Signal-Amplifying Conjugated Polymer–DNA Hybrid Chips

Trinh Pham

doi:10.1002/ANIE.200700419

Outline

Signal-Amplifying Conjugated Polymer–DNA Hybrid Chips

Trinh Pham

2007, Angewandte Chemie

https://doi.org/10.1002/ANIE.200700419

visibility

50 views

description

4 pages

Abstract

Bio-/synthetic hybrid materials have recently received considerable attention owing to their potential biomedical applications. [1] The most reliable way of identifying any biological target is through its genetic code. However, the current commercial DNA microarray requires costly and time-consuming PCR to multiply the number of analyte DNA molecules and label the analyte DNA with a fluorescent dye because of the low detection limit. In this context, devising self-signal-amplifying DNA microarrays can realize low-cost, fast, and reliable detection of nucleic acids. Herein, we report signal-amplifying DNA chips fabricated by on-chip DNA synthesis on a thin film of a newly developed conjugated polymer ( and the chemical structure in Figure 2 a).

DNA Microarrays DOI: 10.1002/anie.200700419 Signal-Amplifying Conjugated Polymer–DNA Hybrid Chips** Kangwon Lee, Jean-Marie Rouillard, Trinh Pham, Erdogan Gulari, and Jinsang Kim* Bio-/synthetic hybrid materials have recently received con- siderable attention owing to their potential biomedical applications. [1] The most reliable way of identifying any biological target is through its genetic code. [2] However, the current commercial DNA microarray requires costly and time-consuming PCR to multiply the number of analyte DNA molecules and label the analyte DNA with a fluorescent dye because of the low detection limit. In this context, devising self-signal-amplifying DNA microarrays can realize low-cost, fast, and reliable detection of nucleic acids. Herein, we report signal-amplifying DNA chips fabricated by on-chip DNA synthesis on a thin film of a newly developed conjugated polymer (Figure 1 and the chemical structure in Figure 2a). Conjugated polymer-based biosensors are an attractive approach to improve the detection limit because an environ- mental change at a single site can affect the properties of the collective system, producing large signal amplification. [3] Therefore, if one devises a strategy combining the signal- amplification scheme of conjugated polymers and efficient on-chip DNA synthesis, signal-amplifying DNA microarrays can be conveniently prepared. On-chip oligonucleotide synthesis [2d, 4] has the unique advantage of being performed in a parallel fashion, is flexible in sequence design, easy to manufacture, and has a high sequence fidelity compared with other recently developed methods, such as the pin micro- dotting method, [2c] the ink-jet microdropping method, [5] and the electrostatic addressing method. [6] Almost all the on-chip DNA synthesis technologies, however, require harsh condi- tions such as long exposure to UV light and/or to strong acids. Under these harsh conditions, conventional conjugated polymers will be photobleached or chemically degraded. We have developed a novel conjugated polymer with a strong fluorescence emission and unique stability under the above-mentioned harsh conditions. Figure 2 a shows the chemical structure of the poly(oxadiazole-co-phenylene-co- fluorene) P1 with oxadiazole units and amine side chains. All monomer units of P1 were designed to have their own contribution to the final property of P1 and synthesized through multiple synthetic steps (see the Supporting Infor- mation). Oxadiazole is an electron-poor heterocyclic mole- cule that has been used in polymer design in which the improvement of electron transport and/or stability of the polymer is required. [7] We designed an oxadiazole-containing monomer (M3) and incorporated this unit into the conjugated polymer backbone by using a Pd-based Suzuki coupling method. [8] The oxadiazole-containing monomer unit M3 of P1 has an intense blue fluorescence emission at 413 nm in a chloroform solution and is stable when exposed to strong UV irradiation and a strong acidic environment. The amine groups on the phenylene unit (M1) of P1 serve as functional groups for immobilization of P1 on a glass substrate as well as linkers for direct on-chip synthesis of oligonucleotides on the resulting thin-layer film of P1. The fluorene unit (M2) of P1 is incorporated to provide good solubility in organic solvents and to ensure a good spectral overlap with commonly used organic dyes for an efficient fluorescence resonance energy transfer (FRET). Figure 2 b shows the absorption (UV) and photoluminescence spectra (PL) of P1 in chloroform and incorporated in the film. The absolute quantum yield of P1 solution in chloroform (1 mg L À1 ), measured in an integrating sphere (PTI technologies, Inc.), was 94%. We investigated the stability of P1 compared with commonly used conjugated polymers, such as poly(p-phenylene-ethynylene)s and poly(3- hexylthiophene), under strong UV irradiation and highly acidic conditions. None of the compounds except P1 survived these tests (data not shown). The fluorescence of the conven- tional conjugated polymers was completely quenched by degradation of polymers under these harsh conditions. How- ever, P1 showed unique stability against the exposure to UV irradiation and acid treatments both in the solution and solid state. The unique stability of P1 made possible on-chip DNA synthesis directly on a thin film of the conjugated polymer. The preparation of P1-coated glass substrates is described in Figure 3a. We covalently linked P1 to a glass substrate to Figure 1. Schematic representation of the signal-amplifying conjugated polymer-based DNA chip. a) P1-coated glass slide by covalent bond- ing; b) light-directed on-chip oligonucleotide synthesis; c) hybridiza- tion with a target DNA results in large emission enhancement of the fluorescent dye through efficient Förster resonance energy transfer. [*] K. Lee, Prof. J. Kim Department of Materials Science and Engineering University of Michigan 2300 Hayward St., Ann Arbor, MI 48109 (USA) Fax: (+ 1)734-936-4681 E-mail: jinsang@umich.edu Dr. J.-M. Rouillard, T. Pham, Prof. E. Gulari, Prof. J. Kim Department of Chemical Engineering University of Michigan 2300 Hayward St., Ann Arbor, MI 48109 (USA) [**] We appreciate financial support from the National Science Foun- dation (BES 0428010) and the equipment grant from the University of Michigan College of Engineering. J.K. and K.L. also acknowledge the Ilju Foundation for the Ilju scholarship. Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. Angewandte Chemie 4667 Angew. Chem. Int. Ed. 2007, 46, 4667 –4670 # 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

prevent any loss of P1 during the on-chip DNA synthesis. To do so, isothiocyanate-functionalized glass substrates were prepared by using a slightly modified literature procedure. [3e] First, aminopropyl groups were introduced onto a glass substrate by first cleaning with pirahna solution (H 2 O 2 / H 2 SO 4 3:7 (v/v)) followed by an aminopropyltrimethoxysi- lane (APTMS) coating. 1,4-Phenylenediisothiocyanate was then reacted with the amine of APTMS to form a reactive linker for P1. Finally, P1 was chemically bound onto the glass substrate. After immobilization of P1, the derived UV spectrum of the glass substrate showed a new broad band at 350–400 nm, which corresponds to P1 absorption. Fluores- cence spectroscopy also showed a well-defined fluorescence emission spectrum of P1 from the glass substrate. The on-chip DNA synthesis [9] on the P1-coated glass substrate was conducted by using a modified automatic oligosynthesizer equipped with a UV patterning device. The synthesis is carried out by using 5’-(4,4’-dimethoxytrityl) (DMT) nucleophosphoramidite monomers as the building blocks and each synthesis cycle consists of a deprotection step by using photogenerated acids, coupling of a DMT-protected monomer, capping of unreacted terminal OH groups, and oxidation of the phosphite to phosphatetriester at internu- cleotide linkages. [4b, 10] Various sequences of DNA can be synthesized at different locations on the chip by generating a strong acid at the desired locations by UV-induced decom- position of a photoacid generator (PAG). The photogener- ated acid (PGA) then catalyzes the deprotection reaction, producing a 5’-OH group, which is available for the next monomer. We synthesized two different sequences. The first sequence was 5’-ACATCC GTG ATG TGT T-glass-3’ (the 3’ T is a spacer), which was used for hybridization with the complementary sequence with hexachlorofluorescein (HEX) dye, and the second sequence was 5-ACAG AAG CAT TAT TTC T-glass-3’ for the Cy5-labeled complementary sequence. Figure 3 b shows the fluorescence image of the synthe- sized DNA on the P1-coated glass substrate after hybrid- Figure 2. a) Chemical structure of P1. b) UV/PL spectra of P1 in chloroform (black = UV, blue = PL) and solid film (green = UV, red = PL). Communications 4668 www.angewandte.org # 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2007, 46, 4667 –4670

ization with two different dye-labeled complementary DNA molecules. Selective fluorescent patterns of green (HEX) and red (Cy-5) dots are clearly shown in Figure 3b. This result demonstrates that direct on-chip DNA synthesis onto a P1- coated glass slide was macroscopically accomplished. More- over, during the harsh DNA synthesis procedures, the emissive property of P1 was maintained. We prepared a control sample to conduct quantitative analysis of signal amplification by P1. The control sample had the same 16-base DNA sequence (5’-ACATCC GTG ATG TGT T-glass-3’) as was synthesized on an amine-functionalized glass slide, but without P1. The density of the synthesized oligonucleotide (2.44 pmol cm À2 ) on the conventional control slide was the same as that of the oligonucleotide on the P1-coated slide. This was confirmed by UV absorption at 410 nm (see the Supporting Information). We used a 15-base HEX-labeled complementary DNA sequence to observe the FRET effect from P1 to HEX dye. FRET involves a nonradiative transmission of fluorescence energy from a donor molecule to the acceptor molecule. P1 has a good spectral overlap with HEX, satisfying the require- ment for efficient FRET. Figure 4a shows the fluorescent emission spectrum of the P1-coated DNA chip and the control slide before and after hybridization with the HEX- labeled complementary DNA (c-DNA-HEX). Upon hybrid- ization tests with c-DNA-HEX on the signal-amplifying P1- immobilized DNA chip, one can observe a large signal amplification. The fluorescence emission of P1 was decreased when excited at 380 nm, whereas the emission of HEX was significantly amplified. Direct excitation of HEX at 535 nm produced only a weak fluorescence emission as shown in Figure 4a. This large signal amplification clearly indicates an efficient fluorescence resonance energy transfer from P1 to HEX. The detection limit of our signal-amplifying DNA microarray is 10 À10 m (see the Supporting Information). We conducted the same hybridization test on the control slide. Direct excitation of HEX at its absorption maximum (l max ) of 535 nm produced the same weak fluorescence emission as obtained from the direct excitation of the P1-immobilized DNA chip at 535 nm. A selectivity test was also done with HEX-labeled one-mismatch DNA (5’-HEX-ACA CAT CTC GGA TGT-3’) and HEX-labeled noncomplemen- Figure 3. a) Schematic representation of the light-directed parallel on-chip DNA synthesis on P1-immobilized glass: i) APTMS, ii) 1,4-phenyl- enediisothiocyanate, iii) polymer (P1), and iv) cyclic procedures of oligo synthesis. b) A fluorescence image of a patterned signal-amplifying DNA microarray with two different DNA sequences after hybridization with a mixture of c-DNA-HEX (green) and c-DNA-Cy5 (red; scale bar: 200 mm). Angewandte Chemie 4669 Angew. Chem. Int. Ed. 2007, 46, 4667 –4670 # 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org

tary DNA (5’-HEX-TGT GTA GTG CCT ACA-3’). Fig- ure 4 b shows the relative fluorescence intensity of HEX on the complementary and one-mismatch DNA compared with that of the noncomplementary DNA, demonstrating the selectivity of the signal-amplifying conjugated polymer-based DNA microarray. In summary, we have established a fast and readily applicable strategy to make a signal-amplifying DNA micro- array by developing a novel conjugated polymer and combin- ing it with an efficient and convenient on-chip DNA synthesis. The newly developed conjugated poly(oxadiazole-co-phenyl- ene-co-fluorene) is highly emissive and has unique stability in harsh environments. DNA hybridization tests showed a good selectivity and a large signal amplification achieved by efficient FRET from the emissive conjugated polymer to the dye-labeled target DNA. The results provide a design principle for further development of self-signal-amplifying DNA microarrays that possibly allow PCR-free DNA detec- tion through a large signal amplification. Received: January 30, 2007 Revised: April 6, 2007 . Keywords: conjugated polymers · DNA structures · FRET (fluorescence resonant energy transfer) · polyoxadiazoles · signal amplification [1] a) R. Ranger, D. A. Tirrell, Nature 2004, 428, 487 – 492; b) J. J. Storhoff, C. A. Mirkin, Chem.Rev. 1999, 99, 1849 – 1862; c) A. B. Sanghvi, K. P.-H. Miller, A.M. Belcher, C. E. Schmidt, Nat. Mater. 2005, 4, 496 – 502. [2] a) R. B. Breaker, Nature 2004, 432, 838 – 845; b) R. F. Service, Science 1998, 282, 396 –399; c) M. Schena, D. Shalon, R. W. Davis, P. O. Brown, Science 1995, 270, 467 – 470; d) G. McGall, J. Labadie, P. Brock, G. Wallraff, T. Nguyen, W. Hinsberg, Proc. Natl. Acad. Sci. USA 1996, 93, 13 555 – 13 560. [3] a) D. T. McQuade, A. E. Pullen, T. M. Swager, Chem. Rev. 2000, 100, 2537– 2574; b) P. S. Heeger, A. J. Heeger, Proc. Natl. Acad. Sci. USA 1999, 96, 12219 – 12221; c) K. Peter, R. Nilsson, O. Inganäs, Nat. Mater. 2003, 2, 419 – 424 ; d) H.-A. Ho, M. Boissinot, M.G. Bergeron, G. Corbeil, K. DorØ, D. Boudreau, M. Leclerc, Angew. Chem. 2002, 114, 1618 – 1621; Angew. Chem. Int. Ed. 2002, 41, 1548– 1551; e) B. Liu, G. C. Bazan, Proc. Natl. Acad. Sci. USA 2005, 102, 589 – 593 ; f) L. Chen, D. W. McBranch, H.-L. Wang, R. Helgeson, F. Wudl, D.G. Whitten, Proc. Natl. Acad. Sci. USA 1999, 96, 12 287 – 12 292; g) K. Haskins-Glusac, M.R. Pinto, C. Tan, K. S. Schanze, J. Am. Chem. Soc. 2004, 126, 14 964 – 14971; h) C. C. Pun, K. Lee, H.-J. Kim, J. Kim, Macromolecules , 2006, 39, 7461 – 7463. [4] a) S. P. Fodor, J. L. Read, M. C. Pirrung, L. Stryer, A. T. Lu, D. Solas, Science 1991, 251, 767 – 773; b) X. Gao, E. LeProust, H. Zhang, O. Srivannavit, E. Gulari, P. Yu, C. Nishiguchi, Q. Xiang, X. Zhou, Nucleic Acids Res. 2001, 29, 4744 – 4750 ; c) K. Komolpis, O. Srivannavit, E. Gulari, Biotechnol. Prog. 2002, 18, 641 – 646; d) J.-M. Rouillard, M. Zuker, E. Gulari, Nucleic Acids Res. 2003, 31, 3057 – 3062; e) X. Gao, X. Zhou, E. Gulari, Proteomics 2003, 3, 2135 – 2141. [5] A. P. Blanchard, R. J. Kaiser, L. E. Hood, Biosens. Bioelectron. 1996, 11, 687. [6] J. Cheng, E.L. Sheldon, L. Wu, A. Uribe, L.O. Gerrue, J. Carrino, M. J. Heller, J. P. O)Connell, Nat. Biotechnol. 1998, 16, 541 – 546. [7] a)O. J. Bolton, J. Kim, J. Mater. Chem. 2007, 17, 1981 – 1988; b) X.-C. Li, G. C. W. Spencer, A. B. Holmes, S. C. Moratti, F. Cacialli, R. H. Friend, Synth. Met. 1996, 76, 153 – 156 ; c) Z. Peng, Z. Bao, M. E. Galvin, Adv. Mater. 1998, 10, 680 – 684. [8] N. Miyaura, A. Suzuki, Chem. Rev. 1995, 95, 2457 – 2483. [9] L. J. McBride, M. H. Caruthers, Tetrahedron Lett. 1983, 24, 245 – 248. [10] X. Gao, P. Yu, E. Proust, L. Sonigo, J. P. Pellois, H. Zhang, J. Am. Chem. Soc. 1998, 120, 12 689 – 12 699. Figure 4. a) PL emission spectra of P1 substrate before (black) and after hybridization when excited at 380 nm (red) and 535 nm (blue); PL emission of the control (pink, excited at 535 nm). Note that the blue and pink lines are essentially superimposed and appear around 550–625 nm. Inset: Comparison of fluorescence intensity upon excita- tion at 535 nm (left) and 405 nm (right). Scale bar: 25 mm. b) Selectiv- ity test: A) perfect match, B) one mismatch, and C) random sequence. Inset: A microscanned image, from the top row down: perfect match, one mismatch, and random sequence. The spot diameter is 55 mm. I f = fluorescence intensity. Communications 4670 www.angewandte.org # 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2007, 46, 4667 –4670

Sorry, preview is currently unavailable. You can download the paper by clicking the button above.

References (28)

R. Ranger, D. A. Tirrell, Nature 2004, 428, 487 -492;
J. J. Storhoff, C. A. Mirkin, Chem. Rev. 1999, 99, 1849 -1862;
A. B. Sanghvi, K. P.-H. Miller, A. M. Belcher, C. E. Schmidt, Nat. Mater. 2005, 4, 496 -502.
R. B. Breaker, Nature 2004, 432, 838 -845;
R. F. Service, Science 1998, 282, 396 -399;
M. Schena, D. Shalon, R. W. Davis, P. O. Brown, Science 1995, 270, 467 -470;
G. McGall, J. Labadie, P. Brock, G. Wallraff, T. Nguyen, W. Hinsberg, Proc. Natl. Acad. Sci. USA 1996, 93, 13 555 -13 560.
D. T. McQuade, A. E. Pullen, T. M. Swager, Chem. Rev. 2000, 100, 2537 -2574;
P. S. Heeger, A. J. Heeger, Proc. Natl. Acad. Sci. USA 1999, 96, 12 219 -12 221;
K. Peter, R. Nilsson, O. Inganäs, Nat. Mater. 2003, 2, 419 -424;
H.-A. Ho, M. Boissinot, M. G. Bergeron, G. Corbeil, K. DorØ, D. Boudreau, M. Leclerc, Angew. Chem. 2002, 114, 1618 -1621; Angew. Chem. Int. Ed. 2002, 41, 1548 -1551;
B. Liu, G. C. Bazan, Proc. Natl. Acad. Sci. USA 2005, 102, 589 -593;
L. Chen, D. W. McBranch, H.-L. Wang, R. Helgeson, F. Wudl, D. G. Whitten, Proc. Natl. Acad. Sci. USA 1999, 96, 12 287 -12 292;
K. Haskins-Glusac, M. R. Pinto, C. Tan, K. S. Schanze, J. Am. Chem. Soc. 2004, 126, 14 964 -14 971;
C. C. Pun, K. Lee, H.-J. Kim, J. Kim, Macromolecules, 2006, 39, 7461 -7463.
S. P. Fodor, J. L. Read, M. C. Pirrung, L. Stryer, A. T. Lu, D. Solas, Science 1991, 251, 767 -773;
X. Gao, E. LeProust, H. Zhang, O. Srivannavit, E. Gulari, P. Yu, C. Nishiguchi, Q. Xiang, X. Zhou, Nucleic Acids Res. 2001, 29, 4744 -4750;
K. Komolpis, O. Srivannavit, E. Gulari, Biotechnol. Prog. 2002, 18, 641 -646;
J.-M. Rouillard, M. Zuker, E. Gulari, Nucleic Acids Res. 2003, 31, 3057 -3062;
X. Gao, X. Zhou, E. Gulari, Proteomics 2003, 3, 2135 -2141.
A. P. Blanchard, R. J. Kaiser, L. E. Hood, Biosens. Bioelectron. 1996, 11, 687.
J. Cheng, E. L. Sheldon, L. Wu, A. Uribe, L. O. Gerrue, J. Carrino, M. J. Heller, J. P. OConnell, Nat. Biotechnol. 1998, 16, 541 -546.
O. J. Bolton, J. Kim, J. Mater. Chem. 2007, 17, 1981 -1988;
X.-C. Li, G. C. W. Spencer, A. B. Holmes, S. C. Moratti, F. Cacialli, R. H. Friend, Synth. Met. 1996, 76, 153 -156;
Z. Peng, Z. Bao, M. E. Galvin, Adv. Mater. 1998, 10, 680 -684.
N. Miyaura, A. Suzuki, Chem. Rev. 1995, 95, 2457 -2483.
L. J. McBride, M. H. Caruthers, Tetrahedron Lett. 1983, 24, 245 - 248.
X. Gao, P. Yu, E. Proust, L. Sonigo, J. P. Pellois, H. Zhang, J. Am. Chem. Soc. 1998, 120, 12 689 -12 699.

A conjugated polyelectrolyte (CPE) [1,2] a conjugated polymer containing a charged (anionic or cationic) group, has received considerable attention for its bio-applications such as solution-based DNA sensor, [3-11] DNA microarray, [12-14] protein sensor, [15-24] bioimaging [25-28] as well as optoelectronic applications such as organic semiconductors, [29-31] light-emitting devices, [32,33] and actuators. [34] The ionic side group plays an important role to provide CPEs with water-solubility that is central to many biological applications. In addition, maintaining the emissive property of a CPE in aqueous solution is another requirement for many biosensor applications because the merit of using conjugated polymers as a sensor is their amplified fluorescence signaling property upon environmental changes. [35-45] However, in this regard, CPE inherently has a critical solubility limitation in aqueous environment because the π-conjugated poly mer backbone is chemically hydrophobic and structurally rigid. The rigid-rod and hydrophobic nature of CPEs induces polymer aggregation via intermolecular hydrophobic interaction among the poly mer backbones in aqueous environment. [37,38,46] Therefore, the solubility of CPE in water is significantly low, consequently inducing significant decrease in the fluorescence quantum yield due to the aggregation-induced self-quenching. [47-51] Moreover, once CPEs are completely dried, it is tremendously difficult to re-dissolve them in water due to the rigid hydrophobic nature of the backbone and resulting strong and cohesive aggregation. Besides the solubility issue, there is another requirement for CPE to be useful for biological applications. To achieve efficient and convenient bio-conjugation between the CPE and a biological molecule, CPEs should have an efficient and convenient functional group such as a carboxylic acid group or an amine group. [52,53] Due to these demanding requirements, it remains a difficult task to develop highly emissive and completely water-soluble functional CPEs. Several research groups have developed CPE-based functional systems by utilizing the emissive property of CPEs. Leclerc et al. developed DNA sensors using cationically charged polythiophene derivatives. Charge-charge interaction between the cationic CPE and a single strand DNA and subsequent detection of the complementary DNA produces a conformation change of the CPE and consequent color change as a sensory signal. [7,15,54-57] Bazan and co-workers have reported signal amplifying biosensors based on cationically charged CPEs and fluorescence resonance energy transfer (FRET). [4] Schanze et al. investigated CPE systems and reported amplified fluorescence quenching of sulfonated CPEs due to π-π aggregation of the rigid linear CPEs in aqueous media. [37,38,40] Bunz and coworkers have investigated pH-dependent optical properties of CPEs by modulating carboxylic acid side-chain. [58] Furthermore, cationic gold nanoparticles conjugated with carboxylated CPEs

Signal-Amplifying Conjugated Polymer–DNA Hybrid Chips

Sign up for access to the world's latest research

Abstract

Related papers

References (28)

Related papers

Related topics

Cited by