Template:Team:TU Eindhoven/Experimental Approach HTML
Experimental approach
To test the viability of the designed system, we have designed a number of experiments. These experiments are conducted to verify whether the individual elements of our device work. An overview of the experiments is given below.
Verifying the click reaction
A vital aspect of our device is clicking the aptamers to the membrane proteins. For this click, we make use of the exact same click chemistry used by iGEM TU Eindhoven 2014. iGEM TU Eindhoven 2014 has used the click reaction C-terminally. To analyze whether the localization of the azide-functionalized amino acid within the loops of OmpX impedes the click reaction, we will click a DBCO-functionalized fluorophore to the OmpX. With today’s technology and knowledge, ground-breaking drug discoveries are at the forefront of the medical sciences and society. For many diseases, however, the foundation of curing lies not exclusively in the availability of these sophisticated drugs, but rather in an accurate and early diagnosis. For colon cancer, for example, the survival rate of patients diagnosed at the early stage is 90%, whereas the survival rate of patients diagnosed in the critical stage is a mere 13% [1]. Similar figures hold for many more diseases.
Without diving too much in the details, one can see that a simple Google search already yields many reports underlining the importance of an early detection of disease for Alzheimer’s disease and Multiple Sclerosis.
Making an early biomedical diagnosis is thus often of vital importance.
Such diagnoses can be made in multiple ways. Often, they are made using analytical instruments, such as MRI scanners, NMR and mass spectrometry. These instrumentation methods can provide rich information on both the structure as well as the concentration of disease markers [2]. Even though these instruments can come to a sound diagnosis, they have a profound disadvantage: samples often need to be pre-treated and diagnoses cannot be made on-site, leading to prolonged processing times.
Due to this disadvantage, biosensors have found their way into society. In contrast to analytical instruments, biosensors can quickly diagnose a disease, can be used on-site and are often easy to use. iGEM TU Eindhoven has devised to develop a universal platform for biosensors. The designed platform is constructed from three major elements, being the recognition element which is used to detect disease markers, the signaling components which translate the detection into a measurable signal and a scaffold which joins the signaling components and recognition elements. An overview of the elements is presented below.
[1] American Cancer Society. Colorectal Cancer Facts & Figures 2011-2013. Atlanta: American Cancer Society, 2011.
[2] W. Zhou, P.-J. J. Huang, J. Ding, and J. Liu, “Aptamer-based biosensors for biomedical diagnostics.,” Analyst, vol. 139, no. 11, pp. 2627–40, 2014.
[3] C. Tuerk and L. Gold, “Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.,” Science, vol. 249, no. 4968, pp. 505–10, Aug. 1990.
[4] D. Musumeci and D. Montesarchio, “Polyvalent nucleic acid aptamers and modulation of their activity: A focus on the thrombin binding aptamer,” Pharmacol. Ther., vol. 136, no. 2, pp. 202–215, 2012.
[5] A. Cibiel, C. Pestourie, and F. Ducongé, “In vivo uses of aptamers selected against cell surface biomarkers for therapy and molecular imaging,” Biochimie, vol. 94, no. 7, pp. 1595–1606, 2012.
[6] L. H. Lauridsen and R. N. Veedu, “Nucleic acid aptamers against biotoxins: a new paradigm toward the treatment and diagnostic approach.,” Nucleic Acid Ther., vol. 22, no. 6, pp. 371–9, 2012.
[7] A. Rhouati, C. Yang, A. Hayat, and J. L. Marty, “Aptamers: A promosing tool for ochratoxin a detection in food analysis,” Toxins (Basel)., vol. 5, no. 11, pp. 1988–2008, 2013.
[8] F. Radom, P. M. Jurek, M. P. Mazurek, J. Otlewski, and F. Jeleń, “Aptamers: Molecules of great potential,” Biotechnol. Adv., vol. 31, no. 8, pp. 1260–1274, 2013.
[9] B. J. Hicke, A. W. Stephens, T. Gould, Y.-F. Chang, C. K. Lynott, J. Heil, S. Borkowski, C.-S. Hilger, G. Cook, S. Warren, and P. G. Schmidt, “Tumor Targeting by an Aptamer,” J. Nucl. Med., vol. 47, no. 4, pp. 668–678, Apr. 2006.
[10] Y. Wu, K. Sefah, H. Liu, R. Wang, and W. Tan, “DNA aptamer-micelle as an efficient detection/delivery vehicle toward cancer cells.,” Proc. Natl. Acad. Sci. U. S. A., vol. 107, no. 1, pp. 5–10, 2010.
[11] A. D. Kent, N. G. Spiropulos, and J. M. Heemstra, “General approach for engineering small-molecule-binding DNA split aptamers,” Anal. Chem., vol. 85, no. 20, pp. 9916–9923, 2013.
[12] J. J. Rice, A. Schohn, P. H. Bessette, K. T. Boulware, and P. S. Daugherty, “Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands.,” Protein Sci., vol. 15, no. 4, pp. 825–36, Apr. 2006.
[13] J. Vogt and G. E. Schulz, “The structure of the outer membrane protein OmpX from Escherichia coli reveals possible mechanisms of virulence.,” Structure, vol. 7, no. 10, pp. 1301–9, Oct. 1999.
[14] W. Alberts, Johnson, Lewis, Raff, Roberts, Molecular Biology of The Cell. Pearson, 2005.
[15] I. Medintz and N. Hildebrandt, Eds., FRET - Förster Resonance Energy Transfer. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2013.
[2] W. Zhou, P.-J. J. Huang, J. Ding, and J. Liu, “Aptamer-based biosensors for biomedical diagnostics.,” Analyst, vol. 139, no. 11, pp. 2627–40, 2014.
[3] C. Tuerk and L. Gold, “Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.,” Science, vol. 249, no. 4968, pp. 505–10, Aug. 1990.
[4] D. Musumeci and D. Montesarchio, “Polyvalent nucleic acid aptamers and modulation of their activity: A focus on the thrombin binding aptamer,” Pharmacol. Ther., vol. 136, no. 2, pp. 202–215, 2012.
[5] A. Cibiel, C. Pestourie, and F. Ducongé, “In vivo uses of aptamers selected against cell surface biomarkers for therapy and molecular imaging,” Biochimie, vol. 94, no. 7, pp. 1595–1606, 2012.
[6] L. H. Lauridsen and R. N. Veedu, “Nucleic acid aptamers against biotoxins: a new paradigm toward the treatment and diagnostic approach.,” Nucleic Acid Ther., vol. 22, no. 6, pp. 371–9, 2012.
[7] A. Rhouati, C. Yang, A. Hayat, and J. L. Marty, “Aptamers: A promosing tool for ochratoxin a detection in food analysis,” Toxins (Basel)., vol. 5, no. 11, pp. 1988–2008, 2013.
[8] F. Radom, P. M. Jurek, M. P. Mazurek, J. Otlewski, and F. Jeleń, “Aptamers: Molecules of great potential,” Biotechnol. Adv., vol. 31, no. 8, pp. 1260–1274, 2013.
[9] B. J. Hicke, A. W. Stephens, T. Gould, Y.-F. Chang, C. K. Lynott, J. Heil, S. Borkowski, C.-S. Hilger, G. Cook, S. Warren, and P. G. Schmidt, “Tumor Targeting by an Aptamer,” J. Nucl. Med., vol. 47, no. 4, pp. 668–678, Apr. 2006.
[10] Y. Wu, K. Sefah, H. Liu, R. Wang, and W. Tan, “DNA aptamer-micelle as an efficient detection/delivery vehicle toward cancer cells.,” Proc. Natl. Acad. Sci. U. S. A., vol. 107, no. 1, pp. 5–10, 2010.
[11] A. D. Kent, N. G. Spiropulos, and J. M. Heemstra, “General approach for engineering small-molecule-binding DNA split aptamers,” Anal. Chem., vol. 85, no. 20, pp. 9916–9923, 2013.
[12] J. J. Rice, A. Schohn, P. H. Bessette, K. T. Boulware, and P. S. Daugherty, “Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands.,” Protein Sci., vol. 15, no. 4, pp. 825–36, Apr. 2006.
[13] J. Vogt and G. E. Schulz, “The structure of the outer membrane protein OmpX from Escherichia coli reveals possible mechanisms of virulence.,” Structure, vol. 7, no. 10, pp. 1301–9, Oct. 1999.
[14] W. Alberts, Johnson, Lewis, Raff, Roberts, Molecular Biology of The Cell. Pearson, 2005.
[15] I. Medintz and N. Hildebrandt, Eds., FRET - Förster Resonance Energy Transfer. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2013.