), followed by 459AA units of vitronectin V65 subunit chain (Sequence no: 20-398). Contained within this chain is a 44AA unit (Sequence no: 20-63) Somatomedin-B peptide accountable for any protease inhibiting activity exhibited by vitronectin (Fryklund & Sievertsson 1978){Fryklund, 1978 #39}. The same somatomedin-B peptide is illustrated in the figure below. The protein is concluded with a vitronectin V10 subunit chain consisting of 80AA units (Sequence no: 399-478). The functioning of vitronectin can be modulated with the aid of proteolytic enzymes (Suzuki et al. 1985).

Figure 2 of the Somatomedin-B domain of Vitronectin molecule produced
using PyMOL software. Source: Research Collaboratory for Structural
Bioinformatics-Protein Data Bank-1S4G.
Vitronectin, being analogous with fibronectin has been proved to play a major role in cell adhesion processes due to the presence of cell recognition sequence ‘Arg-Gly-Asp’ [RGD] being readily accepted (Ruoslahti 1996) by ‘αvβ3’[a 2-component, Alpha-v and Beta-3 receptor integrin for vitronectin] integrins (Horton 1997). It is also associated with wound healing activities as it serves as a profibrotic (increased fibrosis) activity marker in the liver, heart and kidney. High expressions of vitronectin in the human body is associated with the likelihood of tumour growth (Schvartz, Seger & Shaltiel 1999).

1.2.2 Transferrin

Also known as serotransferrin; transferrin (TF) is a blood plasma-based glycoprotein of 78000-80000 Dalton molecular weight, comprised of 698AA units. The range of molecular weights is due to the presence of many isoforms of transferrin. It is a molecule of high importance as it forms an essential carrier protein involved in the uptake of iron molecules by red blood cells [RBCs]. Structurally, it is made up of 19AAs signalling peptide (Sequence no: 1-19) and a 679AA serotransferrin chain (Sequence no: 20-698).

Figure 3 of the Transferrin protein molecule produced using PyMOL software.
Source: Research Collaboratory for Structural Bioinformatics-Protein Data Bank-5DYH.
In cell culture systems, transferrin is an essential antioxidant performing the task of uptake of iron from extracellular environments and mediation within cells. The uptake is regulated by the varied expression of transferrin receptors. Under physiological conditions, transferrin binds firmly to iron inhibiting the formation of free radicals to catalyse iron (Testa 2013).
An excess of transferrin in the body can cause extreme disorders such as Haemochromatosis (exuberance of iron) leading to joint pain, arthritis and general malaise. People with this disorder have a bronzing coloration of skin, highly erratic mood swings accompanied by frequent abdominal pain (Blanc & Vannotti 1966). While contrastingly, a transferrin deficiency will result in a condition called Atransferrinemia or Hypotransferrinemia causing microcytic and hypochromic anaemia (abnormally small and decolourised RBC) with insufficient iron in the blood due to Hemosiderosis (accumulation of iron) occurring the heart and liver systems. This leads to individuals being prone to recurrent infections and finally heart failure (Knisely, Beutler & Gelbart 2004).
Luckily, both of these disorders are very rare and autosomal recessive disorders. These disorders are a result of mutations in the exons (coding regions) for the DNA coding for transferrin (Testa 2013).

1.3 Cell Culture & Substrates

The substrate surface properties plays a crucial role in the cellular processes of adhesion, mobility, proliferation and in controlling the phenotype and functioning (Koegler et al. 2012). Investigating cell culture on synthetic substrates presents us with an opportunity to explore the physical and chemical properties of solid matter upon cell function. This allows us to gain an understanding of the interaction between the substrate and cell, and observe any changes in responses with respect to the substrate.

Figure 4: Representation of the substrate forming the hub between physical properties
and cellular functioning. Source: (Ross et al. 2011).
There is a delicate balance between the substrate properties and the characteristic cell responses as illustrated in figure 4. Different cells respond in their own characteristic ways when placed in unfamiliar environments. This stems from the multitude of mechanisms that are instrumental in cellular behaviour (Ross et al. 2011).

1.4 Surface Topography & Modification

Surface topography can be defined as the characteristics of a surface, including its profile and finish. This is limited to a depth of 10nm (0.01 µm or 10-5 mm) from the apex of the interface. It can be broken down into three main aspects.

  1. Lay: The Predominant surface patterning in a direction
  2. Waviness: Widely spaced irregularities
  3. Roughness: Closely distributed irregularities

Surface topography has a substantial effect on the bulk properties of a material such as density, pressure, crystalline uniformity, bulk modulus, etc. Therefore it is essential to maintain the regularity of a surface as it is highly influenced by the adsorption of impurities (Kohli 2014). This can be prevented by surface functionalisation of chemical groups with high specificity.
Based upon application and need, surface topography of all solid matter can be modified either by a top-down or bottom-up approach to incorporate certain characteristics such as surface roughness, surface charge, surface patterning, biocompatibility, hydrophobicity and reactivity (Mittal 2012). All physical solid matter is well known to have two different length scales: – namely, macroscale and microscale, where the nature of the material changes as we decrease its physical size down to the micro and nano-scale. The mechanical (layout) and chemical (functional groups) topographic features of substrate surfaces hold dominion over the degree of physical and chemical interactions of biological material. Measurement of surface topography through various non-contact radiation based methods such as interferometry, confocal microscopy and electron microscopy provide a high-resolution picture with visible characteristics.
Several techniques exist for surface modification employing a top-down approaches such as lithography by which material is gradually etched away using radiation, electrochemical etching by use of a chemical agent to implement desired structure and chemical groups, producing nanofibers by electrospinning or nanoimprinting using templates. All of these techniques require specialised high-tech equipment and are not convenient for high throughput patterning and fabrication. In contrast, the bottom-up methods are comprised of molecular self-assembly, and colloidal particle assembly (Ogaki, Alexander & Kingshott 2010).
Surface functionalisation is a chemical approach to surface modification based on achieving a desired surface property (Williams 2010). Control of spatial growth and interaction of cells on a substrate can be achieved through deposition of nanoscale thin films. The most commonly employed methods for creating surface films of different chemistry are spin-coating and vapour phase deposition as they are relatively easy and highly controllable in terms of layer thickness and functionality properties. This can be either in the form of physical adsorption [weak electrostatic interactions] or chemical immobilisation [strong covalent bonding] depending on requirements.
Cell culture has exhibited modulation and growth patterns in accordance with surface topographic variations with an affinity towards hydrophilic areas in contrast to hydrophobic regions (Ogaki, Alexander & Kingshott 2010). This confirms that geometrical shapes and patterns on substrates have an obvious influence on cell/tissue orientation and stress fibre development [contact guidance] with heightened proliferation rates seen on continuous surfaces with recession seen on discontinuous surfaces or microporous materials with greater than 5µm feature sizes. Nano-topographically rough surfaces show signs for increased structural and functional connection between living tissues and biomedical implants with reduced fibrous encapsulation (Ito 1999).
From the multitude of substrates available today for cell culture, synthetic substrates fabricated from polymers offer a reliable platform for cell culture and studying of other biological in vitro applications (Lopez-Pena et al. 2015). Through the process of addition or condensation polymerisation reactions, substrates with defined characteristics [surface charge and chemical composition] within a highly controlled environment can be crafted. Hydrogels are one such product which is capable of forming 3-dimensional networks. Hydrogels possess high water accommodating capacities. Coupling this with fine tuneable properties becomes ideal in studying cellular behaviour (Ahmed 2015). Quite congruous with the fine tuneable characteristics of hydrogels a relatively new polymer based, colloidal crystal substrate has come to into play with promising in vitro results aimed at making an imprint in biomedical applications (Wang et al. 2016).

1.4.1 Colloidal Crystal Substrates

With respect to the above-mentioned substrate characteristics, binary colloidal crystals (BCCs) show promising in vitro examples to study cell growth behaviour. BCCs are produced from an aqueous combination of two colloidal sized particle solutions with submicron diameters. Combinations used so far in this method are silica [SiO2], poly (methyl methacrylate) [PMMA] and polystyrene [PS] particles with a maximum crystal forming diameter of 5µm for the largest particle size engaged. (Wang et al. 2015)

Figure 5 of cross-sectional view of BCC fabrication process by
EICAA method. (Wang et al. 2015)

Figure 6 of the SEM Figures of BCC substrates with
patterning in view. (Wang et al. 2015).
Using the self-assembly approach, BCCs can be constructed using the process of evaporation induced assembly within a confined area (EICAA). Glass substrates treated with air plasma for increasing the hydrophilicity of the surface are employed in forming a base upon which BCCs can be fabricated. This EICAA approach allows for high degree of uniform patterning over a large area, and it is quite easy to replicate following a simple process that is a cost effective method. The surface chemistry can be controlled with particle surface functionalisation. These BCCs provide a platform for detailed studies of cell culture and protein patterning responses in a controlled environment. (Singh et al. 2011)

1.5 Analysing Protein Adsorption

Protein adsorption on substrates can be studied through a myriad of advanced techniques with a large multitude of sophisticated scientific equipment (Lynch & Dawson 2008). Prevailing techniques are interferometry, surface plasmon resonance, Fluorescence spectroscopy, electron microscopy [AFM, TEM, STM, SEM…], X-ray spectroscopy [EDX, XRD, SAXS, WAXS, XFM, XPS]. With limited availability at hand of instruments and to minimise experimental costing, a combination of scanning electron microscopy (SEM), surface plasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS) and the not so conventional matrix assisted laser desorption ionisation (MALDI) will be engaged to study vitronectin and transferrin adsorption characteristics on substrates.
With a scope of scrutinizing the role of a whole assembly of proteins employed in stem cell reprogramming, vitronectin and transferrin have been chosen as an outset to the analysis of all proteins involved [namely, vitronectin, transferrin, insulin, FGF-2 and the mixture of DMEM/F-12] in the protocols mentioned below:
“TCPS + Vn + Fibroblast(transfected) + E7 media  iPSC cells”
(Guokai et al. 2011)
“BCC + E7 media + Fibroblast(transfected) iPSC cells”
(Wang et al. 2016)
E7 composition (Guokai et al. 2011):-

  1. DMEM/F-12
  2. Transferrin
  3. FGF-2
  4. Insulin
  5. L-ascorbic acid (Vitamin C)
  6. Selenium
  7. Sodium bicarbonate (NaHCO3)

1.5.1 Scanning Electron Microscopy (SEM)

Invented in the late 1930’s, later developed and made commercial by the late 1960’s, the SEM has been a driving force in the world of microscopy by allowing the observation and understanding of micro & nano-structures and microorganisms (Leng 2013).

Figure 7: Representation of electron beam from SEM interacting
with a specimen. (Zhu et al 2014).
A single beam of high-energy electrons is fired at a target to be examined. The penetration of the electron beam up to a few microns into the sample gives various radiation signals such as secondary electrons (SE), backscattered electrons (BSE), characteristic X-rays and cathodoluminescent light. All these signals can be used to compose a picture of the sample under investigation with respective detectors. The BSE’s are used to examine the surface texture of the sample, a simple observation (Zhu et al. 2014). The characteristic X-rays are employed to investigate the chemical and elemental composition and distribution in the sample. With the SEM, a resolution down to the nanometre scale can be achieved without disturbing the surface quality of samples.
The surface topography of a substrate looks much like a landscape under the magnification of an electron microscope. At this scale, we can accurately determine the contouring of the entire surface along with elemental distribution (heavy metals) by utilising the X-ray Energy-dispersive spectroscopy (EDS) if required coupled with dedicated software programs.
Leslie Gunther-Cummins, AIF. Unidentified bacteria collected from a gym floor. (Image taken using the Zeiss Supra 40 FESEM)
Figure 8A of an unidentified bacteria collected from a gym floor.
Frank Macaluso, AIF. Sample courtesy of Sandy Suzuka, Mary Frabry Lab. Mouse red blood cells. Colored by Hillary Guzik, AIF. (Image taken using the JEOL JSM-6400 SEM)
Figure 8B of red blood cells from a mouse.
Before exposure to a SEM, a sample has to be made conductive to aid with imaging and decrease electrostatic charge accumulation on certain resistive areas as all samples will be placed in an inert vacuum based environment. Taking into account the instrument capabilities, we can visualise protein distribution on substrate surfaces. Proteins being powerful in nature are quite delicate biomolecules as they are highly sensitive to their environment, especially temperature, pressure, chemical agents all are accommodated by the protein either by fragmentation or folding process. Considering that there is a need for a coating material for conductivity purposes, there is always a chance of contamination of proteins by changing the chemical integrity or influencing conformational changes (Kurniati, Darmokoesoemo & Puspaningsih 2016). Therefore it can be said that standard SEM examination is not recommended for biological samples but it is an essential tool to visualise structures, determine the distribution and for measurement purposes.
Detection capacity of the SEM is subjected to certain limitations. The resolution of sample images is controlled by the probe (electron beam) size. Magnification is governed by the distance between sample and detector referred to as the working distance which is limited to 10 millimetres or less without contact with the sample. Utilising the secondary electron emission mode, a nice topographic contrast is revealed with resolutions down to 1 nanometre. With the backscattered electron emission mode, an overall elemental composition and distributional images can be obtained. The resolution limit of this is up to 5 nanometres. The highlight of using the SEM will be to look at patterning regularity of BCC substrates, measuring trough and ridge distances between colloidal particles. (Leng 2013)

1.5.2 X-ray Photoelectron Spectroscopy (XPS)

XPS or Electron Spectroscopy for Chemical Analysis (ESCA), is a quantitative spectroscopic technique employed for measuring elemental compositions in the range of parts per thousand. Based on the principle of Planck’s Equation, characteristic photonic energy emitted by an electron jumping from an outer shell to core-shell within an element when irradiated by a high energy source is accounted for to obtain signature elemental spectra. These elemental signatures give valuable data such as the electronic state, the oxidation state and empirical formula for compounds present in the top 10nm surface layer of materials. XPS uses soft X-rays (1.5kV), monochromated (AlKα or MgKα) source, to eject electrons from the surface of a sample. XPS has a large energy detection range of 0-1100 eV making it possible for detection of all the elements present in the periodic table except for hydrogen and helium, both having atomic numbers less than ‘3’ (Leng 2013).
Figure 9 of possible transitions resulting in the emission of X-rays.
Source: ‘X-Rays: Atomic Origins and’.

Figure 10 of a wide angle XPS scan of the experimental substrate. Source: Parameswara, C 2016, Investigation of serum protein adsorption on self-assembled binary colloidal crystal surfaces using MALDI-ToF Mass Spectrometry, CHE30008-Research Project, pp.1-20.
Detection priority through XPS analysis is towards determining the accumulation, binding states and quantification of vitronectin and transferrin proteins respectively from 10µL along with strengths of binding to the fabricated substrates (Perez‐Roldan et al. 2013).

1.5.3 Matrix Assisted Laser Desorption/Ionisation (MALDI)

Since its invention in 1985, starting with atom bombardment and evolving through laser desorption in the absence of matrices, with quasi-molecular ionisation, detection of (M + H)+ signals and determining the identity of essential biomolecules (Karas, Bachmann & Hillenkamp 1985). The concept of employing another molecule that absorbs radiation to ionise another has transformed this into a high-throughput, ultrahigh resolution mass spectrometry technique known as matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). MALDI is a soft ionisation technique employing laser energy to a matrix solution creating ions from extensive biomolecules embedded within the matrix with a relatively minimal extent of fragmentation for mass spectrometric analysis. MALDI analysis is a three stage process:

  1. The sample to be analysed is mixed with a suitable matrix material and applied to a conducting MALDI or glass plate.
  2. The laser irradiates the sample initiating ionisation and desorption of the sample ions that is extracted and mass analysed.
  3. Protonated or deprotonated molecular ions pass through the detector for mass spectrometric analysis and interpretation.

A high power UV laser or nitrogen (N2) laser is customarily employed for rapid soft ionisation. A time-of-flight (ToF) detector offers revelation over a large mass range. ToF detectors allow acceleration of ions in an applied electric field of known strength. This ion with similar charges to be propelled with equivalent kinetic energies with the mass-to-charge ratio dictating the speed ions (heavier particles travel slower). The time taken by each ion from source to detector is accounted to ascertain particle identity (Fuchs, Süß & Schiller 2010).
With technology upgrading the way instruments and devices acquire, analytically resolve and interpret data, we can now study both positive and negative ions. Ions commonly analysed are (M+H+) as positive ionisation is preferred over negative ionisation. Negative ionisation can lead to significant and unwanted sulphate loss resulting in erroneous interpretations. This is concluded as the structural integrity of proteins and peptides is favoured for rapid and distinct identification of bio-molecules (Nimptsch et al. 2012).
The exact solution to protein resolution can sometimes give erroneous or outlying results. This can be attributed to sampling preparation, matrix selection and contamination. To eradicate such hurdles, the MALDI setup must committedly be calibrated and optimised for accurate results (Liu & Schey 2005). Careful consideration must be taken to prevent contamination and matrix selections when analysing complex mixtures.
Taking advantage of this protein identification program, MALDI has found wide applications in bacterial identification and antibody resistance testing. Bacterial identities being a reflection of characteristic ribosomal protein patterns present in an organism hinged on growth attributes and eccentricity. Finally, the microorganism is verified by comparing MALDI results against a spectral database using variable algorithms. (Wieser & Schubert 2016)
MALDI matrices possess a strong photo-ionic property and low sublimation point with effortless crystal formation when associated with analytes. Matrices achieve two essential objectives which include: –

  1. Absorbing photonic energy from the radiation source with the relocation of excitation energy to the analyte.
  2. Function as a solvent to reduce intermolecular forces and agglomeration of the analyte.(Li & Gross 2004)

Figure 11 of MALDI ToF instrumental. Source:
Matrix solutions are commonly strong ionic solvents, usually low in molecular weight and are acidic in nature for the production of (M+H+) ions. Care must be taken in the selection process as matrices determine crystal growth consequences which will be reflected in the mass spectrum generated, especially for peptides and proteins (Cohen & Chait 1996).
One of the matrices of choice is α-cyano-4-hydroxycinnamic acid (HCCA), and its chemical formula is C10H7NO3. With an IUPAC name of ‘(E)-2-cyano-3-(4-hydroxyphenyl) prop-2-enoate, HCCA has a molar mass of 189.17 g/mol and is available in the form of a yellow powder. HCCA is employed as it allows for clear resolution of low molecular weight proteins with less than 10,000Da (Yang et al. 2013).

12A: α-cyano-4-hydroxycinnamic acid (HCCA)   12B: Sinapinic acid (SA)
Figures 12A & 12B: Lewis structures of HCCA and SA
matrices used in MADLI-ToF analysis.
Secondly, Sinapinic acid or Sinapic acid (SA) has the chemical formula C11H12O5, and it has an IUPAC name of 3-(4-hydroxy-3, 5-dimethoxyphenyl) prop-2-enoic acid. The molar mass of SA is 224.21 g/mol and is available in the form of a white powder. SA is best suited for MALDI applications as it caters to detection and assertion of a large range of molecular weight proteins greater than 10,000Da (Yang et al. 2013).
The matrix solution is prepared with a standard recipe (Liu & Schey 2008). To this, about 10 to 20 mg of the matrix is added to form a saturated solution necessary for crystal generation (Boyd et al. 2011). SA individually with VN and TF followed by a combination of VN with TF with both matrices will be examined under MADLI ToF mass spectrometry.

1.5.4 Surface Plasmon Resonance (SPR)

SPR is a non-invasive optical technique for exploring material adsorption and binding interactions on the surface of planar surfaces. First observed by R. W. Wood in 1902 when the uneven distribution of spectral lines was created from a continuous light passing through an optical metallic diffraction grating pattern. It was developed and employed in 1980 onwards for the investigation of thin films including chemical and biological interactions with fixed receptors and analytes in solutions. (Maystre 2012)

Figure 13: Representation of SPR operation. Source: Choosing a suitable method for the identification of replication origins in microbial genomes- DOI: 10.3389/fmicb.2015.01049.
Resonant oscillation of conductive electrons at the interface between a material with positive and negative permittivity’s, when stimulated by incident light is called surface plasmon resonance (SPR). In possession of an ambulatory light source and detector, SPR instruments have the capability of measuring evanescent surface waves or plasmons generated by incident radiation along with any changes in the refractive index of sample material providing examination of crucial information such as the kinetics of binding, layer thickness and quantity of binding within a 1cm2 area of material. (Homola, Yee & Gauglitz 1999)
SPR in combination with MALDI-ToF and XPS become a strong team of instruments in the investigation of the characteristic nature of vitronectin(Hallström et al. 2009) and transferrin(Aubailly et al. 2011) adsorbing onto synthetic substrates which will be investigated further.

1.6 Conclusion

Investigating the affinity, preference of adsorption and interactions of vitronectin and transferrin on four combinations of BCC substrates will be the priority. This will be based on MALDI-ToF MS in conjunction with SPR, XPS and SEM to investigate the relative intercommunications of the individual (VN and TF) and a combination of the proteins. The significance of this project will reveal the complex nature of how proteins interact with the surface of substrates and with each other, giving rise to an exceptional understanding of protein functioning in unknown environments. This will be a decisive inauguration in recognising and understanding cellular behaviour on the surface of substrates. It will extend to a deepened learning of protein personalities during the process of induced stem cell reprogramming processes.
One hypothesis that can be made based on the research question in contemplation is that these proteins undergo conformational changes to suit environments but perform their programmed functions. Understanding the correlation/ affinity of protein attachments on nano-topographic surfaces will pave new roads into long lasting bio-materials and provide new perspective towards tissue engineering and cell reprogramming.

1.7 References

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