Journal of Membrane Science 328 (2009) 97–103 Contents lists available at ScienceDirect Journal of Membrane Science journal homepage: www.elsevier.com/locate/memsci Preparation of fractioned DNA aptamer–Pt complex through ultrafiltration and the colorimetric sensing of thrombin Akon Higuchi a,b,∗ , Siou-Ting Yang a , Yi-Di Siao a , Pei-Vin Hsieh a , Hisashi Fukushima c , Yung Chang d , Wen-Yih Chen a,e,∗∗ a Department of Chemical and Materials Engineering, National Central University, Jhongli, Taoyuan 32001, Taiwan b Department of Reproduction, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan c OneCell, Inc., Nagahama Bio-incubation Center 6, 8-1281 Tamura, Nagahama, Shiga 526-0829, Japan d Department of Chemical Engineering, R&D Center for Membrane Technology, Chung Yuan Christian University, 200 Chung-Bei Rd., Chungli, Taoyuan 320, Taiwan e Institute of Systems Biology and Bioinformatics, National Central University, Jhongli, Taoyuan 32001, Taiwan article info Article history: Received 1 November 2008 Received in revised form 24 November 2008 Accepted 28 November 2008 Available online 3 December 2008 Keywords: DNA Ultrafiltration Aptamer Platinum complex Colorimetric sensing DNAzyme abstract DNA aptamers were combined with platinum complexes to form DNA–Pt complexes, which exhibited peroxidase enzymatic activity while retaining the specific binding ability of aptamers. The DNA–Pt complexes were fractioned by size through ultrafiltration membranes having several molecular weight cut-offs, and the peroxidase enzymatic activities of these fractions were compared. The enzymatic activ- ity was highest in the filtrate of DNA–Pt complex solutions prepared with cisplatin and K 2 [PtCl 4 ] after ultrafiltration through membranes having MWCO=300,000. These showed 1.2-fold and 1.6-fold higher activity, respectively, than the corresponding unfractioned complexes. Sandwich-type DNAzyme-linked aptamer assays (DLAAs) using unfractioned or fractioned DNA–Pt complexes successfully detected the target protein thrombin. DLAAs incorporating a DNA–Pt (K 2 [PtCl 4 ]) complex fractioned through ultrafiltration membranes having MWCO = 300,000 showed the highest sensitivity among DLAAs made using fractioned DNA–Pt complexes, and had a 13-fold higher sensitivity than those made with unfractioned DNA–Pt complexes. © 2008 Elsevier B.V. All rights reserved. 1. Introduction DNA can recognize not only complementary sequences of DNA [1,2], but also low-molecular weight molecules [3,4] and proteins [5,6]. Such DNA, termed DNA aptamers, can bind a variety of target molecules [3–8] and animal cells [9,10] with high affinity and speci- ficity. DNA aptamers have found growing interest as therapeutic and diagnostic agents [11–13], and as recognition sites for biosens- ing [14–16]. DNA aptamers can potentially be used in bioassays in medicine and bioengineering research by labeling and/or combina- tion with hemin [17–20] or nanoparticles [21–24], which generate and amplify signals when they bind to their targets. Guanine-rich DNA aptamers can form hemin-containing DNAzymes, which can act as peroxidases [17–20]. Such DNAzymes ∗ Corresponding author at: Department of Chemical and Materials Engineering, National Central University, Jhongli, Taoyuan 32001, Taiwan. Tel.: +886 34227151x34253; fax: +886 34227151x34253. ∗∗ Corresponding author at: Institute of Systems Biology and Bioinformatics, National Central University, Jhongli, Taoyuan 32001, Taiwan. Tel.: +886 34227151x34253; fax: +886 34227151x34253. E-mail addresses: higuchi@ncu.edu.tw (A. Higuchi), wychen@ncu.edu.tw (W.-Y. Chen). could be used for the colorimetric or chemiluminescent detection of nucleic acids, and to follow the activity of telomerase, a versatile marker for cancer cells [25,26]. Rojas et al. have reported that a complex formed between hemin and a congruous oligonucleotide not only displayed enhanced per- oxidase activity but also resulted in a biocatalyst (DNAzyme) with regio-, enantiomeric, and diastereomeric selectivity for heme [27]. Polsky et al. have developed a method to amplify DNA and protein biosensing by employing Pt nanoparticle labels on DNA and DNA aptamers as catalysts for the reduction of H 2 O 2 [24].A DNA aptamer of thrombin conjugated with Pt nanoparticles was attached to an electrode, allowing the electrocatalytic detection of thrombin with a sensing limit of 1 nmol/L [24]. In our previous studies [28,29], we have reported that a DNA–Pt complex showed peroxidase enzymatic activity. The DNA–Pt com- plex was prepared from the simple reaction of DNA with K 2 [PtCl 4 ] in aqueous solution, and provided peroxidase enzymatic activ- ity without functionalization of DNA with biotin, thiols or other reactive groups. This indicated that any type of DNA could be trans- formed into a DNAzyme by reaction with a Pt complex under appropriate conditions. Therefore, we developed DNA aptamers carrying Pt nanoparticles prepared by the reaction of DNA aptamers (without functionalization with biotin, thiol or other reactive 0376-7388/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2008.11.045