Login | Register

Implementation of a Novel Fucosyltransferase Inhibition Assay on a Digital Microfluidics Device


Implementation of a Novel Fucosyltransferase Inhibition Assay on a Digital Microfluidics Device

Leclerc, Laura (2018) Implementation of a Novel Fucosyltransferase Inhibition Assay on a Digital Microfluidics Device. Masters thesis, Concordia University.

[thumbnail of Leclerc_MASc_F2018.pdf]
Text (application/pdf)
Leclerc_MASc_F2018.pdf - Accepted Version
Available under License Spectrum Terms of Access.


Cell-surface carbohydrates—or glycans—influence growth, differentiation, and immune response mechanisms. Alterations to the glycome can be markers for diseases including diabetes, neurodegenerative disorders, and cancer. Fucosyltransferases catalyze the addition of a fucose sugar residue to specific cell-surface glycans, which are involved in intercellular cell rolling/adhesion interactions such as white blood cells homing to inflammation sites and sperm-egg binding in fertilization. Fucosylated glycans are also implicated in inflammatory disease and cancers. In viral and microbial infections, fucosyltransferases can play a role in the adhesion and colonization of the host organism, as in the case of Helicobacter pylori α(1,3)-fucosyltransferase (FucT).
To better our understanding of glycome alterations and improve medical diagnostics and treatments, screens for glycosyltransferase activity and inhibition are needed. Efficient screens for specific glycosylations tend toward costly materials, instrumentation, and specialized skillsets- here, we present a novel inhibition assay for FucT using the fluorogenically labeled disaccharide, MU-β-LacNAc. The assay shows good potential for high throughput (Z’=0.78 in 384-well plate), though such an application is not shown here. It was also implemented on a digital microfluidic (DMF) platform, where inhibition curves of FucT by GDP, a product of the glycosyltransferase reaction that exhibits an inhibitory feed-back loop, were generated on-device. Results of the assay on DMF (IC50 = 0.093 mM ± 0.037) were shown to be comparable to results in a 384-well plate (IC50 = 0.114 mM ± 0.086), achieving a 87.5% reduction in reaction volume and setting the groundwork for future fully automated screens for potential inhibitors of glycosyltransferases.

Divisions:Concordia University > Faculty of Arts and Science > Biology
Item Type:Thesis (Masters)
Authors:Leclerc, Laura
Institution:Concordia University
Degree Name:M.A. Sc.
Date:1 September 2018
Thesis Supervisor(s):Kwan, David and Leclerc, Laura
Keywords:fucosyltransferase,α(1,3)-fucosyltransferase,FucT,inhibition,microfluidics,dmf,digital microfluidics,MU-β-LacNAc,LewisX,LeX,Lewis-X,glycosylation assay
ID Code:984453
Deposited On:16 Nov 2018 17:05
Last Modified:20 Dec 2018 01:00


1. Varki, A.J.D.E.K.J.C. Essentials of Glycobiology. Ch3: Cellular organization of glycosylation, Edn. 2. (Cold Spring Harbor Laboratory Press, NY; 2009).
2. Weerapana, E. & Imperiali, B. Asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems. Glycobiology 16, 91R-101R (2006).
3. Messner, P. Prokaryotic glycoproteins: unexplored but important. J Bacteriol 186, 2517-2519 (2004).
4. Apweiler, R., Hermjakob, H. & Sharon, N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta 1473, 4-8 (1999).
5. Khoury, G.A., Baliban, R.C. & Floudas, C.A. Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database. Sci Rep 1 (2011).
6. Macedo, C.S., Schwarz, R.T., Todeschini, A.R., Previato, J.O. & Mendonca-Previato, L. Overlooked post-translational modifications of proteins in Plasmodium falciparum: N- and O-glycosylation -- a review. Mem Inst Oswaldo Cruz 105, 949-956 (2010).
7. 'Committee on Assessing the Importance and Impact of Glycomics and Glycosciences, B.o., Chemical Sciences and Technology, B.o.L.S., Division on Earth and Life Studies, National & Council', R. in Transforming Glycoscience: A Roadmap for the Future. (eds. T. National & A. Press) (Washington (DC); 2012).
8. Bourne, Y. & Henrissat, B. Glycoside hydrolases and glycosyltransferases: families and functional modules. Curr Opin Struct Biol 11, 593-600 (2001).
9. Varki, A.L., John B. Essentials of Glycobiology. Ch6: Biological Roles of Glycans, Edn. 2. (Cold Spring Harbor Laboratory Press, NY; 2009).
10. Perez, S., Sarkar, A., Rivet, A., Breton, C. & Imberty, A. Glyco3D: a portal for structural glycosciences. Methods Mol Biol 1273, 241-258 (2015).
11. Marth, J.D. Essentials of Glycobiology. Ch8: O-Glycans, Edn. 2. (Cold Spring Harbor Laboratory Press, NY; 2009).
12. Varki, A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3, 97-130 (1993).
13. Deddish, P.A. et al. Carboxypeptidase M in Madin-Darby canine kidney cells. Evidence that carboxypeptidase M has a phosphatidylinositol glycan anchor. J Biol Chem 265, 15083-15089 (1990).
14. Piesecki, S. & Alhadeff, J.A. The effect of carbohydrate removal on the properties of human liver alpha-L-fucosidase. Biochim Biophys Acta 1119, 194-200 (1992).
15. Endreffy, I. et al. Plasma alpha-L-fucosidase activity in chronic inflammation and autoimmune disorders in a pediatric cohort of hospitalized patients. Immunol Res 65, 1025-1030 (2017).
16. Lau, K.S. et al. Complex N-glycan number and degree of branching cooperate to regulate cell proliferation and differentiation. Cell 129, 123-134 (2007).
17. Scott, J.E. Supramolecular organization of extracellular matrix glycosaminoglycans, in vitro and in the tissues. Fed Am Soc Exp Biol J 6, 2639-2645 (1992).
18. Jiang, D., Liang, J. & Noble, P.W. Regulation of non-infectious lung injury, inflammation, and repair by the extracellular matrix glycosaminoglycan hyaluronan. Anat Rec (Hoboken) 293, 982-985 (2010).
19. Hakomori, S. Structure, organization, and function of glycosphingolipids in membrane. Curr Opin Hematol 10, 16-24 (2003).
20. Whitlow, M., Iida, K., Marshall, P., Silber, R. & Nussenzweig, V. Cells lacking glycan phosphatidylinositol-linked proteins have impaired ability to vesiculate. Blood 81, 510-516 (1993).
21. Helenius, A. & Aebi, M. Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73, 1019-1049 (2004).
22. Kornfeld, R. & Kornfeld, S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem 54, 631-664 (1985).
23. Bause, E. Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes. Biochem J 209, 331-336 (1983).
24. Paulson, J.C. & Colley, K.J. Glycosyltransferases. Structure, localization, and control of cell type-specific glycosylation. J Biol Chem 264, 17615-17618 (1989).
25. Breton, C., Snajdrova, L., Jeanneau, C., Koca, J. & Imberty, A. Structures and mechanisms of glycosyltransferases. Glycobiology 16, 29R-37R (2006).
26. Sharon, N. & Lis, H. Lectins as cell recognition molecules. Science 246, 227-234 (1989).
27. Velupillai, P. & Harn, D.A. Oligosaccharide-specific induction of interleukin 10 production by B220+ cells from schistosome-infected mice: a mechanism for regulation of CD4+ T-cell subsets. Proc Natl Acad Sci U S A 91, 18-22 (1994).
28. Dube, D.H. & Bertozzi, C.R. Glycans in cancer and inflammation--potential for therapeutics and diagnostics. Nat Rev Drug Discov 4, 477-488 (2005).
29. Mahal, L.K., Yarema, K.J. & Bertozzi, C.R. Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 276, 1125-1128 (1997).
30. Tu, Z., Lin, Y.N. & Lin, C.H. Development of fucosyltransferase and fucosidase inhibitors. Chem Soc Rev 42, 4459-4475 (2013).
31. Ledesma, M.D., Bonay, P., Colaco, C. & Avila, J. Analysis of microtubule-associated protein tau glycation in paired helical filaments. J Biol Chem 269, 21614-21619 (1994).
32. Sasaki, N. et al. Advanced glycation end products in Alzheimer's disease and other neurodegenerative diseases. Am J Pathol 153, 1149-1155 (1998).
33. Vitek, M.P. et al. Advanced glycation end products contribute to amyloidosis in Alzheimer disease. Proc Natl Acad Sci U S A 91, 4766-4770 (1994).
34. Wang, J.Z., Grundke-Iqbal, I. & Iqbal, K. Glycosylation of microtubule-associated protein tau: an abnormal posttranslational modification in Alzheimer's disease. Nat Med 2, 871-875 (1996).
35. Furukawa, K. & Fukuda, M. Glycosignals in Cancer: Mechanisms of Malignant Phenotypes. (2016).
36. Chang, F., Li, R. & Ladisch, S. Shedding of gangliosides by human medulloblastoma cells. Exp Cell Res 234, 341-346 (1997).
37. Hakomori, S. Aberrant glycosylation in cancer cell membranes as focused on glycolipids: overview and perspectives. Cancer Res 45, 2405-2414 (1985).
38. Aarnoudse, C.A., Garcia Vallejo, J.J., Saeland, E. & van Kooyk, Y. Recognition of tumor glycans by antigen-presenting cells. Curr Opin Immunol 18, 105-111 (2006).
39. Block, T.M. et al. Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans. Proc Natl Acad Sci U S A 102, 779-784 (2005).
40. Schnaar, R.L. Glycobiology simplified: diverse roles of glycan recognition in inflammation. J Leukoc Biol 99, 825-838 (2016).
41. Kogan, G. et al. Structural and immunochemical characterization of the type VIII group B Streptococcus capsular polysaccharide. J Biol Chem 271, 8786-8790 (1996).
42. Stowell, S.R. et al. Innate immune lectins kill bacteria expressing blood group antigen. Nat Med 16, 295-301 (2010).
43. van Die, I. & Cummings, R.D. Glycan gimmickry by parasitic helminths: a strategy for modulating the host immune response? Glycobiology 20, 2-12 (2010).
44. Summers, R.W., Elliott, D.E., Urban, J.F., Jr., Thompson, R. & Weinstock, J.V. Trichuris suis therapy in Crohn's disease. Gut 54, 87-90 (2005).
45. Zheng, X. et al. Soluble egg antigen from Schistosoma japonicum modulates the progression of chronic progressive experimental autoimmune encephalomyelitis via Th2-shift response. J Neuroimmunol 194, 107-114 (2008).
46. Astronomo, R.D. & Burton, D.R. Carbohydrate vaccines: developing sweet solutions to sticky situations? Nat Rev Drug Discov 9, 308-324 (2010).
47. Yin, X.G. et al. IgG Antibody Response Elicited by a Fully Synthetic Two-Component Carbohydrate-Based Cancer Vaccine Candidate with alpha-Galactosylceramide as Built-in Adjuvant. Org Lett 19, 456-459 (2017).
48. Uro-Coste, E. et al. Cerebral amyloid angiopathy and microhemorrhages after amyloid beta vaccination: case report and brief review. Clin Neuropathol 29, 209-216 (2010).
49. Yamamoto, F. Review: ABO blood group system--ABH oligosaccharide antigens, anti-A and anti-B, A and B glycosyltransferases, and ABO genes. Immunohematology 20, 3-22 (2004).
50. Kwan, D.H., Ernst, S., Kotzler, M.P. & Withers, S.G. Chemoenzymatic Synthesis of a Type 2 Blood Group A Tetrasaccharide and Development of High-throughput Assays Enables a Platform for Screening Blood Group Antigen-cleaving Enzymes. Glycobiology 25, 806-811 (2015).
51. Sturla, L. et al. Differential terminal fucosylation of N-linked glycans versus protein O-fucosylation in leukocyte adhesion deficiency type II (CDG IIc). J Biol Chem 278, 26727-26733 (2003).
52. Marquardt, T. et al. Leukocyte adhesion deficiency II syndrome, a generalized defect in fucose metabolism. J Pediatr 134, 681-688 (1999).
53. Hidalgo, A. et al. Insights into leukocyte adhesion deficiency type 2 from a novel mutation in the GDP-fucose transporter gene. Blood 101, 1705-1712 (2003).
54. Block, T.M. et al. Treatment of chronic hepadnavirus infection in a woodchuck animal model with an inhibitor of protein folding and trafficking. Nat Med 4, 610-614 (1998).
55. Block, T.M. et al. Secretion of human hepatitis B virus is inhibited by the imino sugar N-butyldeoxynojirimycin. Proc Natl Acad Sci U S A 91, 2235-2239 (1994).
56. Cox, T. et al. Novel oral treatment of Gaucher's disease with N-butyldeoxynojirimycin (OGT 918) to decrease substrate biosynthesis. Lancet 355, 1481-1485 (2000).
57. Cameron, H.S., Szczepaniak, D. & Weston, B.W. Expression of human chromosome 19p alpha(1,3)-fucosyltransferase genes in normal tissues. Alternative splicing, polyadenylation, and isoforms. J Biol Chem 270, 20112-20122 (1995).
58. Wang, Y. et al. Loss of alpha1,6-fucosyltransferase suppressed liver regeneration: implication of core fucose in the regulation of growth factor receptor-mediated cellular signaling. Sci Rep 5, 8264 (2015).
59. Moriwaki, K. & Miyoshi, E. Fucosylation and gastrointestinal cancer. World J Hepatol 2, 151-161 (2010).
60. Pang, P.C. et al. Human sperm binding is mediated by the sialyl-Lewis(x) oligosaccharide on the zona pellucida. Science 333, 1761-1764 (2011).
61. Foxall, C. et al. The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis(x) oligosaccharide. J Cell Biol 117, 895-902 (1992).
62. Takada, A. et al. Contribution of carbohydrate antigens sialyl Lewis A and sialyl Lewis X to adhesion of human cancer cells to vascular endothelium. Cancer Res 53, 354-361 (1993).
63. Grabenhorst, E., Nimtz, M., Costa, J. & Conradt, H.S. In vivo specificity of human alpha1,3/4-fucosyltransferases III-VII in the biosynthesis of LewisX and Sialyl LewisX motifs on complex-type N-glycans. Coexpression studies from bhk-21 cells together with human beta-trace protein. J Biol Chem 273, 30985-30994 (1998).
64. Cheng, L. et al. FUT family mediates the multidrug resistance of human hepatocellular carcinoma via the PI3K/Akt signaling pathway. Cell Death Dis 4, e923 (2013).
65. Carrascal, M.A. et al. Inhibition of fucosylation in human invasive ductal carcinoma reduces E-selectin ligand expression, cell proliferation, and ERK1/2 and p38 MAPK activation. Mol Oncol 12, 579-593 (2018).
66. Desiderio, V. et al. Increased fucosylation has a pivotal role in invasive and metastatic properties of head and neck cancer stem cells. Oncotarget 6, 71-84 (2015).
67. Cordel, S., Goupille, C., Hallouin, F., Meflah, K. & Le Pendu, J. Role for alpha1,2-fucosyltransferase and histo-blood group antigen H type 2 in resistance of rat colon carcinoma cells to 5-fluorouracil. Int J Cancer 85, 142-148 (2000).
68. Hallouin, F., Goupille, C., Bureau, V., Meflah, K. & Le Pendu, J. Increased tumorigenicity of rat colon carcinoma cells after alpha1,2-fucosyltransferase FTA anti-sense cDNA transfection. Int J Cancer 80, 606-611 (1999).
69. Aubert, M. et al. Restoration of alpha(1,2) fucosyltransferase activity decreases adhesive and metastatic properties of human pancreatic cancer cells. Cancer Res 60, 1449-1456 (2000).
70. Mathieu, S. et al. Transgene expression of alpha(1,2)-fucosyltransferase-I (FUT1) in tumor cells selectively inhibits sialyl-Lewis x expression and binding to E-selectin without affecting synthesis of sialyl-Lewis a or binding to P-selectin. Am J Pathol 164, 371-383 (2004).
71. Hao, Y.Y. et al. alpha1,2-fucosyltransferase gene transfection influences on biological behavior of ovarian carcinoma-derived RMG-I cells. J. Mol. Cell Biol. (Shanghai, China) 41, 435-442 (2008).
72. Che, Y. et al. Critical involvement of the alpha(1,2)-fucosyltransferase in multidrug resistance of human chronic myeloid leukemia. Oncol Rep 35, 3025-3033 (2016).
73. Sakuma, K., Aoki, M. & Kannagi, R. Transcription factors c-Myc and CDX2 mediate E-selectin ligand expression in colon cancer cells undergoing EGF/bFGF-induced epithelial-mesenchymal transition. Proc Natl Acad Sci U S A 109, 7776-7781 (2012).
74. Hiller, K.M. et al. Transfection of alpha(1,3)fucosyltransferase antisense sequences impairs the proliferative and tumorigenic ability of human colon carcinoma cells. Mol Carcinog 27, 280-288 (2000).
75. Yang, X.S. et al. Overexpression of fucosyltransferase IV promotes A431 cell proliferation through activating MAPK and PI3K/Akt signaling pathways. J Cell Physiol 225, 612-619 (2010).
76. Li, J. et al. Human fucosyltransferase 6 enables prostate cancer metastasis to bone. Br J Cancer 109, 3014-3022 (2013).
77. Liu, F., Qi, H.L. & Chen, H.L. Regulation of differentiation- and proliferation-inducers on Lewis antigens, alpha-fucosyltransferase and metastatic potential in hepatocarcinoma cells. Br J Cancer 84, 1556-1563 (2001).
78. Zhang, J., Ju, N., Yang, X., Chen, L. & Yu, C. The alpha1,3-fucosyltransferase FUT7 regulates IL-1beta-induced monocyte-endothelial adhesion via fucosylation of endomucin. Life Sci 192, 231-237 (2018).
79. Laubli, H., Stevenson, J.L., Varki, A., Varki, N.M. & Borsig, L. L-selectin facilitation of metastasis involves temporal induction of Fut7-dependent ligands at sites of tumor cell arrest. Cancer Res 66, 1536-1542 (2006).
80. Kang, X. et al. Glycan-related gene expression signatures in human metastatic hepatocellular carcinoma cells. Exp Ther Med 3, 415-422 (2012).
81. Ji, J. et al. Expression of alpha 1,6-fucosyltransferase 8 in hepatitis B virus-related hepatocellular carcinoma influences tumour progression. Dig Liver Dis 45, 414-421 (2013).
82. Norden, R., Samuelsson, E. & Nystrom, K. NFkappaB-mediated activation of the cellular FUT3, 5 and 6 gene cluster by herpes simplex virus type 1. Glycobiology 27, 999-1005 (2017).
83. McGovern, D.P. et al. Fucosyltransferase 2 (FUT2) non-secretor status is associated with Crohn's disease. Hum Mol Genet 19, 3468-3476 (2010).
84. Ronchetti, F. et al. ABO/Secretor genetic complex and susceptibility to asthma in childhood. Eur Respir J 17, 1236-1238 (2001).
85. Kannagi, R. Transcriptional regulation of expression of carbohydrate ligands for cell adhesion molecules in the selectin family. Adv Exp Med Biol 491, 267-278 (2001).
86. Nystrom, K. et al. Induction of sialyl-Lex expression by herpes simplex virus type 1 is dependent on viral immediate early RNA-activated transcription of host fucosyltransferase genes. Glycobiology 19, 847-859 (2009).
87. Norden, R., Nystrom, K. & Olofsson, S. Inhibition of protein deacetylation augments herpes simplex virus type 1-activated transcription of host fucosyltransferase genes associated with virus-induced sLex expression. Arch Virol 155, 305-313 (2010).
88. Nystrom, K. et al. Virus-induced transcriptional activation of host FUT genes associated with neo-expression of Ley in cytomegalovirus-infected and sialyl-Lex in varicella-zoster virus-infected diploid human cells. Glycobiology 17, 355-366 (2007).
89. Blaser, M.J., Chyou, P.H. & Nomura, A. Age at Establishment of Helicobacter pylori Infection and Gastric Carcinoma, Gastric Ulcer, and Duodenal Ulcer Risk. Cancer Research 55, 562-565 (1995).
90. Crespo, A. & Suh, B. Helicobacter pylori infection: epidemiology, pathophysiology, and therapy. Arch Pharm Res 24, 485-498 (2001).
91. Forman, D. et al. Association between infection with Helicobacter pylori and risk of gastric cancer: evidence from a prospective investigation. BMJ 302, 1302-1305 (1991).
92. Rauws, E.A.J. & Tytgat, G.N.J. Cure of duodenal ulcer associated with eradication of Helicobacter pylori. Lancet 335, 1233-1235 (1990).
93. Appelmelk, B.J., Monteiro, M.A., Martin, S.L., Moran, A.P. & Vandenbroucke-Grauls, C.M. Why Helicobacter pylori has Lewis antigens. Trends Microbiol 8, 565-570 (2000).
94. Edwards, N.J. et al. Lewis X structures in the O antigen side-chain promote adhesion of Helicobacter pylori to the gastric epithelium. Mol Microbiol 35, 1530-1539 (2000).
95. Moran, A.P., Prendergast, M.M. & Appelmelk, B.J. Molecular mimicry of host structures by bacterial lipopolysaccharides and its contribution to disease. FEMS Immunol Med Microbiol 16, 105-115 (1996).
96. Larkin, M. et al. Spectrum of sialylated and nonsialylated fuco-oligosaccharides bound by the endothelial-leukocyte adhesion molecule E-selectin. Dependence of the carbohydrate binding activity on E-selectin density. J Biol Chem 267, 13661-13668 (1992).
97. Galustian, C., Elviss, N., Chart, H., Owen, R. & Feizi, T. Interactions of the gastrotropic bacterium Helicobacter pylori with the leukocyte-endothelium adhesion molecules, the selectins--a preliminary report. FEMS Immunol Med Microbiol 36, 127-134 (2003).
98. Qasba, P.K., Ramakrishnan, B. & Boeggeman, E. Substrate-induced conformational changes in glycosyltransferases. Trends Biochem Sci 30, 53-62 (2005).
99. Collins, B.E. & Paulson, J.C. Cell surface biology mediated by low affinity multivalent protein-glycan interactions. Curr Opin Chem Biol 8, 617-625 (2004).
100. Makita, Z., Vlassara, H., Cerami, A. & Bucala, R. Immunochemical detection of advanced glycosylation end products in vivo. J Biol Chem 267, 5133-5138 (1992).
101. Schjoldager, K.T. et al. A systematic study of site-specific GalNAc-type O-glycosylation modulating proprotein convertase processing. J Biol Chem 286, 40122-40132 (2011).
102. Morais, V.A. et al. Expression and characterization of recombinant human alpha-3/4-fucosyltransferase III from Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) cells using the baculovirus expression system. Biochem J 353, 719-725 (2001).
103. Lloyd, K.O. & Furukawa, K. Biosynthesis and functions of gangliosides: recent advances. Glycoconj J 15, 627-636 (1998).
104. Christiansen, M.N. et al. Cell surface protein glycosylation in cancer. Proteomics 14, 525-546 (2014).
105. Malissard, M., Zeng, S. & Berger, E.G. The yeast expression system for recombinant glycosyltransferases. Glycoconj J 16, 125-139 (1999).
106. Jarvis, D.L. Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production. Virology 310, 1-7 (2003).
107. Jenkins, N., Parekh, R.B. & James, D.C. Getting the glycosylation right: implications for the biotechnology industry. Nat Biotechnol 14, 975-981 (1996).
108. Yin, B. et al. Glycoengineering of Chinese hamster ovary cells for enhanced erythropoietin N-glycan branching and sialylation. Biotechnol Bioeng 112, 2343-2351 (2015).
109. Hamilton, S.R. & Gerngross, T.U. Glycosylation engineering in yeast: the advent of fully humanized yeast. Curr Opin Biotechnol 18, 387-392 (2007).
110. Bill, R.M. Playing catch-up with Escherichia coli: using yeast to increase success rates in recombinant protein production experiments. Front Microbiol 5, 85 (2014).
111. Widmann, M. & Christen, P. Comparison of folding rates of homologous prokaryotic and eukaryotic proteins. J Biol Chem 275, 18619-18622 (2000).
112. Plante, O.J., Palmacci, E.R. & Seeberger, P.H. Automated solid-phase synthesis of oligosaccharides. Science 291, 1523-1527 (2001).
113. Hanson, S., Best, M., Bryan, M.C. & Wong, C.H. Chemoenzymatic synthesis of oligosaccharides and glycoproteins. Trends Biochem Sci 29, 656-663 (2004).
114. Blixt, O. et al. Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides GD3, GT3, GM2, GD2, GT2, GM1, and GD1a. Carbohydr Res 340, 1963-1972 (2005).
115. Shinohara, Y., Furukawa, J., Niikura, K., Miura, N. & Nishimura, S. Direct N-glycan profiling in the presence of tryptic peptides on MALDI-TOF by controlled ion enhancement and suppression upon glycan-selective derivatization. Anal Chem 76, 6989-6997 (2004).
116. Murray, B.W., Takayama, S., Schultz, J. & Wong, C.H. Mechanism and specificity of human alpha-1,3-fucosyltransferase V. Biochemistry 35, 11183-11195 (1996).
117. De Vries, T., Palcic, M.P., Schoenmakers, P.S., Van Den Eijnden, D.H. & Joziasse, D.H. Acceptor specificity of GDP-Fuc:Gal beta 1-->4GlcNAc-R alpha 3-fucosyltransferase VI (FucT VI) expressed in insect cells as soluble, secreted enzyme. Glycobiology 7, 921-927 (1997).
118. Lee, L.V. et al. A potent and highly selective inhibitor of human alpha-1,3-fucosyltransferase via click chemistry. J Am Chem Soc 125, 9588-9589 (2003).
119. Rillahan, C.D., Brown, S.J., Register, A.C., Rosen, H. & Paulson, J.C. High-throughput screening for inhibitors of sialyl- and fucosyltransferases. Angew Chem Int Ed Engl 50, 12534-12537 (2011).
120. Hosoguchi, K. et al. An efficient approach to the discovery of potent inhibitors against glycosyltransferases. J Med Chem 53, 5607-5619 (2010).
121. Lin, T.W., Chang, W.W., Chen, C.C. & Tsai, Y.C. Stachybotrydial, a potent inhibitor of fucosyltransferase and sialyltransferase. Biochem Biophys Res Commun 331, 953-957 (2005).
122. Gonzalo, P., Sontag, B., Guillot, D. & Reboud, J.P. Fluorometric assay of GTPase activity: application to the couple elongation factor eEF-2-ribosome. Anal Biochem 225, 178-180 (1995).
123. Sista, R. et al. Development of a digital microfluidic platform for point of care testing. Lab Chip 8, 2091-2104 (2008).
124. Wulff-Burchfield, E. et al. Microfluidic platform versus conventional real-time polymerase chain reaction for the detection of Mycoplasma pneumoniae in respiratory specimens. Diagn Microbiol Infect Dis 67, 22-29 (2010).
125. Agresti, J.J. et al. Ultrahigh-throughput screening in drop-based microfluidics for directed evolution. Proc Natl Acad Sci U S A 107, 4004-4009 (2010).
126. Kintses, B. et al. Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution. Chem Biol 19, 1001-1009 (2012).
127. Khanafer, K., Vafai, K. & Lightstone, M. Buoyancy-driven heat transfer enhancement in a two-dimensional enclosure utilizing nanofluids. Int J Heat Mass Transfer 46, 3639-3653 (2003).
128. Hamilton, R.L.C., O.K. Thermal conductivity of heterogeneous two-component systems. I & EC Fundam. 1, 182-191 (1962).
129. Liu, Y. et al. Multilayer-assembled microchip for enzyme immobilization as reactor toward low-level protein identification. Anal Chem 78, 801-808 (2006).
130. Zhao, X., Cheng, K. & Liu, D. Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis. Appl Microbiol Biotechnol 82, 815-827 (2009).
131. Zou, Y., Xiang, C., Sun, L.X. & Xu, F. Glucose biosensor based on electrodeposition of platinum nanoparticles onto carbon nanotubes and immobilizing enzyme with chitosan-SiO(2) sol-gel. Biosens Bioelectron 23, 1010-1016 (2008).
132. Zeng, J., Chen, X., Liang, Q., Xu, X. & Jing, X. Enzymatic degradation of poly(L-lactide) and poly(epsilon-caprolactone) electrospun fibers. Macromol Biosci 4, 1118-1125 (2004).
133. Araci, I.E. & Quake, S.R. Microfluidic very large scale integration (mVLSI) with integrated micromechanical valves. Lab Chip 12, 2803-2806 (2012).
134. Thorsen, T., Maerkl, S.J. & Quake, S.R. Microfluidic large-scale integration. Science 298, 580-584 (2002).
135. Beebe, D.J., Mensing, G.A. & Walker, G.M. Physics and applications of microfluidics in biology. Annu Rev Biomed Eng 4, 261-286 (2002).
136. Doshi, N. et al. Flow and adhesion of drug carriers in blood vessels depend on their shape: a study using model synthetic microvascular networks. J Control Release 146, 196-200 (2010).
137. Song, H., Chen, D.L. & Ismagilov, R.F. Reactions in droplets in microfluidic channels. Angew Chem Int Ed Engl 45, 7336-7356 (2006).
138. Song, H., Tice, J.D. & Ismagilov, R.F. A microfluidic system for controlling reaction networks in time. Angew Chem Int Ed Engl 42, 768-772 (2003).
139. Ismagilov, R.F. Integrated microfluidic systems. Angew Chem Int Ed Engl 42, 4130-4132 (2003).
140. Ge, L., Wang, S., Song, X., Ge, S. & Yu, J. 3D origami-based multifunction-integrated immunodevice: low-cost and multiplexed sandwich chemiluminescence immunoassay on microfluidic paper-based analytical device. Lab Chip 12, 3150-3158 (2012).
141. Li, W. et al. Multiplex electrochemical origami immunodevice based on cuboid silver-paper electrode and metal ions tagged nanoporous silver-chitosan. Biosens Bioelectron 56, 167-173 (2014).
142. Hu, J. et al. Advances in paper-based point-of-care diagnostics. Biosens Bioelectron 54, 585-597 (2014).
143. Martinez, A.W., Phillips, S.T., Whitesides, G.M. & Carrilho, E. Diagnostics for the developing world: microfluidic paper-based analytical devices. Anal Chem 82, 3-10 (2010).
144. Fobel, R., Fobel, C. & Wheeler, A.R. DropBot: An open-source digital microfluidic control system with precise control of electrostatic driving force and instantaneous drop velocity measurement. Appl Phys Lett 102 (2013).
145. Vo, P.Q.N., Husser, M.C., Ahmadi, F., Sinha, H. & Shih, S.C.C. Image-based feedback and analysis system for digital microfluidics. Lab Chip 17, 3437-3446 (2017).
146. Paik, P.Y., Pamula, V.K. & Chakrabarty, K. Adaptive Cooling of Integrated Circuits Using Digital Microfluidics. IEEE Transactions on VLSI Systems 16, 432-443 (2008).
147. Kalsi, S. et al. Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform. Lab Chip 15, 3065-3075 (2015).
148. Hadwen, B. et al. Programmable large area digital microfluidic array with integrated droplet sensing for bioassays. Lab Chip 12, 3305-3313 (2012).
149. Zeng, X. et al. Chemiluminescence detector based on a single planar transparent digital microfluidic device. Lab Chip 13, 2714-2720 (2013).
150. Lin, L., Evans, R.D., Jokerst, N.M. & Fair, R.B. Integrated optical sensor in a digital microfluidic platform. IEEE Sens. J. 8, 628-635 (2008).
151. Shih, S.C.C., Barbulovic-Nad, I., Yang, X., Fobel, R. & Wheeler, A.R. Digital microfluidics with impedance sensing for integrated cell culture and analysis. Biosens. Bioelectron. 42, 314-320 (2013).
152. Dryden, M.D.M., Fobel, R., Fobel, C. & Wheeler, A.R. Upon the Shoulders of Giants: Open-Source Hardware and Software in Analytical Chemistry. Anal Chem 89, 4330-4338 (2017).
153. Graham, C. et al. Novel application of digital microfluidics for the detection of biotinidase deficiency in newborns. Clin Biochem 46, 1889-1891 (2013).
154. Boles, D.J. et al. Droplet-based pyrosequencing using digital microfluidics. Anal Chem 83, 8439-8447 (2011).
155. Miller, E.M. & Wheeler, A.R. A digital microfluidic approach to homogeneous enzyme assays. Anal Chem 80, 1614-1619 (2008).
156. Luk, V.N., Fiddes, L.K., Luk, V.M., Kumacheva, E. & Wheeler, A.R. Digital microfluidic hydrogel microreactors for proteomics. Proteomics 12, 1310-1318 (2012).
157. Millington, D. et al. Digital microfluidics comes of age: high-throughput screening to bedside diagnostic testing for genetic disorders in newborns. Expert Rev Mol Diagn, 1-12 (2018).
158. Dittrich, P.S. & Manz, A. Lab-on-a-chip: microfluidics in drug discovery. Nat Rev Drug Discov 5, 210-218 (2006).
159. Martin, J.G. et al. Toward an artificial Golgi: redesigning the biological activities of heparan sulfate on a digital microfluidic chip. J Am Chem Soc 131, 11041-11048 (2009).
160. Nguyen, E.P. et al. Hybrid Surface and Bulk Resonant Acoustics for Concurrent Actuation and Sensing on a Single Microfluidic Device. Anal Chem 90, 5335-5342 (2018).
161. Cao, Q., Han, X. & Li, L. Configurations and control of magnetic fields for manipulating magnetic particles in microfluidic applications: magnet systems and manipulation mechanisms. Lab Chip 14, 2762-2777 (2014).
162. Fan, X. & White, I.M. Optofluidic Microsystems for Chemical and Biological Analysis. Nat Photonics 5, 591-597 (2011).
163. Abdelgawad, M., Freire, S.L., Yang, H. & Wheeler, A.R. All-terrain droplet actuation. Lab Chip 8, 672-677 (2008).
164. Fan, S.K., Yang, H. & Hsu, W. Droplet-on-a-wristband: chip-to-chip digital microfluidic interfaces between replaceable and flexible electrowetting modules. Lab Chip 11, 343-347 (2011).
165. Choi, K., Ng, A.H., Fobel, R. & Wheeler, A.R. Digital microfluidics. Annu Rev Anal Chem (Palo Alto Calif) 5, 413-440 (2012).
166. Pollack, M.G., Fair, R.B. & Shenderov, A.D. Electrowetting-based actuation of liquid droplets for microfluidic applications. Applied Physics Letters 77, 1725-1726 (2000).
167. Hsieh, T.F., S. in IEEE 21st International Conference on Micro Electro Mechanical Systems 641-644 (Tucson, Arizona; 2008).
168. Chatterjee, D., Hetayothin, B., Wheeler, A.R., King, D.J. & Garrell, R.L. Droplet-based microfluidics with nonaqueous solvents and solutions. Lab Chip 6, 199-206 (2006).
169. Kang, K.Y. How Electrostatic Fields Change Contact Angle in Electrowetting. Langmuir 18, 10318–10322 (2002).
170. Pohl, H.A. The Motion and Precipitation of Suspensoids in Divergent Electric Fields. Journal of Applied Physics 22, 869-871 (1951).
171. Jones, T.B. Electromechanics of Particles. (Cambridge Univ. Press
Cambridge, U.K.; 1995).
172. Zhao, Y., Yi, U.-C. & Cho, S.K. Microparticle Concentration and Separation by Traveling-Wave Dielectrophoresis (twDEP) for Digital Microfluidics. Journal of Microelectromechanical Systems 16, 1472-1481 (2007).
173. Rice, C.L.W., R. Electrokinetic flow in a narrow cylindrical capillary. J. Phys. Chem. 69, 4017-4024 (1965).
174. Wong, P.K., Wang, T.H., Deval, J.H. & Ho, C.M. Electrokinetics in Micro Devices for Biotechnology Applications. IEEE/ASME Transactions on Mechatronics 9, 366-376 (2004).
175. Castellarnau, M., Errachid, A., Madrid, C., Juarez, A. & Samitier, J. Dielectrophoresis as a tool to characterize and differentiate isogenic mutants of Escherichia coli. Biophys J 91, 3937-3945 (2006).
176. Chatterjee, D., Shepherd, H. & Garrell, R.L. Electromechanical model for actuating liquids in a two-plate droplet microfluidic device. Lab Chip 9, 1219-1229 (2009).
177. Seyrat, E. & Hayes, R.A. Amorphous fluoropolymers as insulators for reversible low-voltage electrowetting. Journal of Applied Physics 90, 1383-1386 (2001).
178. Lin, Y.Y. et al. Low Voltage Electrowetting-on-Dielectric Platform using Multi-Layer Insulators. Sens Actuators B Chem 150, 465-470 (2010).
179. Luk, V.N., Mo, G. & Wheeler, A.R. Pluronic additives: a solution to sticky problems in digital microfluidics. Langmuir 24, 6382-6389 (2008).
180. Au, S.H., Kumar, P. & Wheeler, A.R. A new angle on pluronic additives: advancing droplets and understanding in digital microfluidics. Langmuir 27, 8586-8594 (2011).
181. Ren, H.S., Vijay;B. Fair, Richard in Int. Conf. Solid-State Sens., Actuators Microsyst., 12th, Vol. 1 619-622 (IEEE, 2003).
182. Ren, H. Automated on-chip droplet dispensing with volume control by electro-wetting actuation and capacitance metering. Sensors and Actuators B: Chemical 98, 319-327 (2004).
183. Ren, H. (2004). Electro-wetting based sample preparation: An initial study for droplet transportation, creation and on-chip digital dilution., Duke University. PhD.
184. Singh, A.K. et al. Unravelling the multiple functions of the architecturally intricate Streptococcus pneumoniae beta-galactosidase, BgaA. PLoS Pathog 10, e1004364 (2014).
185. Mark, B.L. et al. Structural and functional characterization of Streptomyces plicatus beta-N-acetylhexosaminidase by comparative molecular modeling and site-directed mutagenesis. J Biol Chem 273, 19618-19624 (1998).
186. Brassard, D., Malic, L., Normandin, F., Tabrizian, M. & Veres, T. Water-oil core-shell droplets for electrowetting-based digital microfluidic devices. Lab Chip 8, 1342-1349 (2008).
187. Vergauwe, N. et al. Controlling droplet size variability of a digital lab-on-a-chip for improved bio-assay performance. Microfluidics and Nanofluidics 11, 25-34 (2011).
188. Kim, Y.W., Lee, S.S., Warren, R.A. & Withers, S.G. Directed evolution of a glycosynthase from Agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire. J Biol Chem 279, 42787-42793 (2004).
189. Cold Spring Harbor Protocols (2006). "Preparation of 0.1 M Potassium Phosphate Buffer at 25°C." Retrieved 2016-11-07, from http://cshprotocols.cshlp.org/content/2006/1/pdb.tab19.

190. Kajihara, Y. et al. Synthesis of 2-[(2-pyridyl)amino]ethyl beta-D-lactosaminide and evaluation of its acceptor ability for sialyltransferase: a comparison with 4-methylumbelliferyl and dansyl beta-D-lactosaminide. Carbohydr Res 339, 1545-1550 (2004).
191. Zeng, J. & Korsmeyer, T. Principles of droplet electrohydrodynamics for lab-on-a-chip. Lab Chip 4, 265-277 (2004).
192. Liechti, C. "PySerial." 2018, from http://pypi.python.org/pypi/pyserial.
193. Zhang, J.H., Chung, T.D. & Oldenburg, K.R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 4, 67-73 (1999).
194. de Vries, T., Knegtel, R.M., Holmes, E.H. & Macher, B.A. Fucosyltransferases: structure/function studies. Glycobiology 11, 119R-128R (2001).
195. Wong, C.H., Dumas, D. P., Ichikawa, Y., Koseki, K., Danishefsky, S. J., Weston, B. W., Lowe, J. B. Specificity, inhibition, and synthetic utility of a recombinant human. alpha.-1, 3-fucosyltransferase. Journal of the American Chemical Society 114, 7321-7322 (1992).
196. Yoon, J.Y. & Garrell, R.L. Preventing Biomolecular Adsorption in Electrowetting-Based Biofluidic Chips. Anal Chem 75, 5097-5102 (2003).
197. Rajabi, N. & Dolatabadi, A. A novel electrode shape for electrowetting-based microfluidics. Colloids and Surfaces A: Physicochemical and Engineering Aspects 365, 230-236 (2010).
198. Chen, J., Yu, Y., Li, J., Lai, Y. & Zhou, J. Size-variable droplet actuation by interdigitated electrowetting electrode. Applied Physics Letters 101 (2012).
199. Pollack, M.G., Shenderov, A.D. & Fair, R.B. Electrowetting-based actuation of droplets for integrated microfluidics. Lab Chip 2, 96-101 (2002).
200. Paik, P., Pamula, V.K., Pollack, M.G. & Fair, R.B. Electrowetting-based droplet mixers for microfluidic systems. Lab Chip 3, 28-33 (2003).
201. Hamadi, F., Latrache, H., Zekraoui, M., Ellouali, M. & Bengourram, J. Effect of pH on surface energy of glass and Teflon and theoretical prediction of Staphylococcus aureus adhesion. Materials Science and Engineering: C 29, 1302-1305 (2009).
202. Berthier, J. & Peponnet, C. A model for the determination of the dimensions of dents for jagged electrodes in electrowetting on dielectric microsystems. Biomicrofluidics 1, 14104 (2007).
203. Lienemann, J., et al. (2003). Electrode shapes for electrowetting arrays. Nanotech 2003, researchgate.net.
204. Fair, R.B.S., V.; Ren, H.; Paik, P.; Pamula, V.K.; Pollack, M.G. in IEEE International Electron Devices Meeting 32.35.31-32.35.34 (2004).
205. Shih, S.C., Barbulovic-Nad, I., Yang, X., Fobel, R. & Wheeler, A.R. Digital microfluidics with impedance sensing for integrated cell culture and analysis. Biosens Bioelectron 42, 314-320 (2013).
206. Calabrese, E.J. & Baldwin, L.A. The frequency of U-shaped dose responses in the toxicological literature. Toxicol Sci 62, 330-338 (2001).
207. Owen, S.C. et al. Colloidal drug formulations can explain "bell-shaped" concentration-response curves. ACS Chem Biol 9, 777-784 (2014).
208. Zegarra-Moran, O. et al. Correction of G551D-CFTR transport defect in epithelial monolayers by genistein but not by CPX or MPB-07. Br J Pharmacol 137, 504-512 (2002).
209. Dienes, A., Shank, C. & Kohn, R. Characteristics of the 4-methylumbelliferone laser dye. IEEE Journal of Quantum Electronics 9, 833-843 (1973).
210. Kongkamnerd, J. et al. The quenching effect of flavonoids on 4-methylumbelliferone, a potential pitfall in fluorimetric neuraminidase inhibition assays. J Biomol Screen 16, 755-764 (2011).
211. Litten, B., Blackett, C., Wigglesworth, M., Goddard, N. & Fielden, P. Artefacts at the liquid interface and their impact in miniaturized biochemical assay. Biomicrofluidics 9, 052607 (2015).
212. Eydelnant, I.A., Betty Li, B. & Wheeler, A.R. Microgels on-demand. Nat Commun 5, 3355 (2014).
213. Toppila, S., Paavonen, T., Laitinen, A., Laitinen, L.A. & Renkonen, R. Endothelial sulfated sialyl Lewis x glycans, putative L-selectin ligands, are preferentially expressed in bronchial asthma but not in other chronic inflammatory lung diseases. Am J Respir Cell Mol Biol 23, 492-498 (2000).
All items in Spectrum are protected by copyright, with all rights reserved. The use of items is governed by Spectrum's terms of access.

Repository Staff Only: item control page

Downloads per month over past year

Research related to the current document (at the CORE website)
- Research related to the current document (at the CORE website)
Back to top Back to top