Counting ssDNA on a Single Nanoparticle F. Delport * , J.-i. Hotta † , A. Deres † , J. Pollet * , B. Sels ‡ , J. Hofkens † and J. Lammertyn * * Dept. of Biosystems - MeBioS, KULeuven, Leuven, Belgium Email: filip.delport@biw.kuleuven.be † Dept. of Chemistry, Molecular and Nanomaterials, KULeuven, Leuven, Belgium ‡ Dept. M 2 S, Centre for Surface Chemistry and Catalysis, KULeuven, Leuven, Belgium Abstract— This paper describes the immobilization and quan- tification of 15base ssDNA on 250nm silica nanoparticles. 1 up to 2400 amine functionalized ssDNA are coupled to a carboxyl functionalized nanoparticle using EDC/NHS chemistry. The amount of ssDNA on the nanoparticles is quantified by bulk fluorescence measurements in a microtiterplate reader on both the nanoparticles in dense solution and the supernatant. To determine few molecules of ssDNA on each nanoparticle Single Molecule detection techniques were applied. Single molecule confocal microscopy focuses a laser on one nanoparticle and pho- tobleaches fluorescent dyes stochastically, thus enabling a precise counting from 1 up to 6 molecules on a single nanoparticle. These measurements revealed 2/3th of the ssDNA present in comparison with the bulk measurements. Wide field total internal reflection fluorescence microscopy showed the 1/3th missing ssDNA which is immobilized perpendicular to the sample surface. Thus, this method suggests a precise, complete and oriented counting of molecules from single molecule up to bulk level on nanoparticles. I. I NTRODUCTION The development of biofunctionalized nanomaterials has been a driving force for innovative applications in the elec- tronic, chemical, biotechnology and medical industries. The control over fabricating and functionalizing these nanoma- terials is paramount when applications are brought to the market. Functionalized nanoparticles (NP) are of great interest in the field of life sciences for their diagnostic properties as a miniaturized biosensor and as drug delivery vessels. NP have been conjugated with a variety of biomolecules such as proteins, enzymes and antibodies [1], [2], [3], [4]. DNA functionalized NP attract the attention because of their inherent specific and reversible base pairing with complementary DNA strands and for use with aptamers. The quantification of few biomolecules on NP is a difficult task. Most quantifications are executed on the supernatant which is difficult, inaccurate or even irrelevant. Measuring directly on the NP is more exact, but more complex. Still nanomaterials are expensive or only available in small quantities. The common direct detection techniques require massive amounts of sample, e.g. TGA, AES, HPLC, IR, .... An answer for characterizing these new materials on a relevant scale is Single Molecule detection techniques. Thus far Single Molecule Confocal Microscopy (SMCM) has been used to detect a number of proteins on a NP [5]. Sofar this approach has not been carried out to quantify the number of ssDNA strands immobilized to an single NP. The objective of this paper is to use Single Molecule Confocal Microscopy and total internal reflection Fluorescence Microscopy to accurately determine the number of immobilized ssDNA molecules at the NP surface. These results will be compared to bulk fluorescence measurements on the supernatant and the NP surface. II. MATERIALS AND METHODS A. Reagents Carboxyl functionalized silica NP (300 nm) were purchased from micromod (Rostock-Warnemuende, Germany). 5’-amine and 3’-Atto 647N functionalized 15base ssDNA from Euro- gentec (Luik, Belgium) is prepared to 5 μM stock solutions. All chemicals were purchased from Sigma-Aldrich (Bornem, Belgium), unless stated otherwise. The coupling reaction was performed with an activator EDC (Pierce Biotechnology, Rockford, USA) and a stabilizer NHS in 25 mM MES buffer (2.13 g 2-(N-morpholino)ethanesulfonic acid in 400 mL MQ water). Ethanolamine (Pierce Biotechnology, Rockford, USA) acted as a back coating layer to reduce non specific binding. PBS buffer was prepared by dissolving a foil pouche phosphate buffered saline in 1L MQ water and adapting the pH to 7.4 with sodium hydroxide. B. Immobilization Procedure The NP are washed by dissolving them in MES or PBS buffer in an overtop shaker PTR-30(Grant-Bio, Cambridge, UK), centrifugating with a Galaxy14D centrifuge (VWR, Haasrode, Belgium) and removing the supernatant. All reac- tions were performed in 500 μL with 2.5 mg nanoparticles. The Carboxyl modified NP were activated in the pH9 MES buffer by 12.5 mg/mL EDC and stabilized by 12.5 mg/mL NHS for 2h in an overtop shaker. All together C6 amine functionalized 15 base length ssDNA was added with the EDC/NHS mixture to react for 2h. To block non-specific bind- ing, after three MES washing rounds, ethanolamine was added to a final concentration of 50 mM for 30 minutes together with a renewed EDC/NHS mixture to quench the reaction and form a hydrophilic, uncharged surface. Subsequently the NP were washed and stored in PBS until the measurements. To quantify the number of DNA molecules bound to the NP, all samples were diluted to 1 mL. Bulk measurements were performed with a microtiterplate reader Spectramax M2e(Molecular Devices, Sunnyvale, USA). All measurements were carried out on the supernatans for the unbound fraction and on the NP in solution for the bound fraction. The Atto 647N labeled DNA is detected in a microtiterplate reader with 4 repeats of 200 μL at excitation wavelength of 600 nm, emission at 664 nm and cut-off filter at 630 nm. 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