Skip to main content
Article thumbnail
Location of Repository

Optical modulation of high-affinity biomolecules function via photochromic dyes : a step towards an artificial control of biological activity

By Bernard Derek Annan


Prior to this study, there has yet to be a clear demonstration of an artificial control of antibody affinity via photochromic dyes. The research described in this thesis sets out to address this by investigating photochromic dyes and their subsequent applications with high affinity biomolecules - primarily to photomodulate the functions of biomolecules. The main avenue of investigation explored the conjugation of photochromic dyes (spiropyrans) to proteins (an enzyme and five different antibodies), to achieve reversible photomodulation of protein function for possible applications in biosensor technology (such as the development of reagentless bio¬reversible sensing systems). A secondary aim involved the investigation of the feasibility of antibody-antigen binding in the presence of ionic liquids. Ionic liquids have recently experienced growing interest as replacements for traditional organic solvents in a number of industrial applications. The practicability of spiropyrans in ionic liquids was also investigated (with the future possibility of photomodulated antibody-antigen interactions in ionic liquids to deliver a variety of improved analytical performances). The synthesis and photoswitching properties of an appropriate range of spiropyran dyes are reported. The spiropyran dyes are synthesised to possess a carboxyl group to aid carbodiimide mediated conjugation to lysine amino groups of proteins. The photochromic behaviour of the spiropyran dyes in various solvents, temperature and pH ranges were observed. Conjugation of carboxylated spiropyran dyes to an enzyme: horseradish peroxidise, was initially observed to aid development of experimental protocol for the target study group i.e. antibodies. Photomodulation of the modified horseradish peroxidase was found to demonstrate ~ 60 % decline in enzyme activity, an effect which was reversible as a result of the photoswitching capabilities of the attached spiropyran dyes. The five different antibodies; anti Atrazine, anti GroEL, anti Phytanic Acid, anti FITC and anti Staphylococcus aureus were modified with spiropyran dyes as with horseradish peroxidase. Reversible antibody affinity photomodulation was observed via their reaction in an ELISA which yielded a decline of ~ 15 %, ~ 40 %, ~ 50 %, ~ 55 % and ~ 65 % in binding signal respectively. A fatigue assessment was conducted on the photoswitching capabilities of both the conjugated and the unconjugated spiropyran dyes. This was expressed as ten photoswitching cycle experiments, the first evenly spaced over ten days and a second over ten weeks. The initial results suggested dye degradation increased with consecutive photoswitching cycles of the conjugated spiropyran dyes. It was observed that the level of degradation for the unconjugated spiropyran dyes was independent to the timing interval between photoswitching cycles, suggesting storage stability of the compound. However the level of degradation for the conjugated spiropyran dyes was dependent to the timing interval between photoswitching cycles, suggesting storage instability. A subsequent study involved the demonstration of the feasibility of antibody-antigen binding in ionic liquids for the first time. Various combination ratios of ionic liquids with aqueous phosphate buffered saline were employed. Initial experimentation of antibody-antigen binding showed that use of solutions with an ionic liquid content of 50 % and below, produced identical results to that of the standard aqueous phosphate buffered saline. At 95 % ionic liquid content, a lower level of binding activity was observed. The possibility of a photomodulated antibody-antigen interactions in ionic liquids did not produce a significant result on this occasion with the observation of spiropyran dyes failure to photoswitch in solutions with as low as 10 % ionic liquid content. In summary, although the development of a reagentless bio-reversible sensing system continues beyond the period of this PhD thesis, significant progress has been made with regards to photochromic antibodies as possible candidates for further studies and applications, also the establishment of antibody-antigen binding in various ionic liquids can serve as ways to further enhance the applicability of such reactions under different environmental conditions

Publisher: Cranfield University
Year: 2008
OAI identifier:
Provided by: Cranfield CERES

Suggested articles


  1. (2003). 2,4,6-Trinitrotoluene detection using recombinant antibodies. doi
  2. (1994). A novel method r measurement of the merocyanine-spiropyran interconversion in non-activated ). Photocontrol of ion permeation through ilayer membranes using an amphiphilic spiropyran. doi
  3. (1973). A theoretical eht study of the coloured isomeric forms of a spiropyran. doi
  4. (1999). A tolan substituted optically active spiropyran. doi
  5. (2000). Ab initio study on the photoisomers of a nitro-substituted spiropyran. doi
  6. (2005). Azobenzene as photoresponsive c
  7. bind tubulin of trypanosomes and inhibit their growth in vitro. doi
  8. (1998). Binding characteristics of anti-atrazine monoclonal antibodies and their fragments synthesised doi
  9. (2003). Biosensors and multiple mycotoxin analysis. doi
  10. (1996). Chapter 3: Bioaffinity Agents for Sensing
  11. (2000). Chapter 6: Bioaffinity, Bioseparation Process Science,
  12. (1986). Chemical Reactivity at an ntibody Binding Site Elicited by Mechanistic Design of a Synthetic Antigen. doi
  13. (1993). Comparative photodegradation study between spiro[indoline-oxazine] and spiro[indoline-pyran] derivatives in solution. doi
  14. (1998). Complexes of spiropyran-derived merocyanines with metal ions - Thermally activated and light-induced processes. doi
  15. (1998). Complexes of spiropyran-derived merocyanines with metal ions: relaxation kinetics, photochemistry and solvent effects. doi
  16. (1994). Conformational Dynamics Associated doi
  17. (2003). Conformations of nitro-substituted spiropyran and merocyanine studied by lowtemperature matrix-isolation infrared spectroscopy and density-functional-theory alculation. doi
  18. (2005). Copper ion-selective fluorescent sensor based on the inner filter effect using a doi
  19. (1995). Correlations between solvatochromism, Lewis acid-base equilibrium and photochromism of an indoline spiropyran. doi
  20. (2001). Current and emerging commercial optical doi
  21. (1999). Cyanogen Bromide and Tryptic Fragments, the omplete Sequence, and Some Structural Characteristics of Horseradish Peroxidase Welton, doi
  22. (2004). eep Eutectic Solvents Formed between Choline Chloride and Carboxylic Acids: ersatile Alternatives to Ionic Liquids. doi
  23. (1996). Enzyme Immunoassays, From Concept to Product Development, Chapman and Hall, doi
  24. (1999). Enzyme modification by polymers with solubilities that change in resp m
  25. (1984). Ferrocene-mediated nzyme electrode for amperometric determination of glucose. Analytical Chemistry, doi
  26. fo 1,3,3-trimethyl spiro-(2h-L-benzopyran-2,2'-indoline) derivatives. doi
  27. h S spiropyran derivative.
  28. (2003). How hazardous are ionic liquids? Structure–activity relationships and biological testing as important elements for sustainability evaluation doi
  29. (1999). Identification and optimastion of regeneration conditions for affinity-based biosensor assays. A multivariate cocktail approach. doi
  30. (2003). Impact of ionic liquid physical properties in lapse activity and stabil
  31. (1995). Investigation of the chelation of a photochromic spiropyran with Cu (II). doi
  32. (1994). Irreversible photochromism of spiropyran films at low temperatures. doi
  33. (2001). Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylal re doi
  34. (2003). Light-induced modulation of selfassembly on spiropyran-capped gold nanoparticles: a potential system for the controlled release of amino acid derivatives. doi
  35. (1999). Molecular Engineering of the Biosensor Interface. Cranfield University. W horseradish peroxidase activity via covalent attachment of carboxylated-spiropyran dyes. doi
  36. (2003). Molecular switching in nano-structured photochromic films of biopolymers. doi
  37. (1999). o the enzyme backbone.
  38. (2000). optical data storage using photochromic materials. doi
  39. (1966). Peroxidase isozymes from orseradish roots. I. Isolation and physical properties.
  40. (1977). Photo control of enzyme activity of α-mylase.
  41. (2004). Photo-, thermo-, solvato-, and electrochromic spiroheterocyclic compounds. doi
  42. (2006). Photo-regenerable surface with otential for optical sensing. doi
  43. photochemical reaction step of unsubstituted indolino-spiropyrans. doi
  44. (2003). Photochromic behavior of spiropyran and fulgide in thin films of blends of PMMA and SBS.
  45. (2004). Photochromic behavior of spiropyran in the photoreactive polymer containing chalcone moieties. doi
  46. (1965). Photochromic chelating agents.
  47. (1995). Photochromic poly (α-amino acid)s: hotomodulation of molecular and supramolecular structure. doi
  48. (1959). Photochromic spiropyrans. doi
  49. (2003). Photochromism of spiropyran in molecular sieve voids: effects of host–guest interaction on isomer status, switching stability and reversibility. doi
  50. (1989). Photomodulation of polypeptide conformation by sunlight in spiropyran-containing poly (L-glutamic acid). doi
  51. (2007). Photonic modulation of surface properties: a novel concept in chemical ensing. doi
  52. (1993). Photoregulation of enzyme activity. Photochromic, transition-state-analogue inhibitors of cysteine and serine roteases. doi
  53. (1991). Photoregulation of papain activity through anchoring photochromic azo groups t A doi
  54. (1991). Photoregulation of α-chymotrypsin by its mobilization in a photochromic azobenzene copolymer. doi
  55. (2001). Photoresponsive polypeptides. doi
  56. (1992). Photoswitchable binding of substrates to proteins: photoregulated binding of α-Dmannopyranose to concanavalin A modified by a thiophenefulgide dye. doi
  57. (1997). Photoswitchable biomaterials: en route to optobioelectronic systems. doi
  58. (1998). Photoswitchable peptide antigens ON solid surfaces. doi
  59. (1998). Photoswitching of doi
  60. (2002). Photoswitching of peroxidase activity by position-specific incorporation of a photoisomerizable non-natural amino doi
  61. (1997). Poly(L-lysine) containing azobenzene li G Science Incorporated, ISBN 0-86542-568-X. Photon-controlled phase partitioning of spiropyrans.
  62. (2002). Potomodulation of conformational states of enylazobenzyloxycarbonyl-L-proline and related peptides. doi
  63. (2001). Preparation of novel, moisture-stable, Lewis-acidic ionic uids containing quaternary ammonium salts with functional side chains. doi
  64. (1996). Preparation of photochromic piropyrans linked to methyl cellulose and photoregulation of their properties. doi
  65. (1994). Proton exchange and isomerisation reactions of photochromic and reverse doi
  66. (2007). Redox potential of azobenzene as an amino acid residue in peptides. doi
  67. (1994). Regeneration of immobalised antibodies on fibre optic probes.
  68. (1996). scFv-kappa: A new single-chain antibody format showing increased expression levels in both procaryotic and eucaryotic systems. doi
  69. (2006). Semibiological molecular machine with an implemented AND logic gate for regulation of protein folding. doi
  70. (2001). Single domain camel antibodies: current status. doi
  71. (1998). Single-chain antibody fragments scAbs., which have a human C-kappa constant domain and a hexa-histidine ification, have been raised against the herbicides paraquat and atrazine nd expressed in transgenic.
  72. (2000). Spiropyrans and spirooxazines for memories and switches. doi
  73. (2001). Stability Engineering of Antibody Single-chain Fv Fragments. doi
  74. (1991). Steady-state photomodulation doi
  75. (2004). Synthesis of functionalized spiropyran and spirooxazine derivatives and their photochromic properties. doi
  76. (2006). The incorporation of a photoisomerizable amino acid into proteins in E. coli. doi
  77. (1990). The primary vans doi
  78. (2005). The specific molecular identification of life experiment (SMILE). doi
  79. (2007). Thermally stable merocyanine form of photochromic spiropyran with aluminum ion as doi
  80. (1996). Towards the photocontrol of bio-affinity systems.
  81. (2003). V(D)J Recombination and the Evolution of the Adaptive Immune System. doi

To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.