Chemistries for patterning robust DNA microbarcodes enable multiplex assays of cytoplasm proteins from singlecancer cells.Shin YS, Ahmad H, Shi Q, Kim H, Pascal TA, Fan R, Goddard WA 3rd, Heath JR. Chemphyschem. 2010, 11(14):3063-9.
The most highly multiplex protein assays are protein microarrays[4-6]. The microarrays face to two challenges: one is the relative instability and the other is scalling miniaturized DNA microarrays. To solve these problems: Fan et al. generate DNA barcode-type arrays, using DNA-encoded antibody library (DEAL) technique to measure highly multiplex panel of proteins from biospeciments[14].
We explore three different flow patterning surface chemistries: two rely upon the electrostatic adsorption of DNA onto a poly-L-lysine (PLL) surface, and the third utilizes flow patterning of dendrimers onto aminated glass substrates, followed by covalent attachment of DNA oligomers onto the dendrimer scaffolds. we find that solvent mixtures which associate counterions more strongly to the negatively charged DNA oligomers yield more reproducible and robust barcodes.
The chip is comprised of a patterned polydimethylsiloxane (PDMS) layer adhered to an aminated or PLL-coated glass substrate. The microchannels are long (about 55 cm),meandering channels that span ca. 0.85 cm*cm, and are used to pattern a DNA barcode over most of the glass surface. After the flow patterning is completed, the PDMS layer is replaced with a second micropatterned PDMS layer that is designed to support a biological assay.
In schemes 1 and 2, using thermal or UV treat DNA to cross-link the deposited DNA to the substrate. In Scheme 1 ssDNA oligomers dissolved in phosphate buffered saline (PBS) are utilized, whereas in Scheme 2 ssDNAs in a 1:1 mixture of 1 PBS and dimethyl sulfoxide (DMSO) are employed. DMSO is used in conventional microarray preparation to improve feature consistency by reducing the rate of solvent evaporation and by denaturing the DNA[15] although, as described below, its role in this process is different. In
Scheme 3 a covalent immobilization method based upon a dendrimer scaffold is utilized.[16]
The highly branched structure of the dendrimers provides a high density of reactive sites for surface attachment and for DNA coupling, thus leading to a high overall binding capacity.
Thirteen discrete channels: five adjacent channels according to Scheme 1, skipped three channels, and then loaded the remaining five channels according to Scheme 2. The use of fluorescently-tagged DNA permitted measurements of the DNA distribution The results show Scheme 2 is more consistent distribution crross the entire chip, but Scheme 1 shows relatively higher fluorescence intensity at the input side of the chip.
Scheme 3, the PAMAM dendrimers are first covalently attached to the aminated glass surface, and then (aminated) ssDNA oligomers are covalently attached to the dendrimers. The aqueous DNA distribution is expected to be uniform in Scheme 3.The CV is defined as the standard deviation divided by the mean and expressed as a percentage. CVs for Schemes 1, 2, and 3 registered 69.8 %, 10.5 %, and 10.9 %, respectively.
Using Scheme 2 to assay the following proteins: (Src), (mTOR), (S6K),(GSK)-3a/b, phospho-p38a, (ERK), and (EGFR) at 10 ng/mL and 1 ng/mL concentrations. This panel samples key nodes of the phosphoinositide 3-kinase (PI3K) signaling pathway within GBM, and are used below for single-cell assays.[23]. Using Scheme 3 to detect three pro-
teins: [interferon (INF)-g, tumor necrosis factor (TNF)a, and in-terleukin (IL)-2] at 100 ng/mL and 10 ng/mL.
sscDNA+1-Ab----proteins from cell lysate----2-Ab+biotin+Cy5-labeled strptavidin.
the absolute (chip-to-chip) consistency of Scheme 3 is hard to guarantee due to its use of the unstable coupling reagents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and NHS.[24]
Experimental Section

The master  mold  was  prepared  using  either  a  negative  photoresist, SU8 2010, with photolithography or an etched silicon mold generated by a deep reactive ion etching (DRIE) process. The mold has long meandering channels with a 20×20 um cross section. The distance from channel to channel is also 20 um, which generates 10 higher density than standard, spotted microarrays. Sylgard PDMS (Corning) prepolymer and curing agent were mixed in a 10:1 ratio (w/w), poured onto the mold, and cured (80 ℃, 1 hour). The cured PDMS  slab  was  released  from  the  mold,  inlet/outlet  holes  were punched, and  the device was  bonded onto a PLL coated (C40–5257 M20, Thermo scientific) or aminated glass slide (48382–220, VWR) to form enclosed channels. The number of microfluidic channels determines the size of the microarray; 13 parallel microchannels were used in this study
    Patterning of DNA Barcode Arrays: a 30-mer DNA oligomer labeled with Cy3  fluorescence tag on the 5’ end (5’-/Cy3/-AAA AAA AAA ATA CGG ACT TAG CTC CAG GAT-3’) in a 1:1 mixture (v/v) of 1 PBS buffer and DMSO or a 1:1 mixture (v/v) of 1 PBS buffer and deionized (DI) water was used. The final DNA concentration was 2.5 mm. DNA solution was pushed into the
channel under a constant pressure (2.5 psi). Immediately after the channels were fully filled, fluorescence images were obtained by confocal microscopy.
      Dendrimer-based microarrays were prepared using aminated substrates.  Generation  4.5  Poly(amidoamine)  (PAMAM)  dendrimers (470457–2.5G, Aldrich), 5 % wt in MeOH, were mixed 1:1 (v/v) with EDC/NHS (0.2 m) in MES buffer (0.1m, pH 6.0). After 5 min of incubation, the activated dendrimers were introduced to the microfluidic channels, and allowed to flow (2 h). Following a brief MeOH rinse  to  remove  unbound  dendrimers,  the  channels  were  filled with EDC/NHS (0.2 m) in MES (0.1 m, pH 5.3) with NaCl (0.5 m). After 0.5 h, 5’ aminated DNA sequences in 1 PBS (200 mm) were introduced to the channels and allowed to flow (2 h). Thereafter, the microfluidic device was removed from the substrate, and the latter was rinsed copiously with DI water. Prepared substrates that were not used immediately were stored in a desiccator.


[1]  J. R. Heath, M. E. Davis, Annu Rev Med. 2008, 59, 251 – 265.
[2]  J. R. Heath, M. E. Phelps, L. Hood, Mol. Imaging Biol. 2003, 5, 312 –325.
[3]  I. G. Khalil, C. Hill, Curr Opin Oncol. 2005, 17, 44 –48.
[4]  G. Zheng, F. Patolsky, Y. Cui, W. U. Wang, C. M. Lieber, Nat. Biotechnol.2005, 23, 1294 –1301.
[5]  C. M. Niemeyer, Nano Today 2007, 2, 42 – 52.
[6]  C. Boozer, J. Ladd, S. Chen, S. Jiang, Anal. Chem. 2006, 78, 1515– 1519.
[7]  R. Wacker, C. M. Niemeyer, ChemBioChem 2004, 5, 453 – 459.
[8]  H. Schroeder, M. Adler, K. Gerigk, B. Muller-Chorus, F. Gotz, C. M. Nie-meyer, Anal. Chem. 2009, 81, 1275 –1279.
[9]  R. Wacker, H. Schrçder, C. M. Niemeyer, Anal. Biochem. 2004, 330, 281 –287.
[10]  E. S. Douglas, R. A. Chandra, C. R. Bertozzi, R. A. Mathies, M. B. Francis,Lab on a Chip. 2007, 7, 1442– 1448.
[11]  R. C. Bailey, G. A. Kwong, C. G. Radu, O. N. Witte, J. R. Heath, J Am Chem.Soc. 2007, 129, 1959– 1967.
[12]  C. M. Niemeyer, T. Sano, C. L. Smith, C. R. Cantor, Nucleic Acids Res. 1994,22, 5530 –5539.
[13]  T. Sano, C. Smith, C. Cantor, Science 1992, 258, 120 –122.
[14]  R.  Fan,  O.  Vermesh,  A.  Srivastava,  B. K.  Yen,  L.  Qin,  H.  Ahmad,  G. A.Kwong, C. C. Liu, J. Gould, L. Hood, J. R. Heath, Nat. Biotechnol. 2008,26, 1373 –1378.
[15]  M. Dufva, Biomol. Eng. 2005, 22, 173 – 184.
[16]  V. Le Berre, E. Trevisiol, A. Dagkessamanskaia, S. Sokol, A. M. Caminade,J. P. Majoral, B. Meunier, J. Francois, Nucleic Acids Res. 2003, 31, 88e.
[17]  A. W. Bosman, H. M. Janssen, E. W. Meijer, Chem. Rev. 1999, 99, 1665 –1688.
[18]  R. Benters, C. M. Niemeyer, D. Drutschmann, D. Blohm, D. Wohrle, Nucle-ic Acids Res. 2002, 30, 10e.
[19]  P. Angenendt, J. Glokler, J. Sobek, H. Lehrach, D. J. Cahill, J. Chromatogr.A 2003, 1009, 97 – 104.
[20]  P. K. Ajikumar, J. K. Ng, Y. C. Tang, J. Y. Lee, G. Stephanopoulos, H. P. Too,Langmuir 2007, 23, 5670 – 5677.
[21]  A.-T. Kuo, C.-H. Chang, H.-H. Wei, Appl. Phys. Lett. 2008, 92, 244102 –244103.
[22]  J. A. Benn, J. Hu, B. J. Hogan, R. C. Fry, L. D. Samson, T. Thorsen, Anal.Biochem. 2006, 348, 284 – 293.
[23]  Cancer Genome Atlas Research Network, Nature 2008, 455, 1061 –1068.
[24]  A.  Kausaite,  M. v.  Dijk,  J.  Castrop,  A.  Ramanaviciene,  J. P.  Baltrus,  J.Acaite, A. Ramanavicius, Biochem. Molec. Biol. Educ. 2007, 35, 57 –63.
[25]  P. O. Krutzik, J. M. Irish, G. P. Nolan, O. D. Perez, Clin. Immunol. 2004, 110,206 – 221.
[26]  Y. Liang, M. Diehn, N. Watson, A. W. Bollen, K. D. Aldape, M. K. Nicholas,K. R. Lamborn, M. S. Berger, D. Botstein, P. O. Brown, M. A. Israel, Proc.Natl. Acad. Sci. USA 2005, 102, 5814– 5819.
[27]  J. C. Lee, I. Vivanco, R. Beroukhim, J. H. Y. Huang, W. L. Feng, R. M. DeBia-si, K. Yoshimoto, J. C. King, P. Nghiemphu, Y. Yuza, Q. Xu, H. Greulich,R. K. Thomas, J. G. Paez, T. C. Peck, D. J. Linhart, K. A. Glatt, G. Getz, R.Onofrio, L. Ziaugra, R. L. Levine, S. Gabriel, T. Kawaguchi, K. O’Neill, H.Khan, L. M. Liau, S. F. Nelson, P. N. Rao, P. Mischel, R. O. Pieper, T. Clough-esy, D. J. Leahy, W. R. Sellers, C. L. Sawyers, M. Meyerson, I. K. Mellingh-off, PLoS Med. 2006, 3, e485.
[28]  T. Thorsen, S. J. Maerkl, S. R. Quake, Science 2002, 298, 580 –584.
[29]  Y. Duan, C. Wu, S. Chowdhury, M. C. Lee, G. Xiong, W. Zhang, R. Yang, P.Cieplak, R. Luo, T. Lee, J. Caldwell, J. Wang, P. Kollman, J. Comput. Chem.2003, 24, 1999 –2012.
[30]  W. D. Cornell, P. Cieplak, C. I. Bayly, I. R. Gould, K. M. Merz, D. M. Ferguson, D. C. Spellmeyer, T. Fox, J. W. Caldwell, P. A. Kollman, J. Am. Chem.Soc. 1995, 117, 5179– 5197.
[31]  S. Plimpton, J. Comput. Phys. 1995, 117, 1 – 19.
[32]  C. S. Tung, E. S. Carter II, Comput. Appl. Biosci. 1994, 10, 427 – 433.
[33]  W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W. Impey, M. L. Klein,J. Chem. Phys. 1983, 79, 926 – 935.
[34]  R. W.  Hockney,  J. W.  Eastwood,  Computer  Simulation  Using  Particles,McGraw-Hill, New York, 1981.

Chemistries for Patterning Robust DNA MicroBarcodes Enable Multiplex Assays of C.pdf

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Chemistries for Patterning Robust DNA MicroBarcodes Enable Multiplex Assays of C.pdf