Colorimetric detection of clinical DNA samples using an intercalator-conjugated polydiacetylene sensor
Graphical abstract
A 9-aminoacridine (9AA) intercalator-modified polydiacetylene (PDA) chromatic sensor containing diacetylene monomer and phospholipid is developed to detect dsDNA amplified from genomic DNA, which is visualized by a distinct color change caused by the insertion of the 9AA head group of PDA into dsDNA. The limits of detection of this method are 20 nM and sizes of around 100 base pairs.
Introduction
DNA biosensors that can be used to analyze clinical samples have been intensively investigated as potential tools for examining diseases caused by genetic defects (Chua et al., 2011, Thaitrong et al., 2010, Sassolas et al., 2008, Teles and Fonseca, 2008, Turner, 2013, Wang, 2000). Genetic analyses are conducted by nucleic acid hybridization (Wang, 2000) or by identifying the product of PCR amplification based on a genomic DNA template (Skuridin et al., 1996). Among the most frequently used methods for DNA detection is agarose gel electrophoresis, in which double-stranded (ds) DNA is visualized in the gel by adding a fluorescent intercalating dye such as ethidium bromide (EtBr) that is inserted between base pairs (Aaij and Borst, 1972, Vardevanyan et al., 2003). However, this method requires skill, time, and specialized equipment to resolve and monitor DNA fluorescence, thereby restricting its utility in point-of-care testing (POCT) or portable diagnostic kits.
Recent advances in DNA detection systems include the development of optical, electrochemical, and mass-sensitive methods (Sassolas et al., 2008, Teles and Fonseca, 2008, Wang, 2000). In particular, optical colorimetric sensors have the advantage of decreasing experimental costs and circumventing the relative complexity inherent in optical imaging and other detection methods by relying on an unaided visual readout instead of complicated instruments, making them amenable to on-site detection in real time (Chua et al., 2011, Sassolas et al., 2008, Teles and Fonseca, 2008). One of the most widely studied colorimetric techniques uses unmodified gold nanoparticles (AuNPs) as a sensor; dsDNA can be distinguished from unfolded, single-stranded (ss) DNA by aggregation-dependent color changes (Li et al., 2009, Li and Rothberg, 2004). However, this approach is still time-consuming and requires extensive training in the synthesis of AuNPs, and is less tolerant to interference than ligand-stabilized AuNPs.
With regards to portable and more efficient analytic sensors for detecting dsDNA in clinical samples, conjugated polymer-based sensors (Ho et al., 2002, Ho et al., 2005, Jung et al., 2008, Jung et al., 2010, Lee et al., 2007, Wang et al., 2006, Wang and Ma, 2005) have the important advantage of amplifying signals in response to external stimuli as compared to conventional sensors that are based on small molecules (Ali and Li, 2009, Du and Tang, 2011, Li et al., 2009, Li and Rothberg, 2004). The versatile and stable conjugated polydiacetylene (PDA) has useful structural and sensing properties when its head group is linked to ligands that can recognize external stressors such as pH, organic solvents, mechanical stress, and specific molecules. The unique and rapid chromatic change from blue to red can be directly perceived by the naked eye, which makes PDA a promising material for dsDNA detection that has the desired characteristics of simplicity and portability. Few studies have detected nucleic acids by exploiting the colorimetric properties of PDA. One group described a colorimetric method for detecting oligonucleotides that involved hybridization with probe oligonucleotide-functionalized PDA vesicles (Wang et al., 2006, Wang and Ma, 2005). However, synthetic oligonucleotides—and not clinical targets—were detected in this study. Only one study, carried out by our group, has directly detected clinical dsDNA amplified from genomic DNA using a primary amine-conjugated PDA colorimetric sensor (Jung et al., 2008). The main shortcoming of this sensor was low specificity for nucleic acids, because the principal sensing mechanism is based on relatively nonspecific ionic interactions between the positively charged PDA liposome and negatively charged nucleic acid.
We present here a novel approach based on intercalation that has greater specificity for clinical dsDNA detection. The positively charged planar aromatic dye 9-aminoacridine (9AA) was selected as a ligand for specific interaction with dsDNA. The 9-AA is inserted orthogonally into dsDNA without forming covalent bonds, and are sandwiched between two adjacent base pairs by both dispersive forces and electrostatic interactions (Ihmels and Otto, 2005, Medhi et al., 1999, Rao and Kollman, 1987). We modified the terminal carboxyl group of a diacetylene monomer with 9AA, and 9AA-functionalized liposomes consisting of diacetylene monomer/phospholipid assemblies were prepared and polymerized. The insertion of the 9AA head group attached to the surface of the resulting PDA vesicles into the PCR products of the Breast Cancer 1 gene (BRCA1) which is the best-known gene associated with breast cancer risk (Levy-Lahad and Friedman, 2007) created stress force on the PDA backbone, resulting in a color transition that was detectable by the naked eye. The sensor was used to assay dsDNA, yielding a linear relationship from 1 to 100 nM and a detection limit of 20 nM in 1 h, indicating satisfactory sensitivity. Morphologic transformations of PDA vesicles caused by interactions with DNA molecules were characterized based on images acquired by transmission electron microscopy (TEM). Importantly, our sensor satisfies the requirements of simplicity, rapidity, and low cost for potential use in POCT.
Section snippets
Materials
The diacetylene monomer 10,12-pentacosadiynoic acid (PCDA; Fig. 1) was purchased from GFS chemicals (Powell, OH, USA). The phospholipid 1,2-dimyristoyl-rac-glycero-3-phosphocholine (DMPC) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and the intercalator 9-aminoacridine (9AA) was obtained from Merck (Darmstadt, Germany). Solvents used in the experiments were of reagent grade. Sequences of primers used for PCR amplification of BRCA1 are shown in Table S1.
Synthesis of the diacetylene monomer PCDA-9AA
The intercalator-modified
Principle
The unique chromatic properties of PDA in response to biological stimuli were used to analyze genomic DNA samples obtained from breast cancer patients. The underlying principle is similar to that of agarose gel electrophoresis, in which DNA can be visualized with an intercalator dye such as EtBr (Aaij and Borst, 1972, Vardevanyan et al., 2003). The insertion of an intercalator-modified PDA head group into the DNA double helix causes stress-induced structural transformations of the PDA backbone
Conclusions
We developed a simple, rapid, and sensitive colorimetric PDA sensor to detect DNA obtained from clinical samples based on a modified intercalating dye. The terminal group of a PCDA monomer conjugated with the 9AA intercalator was used to prepare PDA vesicles composed of PCDA and DMPC. The successful detection of dsDNA by the chromatic sensor was visualized as a distinct color change caused by the insertion of the 9AA head group of PDA into the dsDNA molecule. This method has an LOD of 20 nM and
Acknowledgements
The authors thank Tae Won Kim for providing nucleic acids. This work was supported by the grant from Basic Science Research Program (NRF-2012R1A1A3015259), the BioSynergy Research Project (NRF-2014M3A9C4066457) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, and Center for BioNano Health-Guard funded by the Ministry of Science, ICT & Future Planning (MSIP) of Korea as Global Frontier Project (H-GUARD_2013M3A6B2078964).
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