Carbohydrates onto Glass and Silicon Surfaces
2.1. Chapter Summary
2.5.5. Microtitre Plate Functionalisation 1 Solvent Compatibility Testing
Prior to being able to use the microtitre plates for acrylate functionalisation, their solvent compatibility was investigated. 1 mL of each test solution was added to a different well of the polystyrene plate and the observations were recorded every 10 minutes for the first hour, then every 30 minutes for the following 4 hours, then
92 every hour for the next four hours. Wells showing no signs of damage after this nine hour test period were left for a further 18 hours. The plates were washed, as detailed above and allowed to dry. It must be stressed that the solvent additions were carried out drop wise addition, in order to prevent excess exothermic reactions, particularly in the case of the acryloyl chloride: DMSO mixture.
2.5.5.2. Acrylation of Amine Coated Plates
In a typical procedure acrylic Acid (0.09 mL) and N,N'- Diisopropylcarbodiimide (DIPCDI) (0.28 mL) were added into DMSO (4 mL) and left to activate for 10 minutes (RT). 25 µL of the activated acrylating solution was then added to each well in the amine coated microwell plate, except the bottom row, which was left empty as a control. The plates were left for 30 minutes (RT) then the excess solution was removed from each well by pipette, the plate washed with DMSO three times and allowed to dry. Each well of the plate was then functionalised with 25 µL of the correct thiol solution, as detailed in Table 2.7. The In each case an entire row (12 wells) was functionalised with each solution. After 2 hours the excess solution was removed, the plates washed with deionised Milli-Q three times and allowed to dry. Fluorescently labelled Con A (0.1 mg.mL-1 in HEPES, 25 µL per well) was then added to each of the functionalised well, left for 30 minutes (RT, dark), washed three times with Milli-Q water and dried. The samples were then immediately tested in the microplate reader. The results show the average fluorescence from the 12 data points, with the background fluorescence from the blank wells subtracted, and the standard deviation between them.
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Table 2.7: Functionalisation of the amine coated microplates
First Reagent Added Second Reagent Added Third Reagent Added
Acrylic Acid Thioglycerol (neat)
Fluorescent Con A (0.1 mg.mL-1 in HEPES)
Acrylic Acid Dodecanethiol (neat)
Fluorescent Con A (0.1 mg.mL-1 in HEPES) Acrylic Acid Thioglucose (2 mg.mL-1 in PBS) Fluorescent Con A (0.1 mg.mL-1 in HEPES) Acrylic Acid Thioglucose (2 mg.mL-1 in PBS) + TCEP Fluorescent Con A (0.1 mg.mL-1 in HEPES)
Acrylic Acid None
Fluorescent Con A (0.1 mg.mL-1 in HEPES)
2.5.5.3. Microplate Reader Measurements
Fluorescence measurements for the microplates were taken on a BioTek Synergy HT Microplate reader at excitation/emission wavelengths of 485/528 nm with a sensitivity of 75 nm and the readings were all taken at 20°C. The fluorescence measurements from the microplate reader were then converted into a numerical format by the Gen5 software. The background fluorescence (the average fluorescence reading from 12 completely unfunctionalised and empty wells) was recorded and subtracted from each of the data values.
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2.6.
References
1. C. I. Biggs, S. Edmondson and M. I. Gibson, Biomater. Sci., 2015, 3, 175- 181.
2. H. C. Neu, Science, 1992, 257, 1064-1073.
3. C. R. Bertozzi and L. L. Kiessling, Science, 2001, 291, 2357-2364. 4. R. J. Pieters, Med. Res. Rev., 2007, 27, 796-816.
5. R. Apweiler, H. Hermjakob and N. Sharon, BBA-Gen. Subjects, 1999, 1473, 4-8.
6. N. Laurent, J. Voglmeir and S. L. Flitsch, Chem. Commun., 2008, 4400- 4412.
7. G. Nan, H. Yan, G. Yang, Q. Jian, C. Chen and Z. Li, Curr. Pharm. Biotechnol., 2009, 10, 138-146.
8. N. Kim, C. T. Kim, Y. J. Cho and C. J. Kim, Sens. Actuat. B-Chem., 2011,
160, 563-570.
9. J. C. Tiller, C. J. Liao, K. Lewis and A. M. Klibanov, Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 5981-5985.
10. C. E. Hoyle and C. N. Bowman, Angew. Chem. Int. Ed., 2010, 49, 1540- 1573.
11. A. B. Lowe, Polym. Chem., 2010, 1, 17-36.
12. N. S. Bhairamadgi, S. Gangarapu, M. A. Caipa Campos, J. M. J. Paulusse, C. J. M. van Rijn and H. Zuilhof, Langmuir, 2013, 29, 4535-4542.
13. S. P. Pujari, L. Scheres, A. T. M. Marcelis and H. Zuilhof, Angew. Chem. Int. Ed., 2014, 53, 6322-6356.
14. G. Z. Li, R. K. Randev, A. H. Soeriyadi, G. Rees, C. Boyer, Z. Tong, T. P. Davis, C. R. Becer and D. M. Haddleton, Polym. Chem., 2010, 1, 1196-1204. 15. Y. Nam, D. W. Branch and B. C. Wheeler, Biosens. Bioelectron., 2006, 22,
589-597.
16. J. J. Cras, C. A. Rowe-Taitt, D. A. Nivens and F. S. Ligler, Biosens. Bioelectron., 1999, 14, 683-688.
17. D. Janssen, R. De Palma, S. Verlaak, P. Heremans and W. Dehaen, Thin Solid Films, 2006, 515, 1433-1438.
95 18. R. M. Hensarling, V. A. Doughty, J. W. Chan and D. L. Patton, J. Am. Chem.
Soc., 2009, 131, 14673-14675.
19. S. K. So, W. K. Choi, C. H. Cheng, L. M. Leung and C. F. Kwong, Appl. Phys. A: Mater. Sci. Process., 1999, 68, 447-450.
20. J. R. Vig, J. Vac. Sci. Technol., A, 1985, 3, 1027-1034.
21. S. Baunack and A. Zehe, Phys. Status Solidi A, 1989, 115, 223-227.
22. R. J. Klein, D. A. Fischer and J. L. Lenhart, Langmuir, 2008, 24, 8187-8197. 23. A. E. Ozcam, K. Efimenko, C. Jaye, R. J. Spontak, D. A. Fischer and J.
Genzer, J. Electron Spectrosc. Relat. Phenom., 2009, 172, 95-103.
24. H. G. Tompkins and T. Tiwald, WVASE32 Software Training Manual for IR-VASE, 2009, Available at: https://wiki.kip.uni- heidelberg.de/KIPwiki/images/f/ff/IR-VASE_Training_Manual.pdf
25. NIST, X-ray Photoelectron Spectroscopy Database, Version 4.1, 2014, Available at: http://srdata.nist.gov/xps/
26. M. Mammen, S. K. Choi and G. M. Whitesides, Angew. Chem. Int. Ed., 1998, 37, 2755-2794.
27. O. C. Grant, H. M. K. Smith, D. Firsova, E. Fadda and R. J. Woods,
Glycobiology, 2014, 24, 17-25.
28. S. A. Sundberg, Curr. Opin. Biotechnol., 2000, 11, 47-53.
29. R. B. Merrifield, Adv. Enzymol. Relat. Areas Mol. Biol., 1969, 32, 221-296. 30. Y. Arima and H. Iwata, Biomaterials, 2007, 28, 3074-3082.
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