Talanta 86 (2011) 200–207 Contents lists available at SciVerse ScienceDirect Talanta jo u r n al hom epage: www.elsevier.com/locate/talanta Functional magnetic nanoparticle-based trapping and sensing approaches for label-free fluorescence detection of DNA Ju-Yu Lin, Yu-Chie Chen ∗ Department of Applied Chemistry, National Chiao Tung University, Hsinchu 300, Taiwan a r t i c l e i n f o Article history: Received 18 June 2011 Received in revised form 26 August 2011 Accepted 29 August 2011 Available online 17 September 2011 Keywords: DNA Label free sensing Fluorescence MALDI-MS Magnetic nanoparticles a b s t r a c t In this study, a label-free fluorescence detection method for DNA was designed using functional magnetic nanoparticles (MNPs) as affinity probes. With the advantage of magnetic feature, MNP-based affinity probes can be easily manipulated for trapping and sensing target species. Two types of MNP-based nanoprobes for trapping and detecting target DNAs were fabricated. The basic strategy for this approach is the use of trapping probes to concentrate target DNAs selectively from complex samples. The detec- tion probes are then used as fluorescence reporters to explore the level of the target species. Trapping probes were constructed by covalently immobilizing probe DNA molecules complementary to the tar- get DNA. Detection nanoprobes were made by linking a fluorescent dye, riboflavin-5 ′ -monophosphate (RFMP), onto the surface of the core/shell Fe 3 O 4 @Al 2 O 3 MNPs via Al-phosphate chelation. The fluores- cence derived from RFMP molecules became invisible when molecules were attached onto the MNP surface. However, after phosphorylated species (e.g., DNA molecules) replaced RFMP from the surface of the RFMP-Fe 3 O 4 @Al 2 O 3 MNPs under microwave heating for 15 s, the RFMP molecules released from the MNPs enhanced the fluorescence intensity in the solution. Based on the measurement of the fluorescence intensity, the level of target DNA in the samples was determined. The remaining DNA molecules on the RFMP-Fe 3 O 4 @Al 2 O 3 MNPs were characterized by using matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The detection limit for DNA was as low as 40 pM using this approach. © 2011 Elsevier B.V. All rights reserved. 1. Introduction DNA carries informative genetic codes. Thus, it has been used as the main target in biodiagnostics and biochemical analyses [1–3]. Electrochemistry and optical spectroscopy have been used as analytical tools for DNA detection [1–6]. For high throughput DNA analysis, DNA chips are the typical choice [7–11]. How- ever, fluorescence-labeled nucleic acid probes are usually required [9,10]. Furthermore, amplifying DNA prior to chip analysis may be necessary because of its insufficient sensitivity. Although real-time polymerase chain reaction (PCR) can provide the required sensi- tivity for DNA detection [12], it is generally unsuitable for high throughput analysis because of its time-consuming experimen- tal steps and the high cost of reagents. Alternatively, analytical methods based on label-free fluorescence detection [8,13–15], and molecular beacons [9,16,17] have recently attracted much atten- tion. For example, intercalating dyes like ethidium bromide [15] have been demonstrated as highly sensitive toward double strand DNA. Combining the ability to concentrate traces of target DNA ∗ Corresponding author. Tel.: +886 3 5131527; fax: +886 5723764. E-mail address: yuchie@mail.nctu.edu.tw (Y.-C. Chen). from sample solutions and eliminating unwanted interference are desirable. Developing new methods to improve analysis perfor- mance can be beneficial for DNA analysis. The rapid growth of nanotechnology has opened new avenues for progress in DNA-sensing methods. Functionalized gold nanoparticles (NPs) immobilized with DNA probes have been used extensively in the detection of complementary target DNA based on observations of color changes in NPs [1,6]. Addition- ally, functional magnetic nanoparticle (MNP)-based approaches have recently received much attention [18–21]. We herein pro- pose a new approach, whose basic strategy is the employment of two types of functional MNPs, trapping probes and detec- tion probes, with microwave heating in order to develop a rapid, simple, and sensitive method for DNA analysis. Specifically, two types of MNP-based nanoprobes, one for DNA trapping and the other for DNA detection, were fabricated. With advantages on magnetic property, the trapping nanoprobes were easily used in the selective concentration and isolation of target DNAs from the complex samples. Detection probes were used as activated fluorescence reporters for the target DNA species that were selec- tively concentrated by the magnetic trapping probes. Iron oxide MNP-based nanoprobes for targeting DNA (Fe 3 O 4 @DNA MNPs) were constructed onto the surface of the MNPs by covalently 0039-9140/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2011.08.061