pubs.acs.org/Langmuir © 2009 American Chemical Society
Highly Sensitive and Selective Label-Free Optical Detection of DNA Hybridization Based on Photon Upconverting Nanoparticles Manoj Kumar and Peng Zhang* Department of Chemistry, New Mexico Tech, Socorro, New Mexico 87801 Received March 17, 2009. Revised Manuscript Received April 17, 2009 We report a label-free optical detection scheme for DNA hybridization using photon upconverting nanoparticles. On the basis of luminescence resonance energy transfer between a donor and an acceptor, the method is highly sensitive and can differentiate targets with single base variation. Photon upconverting nanoparticles were used as the donor and an intercalating dye as the acceptor. The sensor could differentiate the perfectly matched target from the single-base mismatched target. The detection limit of this sensor toward perfectly matched target was calculated to be 20 fmol, with no photobleaching. Oligonucleotide sensors of such design demonstrate high sensitivity and specificity without fluorophore labeling.
Introduction Recent advances and demands in the field of molecular diagnostics, molecular medicine, and forensics have propelled the development of highly selective and sensitive nucleotide sensors. Reduced sample volumes and low analyte concentrations have presented a challenge to the current detection technology. DNA hybridization-based detection is a major technique for the diagnosis of genetic disease, where clinical symptoms are linked to DNA alterations.1 DNA hybridization is highly specific and becomes very sensitive when coupled with an optical detection scheme. Optical detection schemes usually require some kind of labeling such as the use of molecular beacons2,3 or fluorophore tagging.4 Typically, organic fluorophores and quantum dots are used for signaling the detection of the hybridization of DNA.5 These fluorophores are usually excited at shorter wavelengths and emit at longer wavelengths. The labeling process could sometimes become very challenging, costly, and time-consuming, and may lead to occlusion of hybridization sites due to steric hindrance. Thus, label-free detection schemes would bring about the obvious benefits of being simple, quick, easy, and cost-effective.6 There have been a number of label-free optical detections reported in the literature, such as well-known surface plasmon resonance (SPR), whispering-gallerymode (WGM) sensing,7 and conjugated polymers amplification with the use of intercalating dye, among others.8,9 Photon upconversion is a phenomenon where, upon the excitation of low-energy photons, high-energy photons are emitted. This is achieved through multiphoton processes. Materials with photon upconverting properties are much less common *Corresponding author. Peng Zhang, Tel: 575-835-6192. Fax: 575-8355364. Email:
[email protected]. (1) Weiss, S. Science 1999, 283, 1676. (2) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303. (3) Zhang, P.; Beck, T.; Tan, W. Angew. Chem., Int. Ed. 2001, 40(2), 402. (4) Zhao, X.; Tapec-Dytioco, R.; Tan, W. J. Am. Chem. Soc. 2003, 125, 11474. (5) Edgar, R.; McKinstry, M.; Hwang, J.; Oppenheim, A. B.; Fekete, R. A.; Giulian, G.; Merril, C.; Nagashima, K.; Adhya, S. Proc. Natl. Acad. Sci. U.S.A. 2006, 103(13), 4841. (6) Cooper, M. A. Anal. Bioanal. Chem. 2003, 377, 834. (7) Arnold, S.; Khoshsima, M.; Teraoka, I.; Holler, S.; Vollmer, F. Opt. Lett. 2003, 28, 272. (8) Wosnick, J. H.; Mello, C. M.; Swager, T. M. J. Am. Chem. Soc. 2005, 127, 3400. (9) He, F.; Tang, Y.; Yu, M.; Feng, F.; An, L.; Sun, H.; Wang, S.; Li, Y.; Zhu, D.; Bazan, G. C. J. Am. Chem. Soc. 2006, 128, 6764.
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DOI: 10.1021/la900936p
than their down-converting counterparts.10 This makes them ideal for the identification of trace amounts of target molecules, since a significantly high signal-to-noise ratio can be achieved when the photon upconverting materials are used for sensing and luminescent reporting. NaYF4:Yb3+ (doped with Er3+ or Tm3+) have been recognized as one of the most efficient photon upconverting phosphors. When they are excited by an infrared (∼980 nm) source, strong visible bands appear around 477, 537, and 650 nm.11 There have been reports of photon upconverting phosphor particles as a means to detect single-stranded nucleic acid, where these particles were used as direct labeling agents.12,13 In our previous reports,14,15 we showed the design of a sandwich assay format based on the energy transfer between the photon upconverting nanoparticles and an appropriate fluorophores, each labeled to a DNA strand. The design was shown to display high sensitivity and could distinguish targets with single-nucleotide variation. In this report, we propose another detection scheme based on photon upconverting nanoparticles, which is simpler with only one capturing probe, without fluorophore labeling, and with higher sensitivity and specificity. The design may have profound implication on a broad spectrum of oligonucleotide assay applications.
Experimental Section Chemicals and Materials. NaF, YCl3 3 6H2O, YbCl3 3 6H2O, Tm(NO3)3 3 5H2O, diethylenetriaminepentaacetic acid (DTPA), and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich. 2-(4-Morpholino)-ethane sulfonic acid (MES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC) were purchased from Fisher Scientific. Phosphate buffer solution was prepared from KH2PO4 and K2HPO4. SYBR Green I was purchased from Invitrogen. All DNA strands were (10) Auzel, F. E. Chem. Rev. 2004, 104, 139. (11) Heer, S.; Kompe, K.; Gudel, H. U.; Haase, M. Adv. Mater. 2004, 16(23), 2102. (12) van de Rijke, F.; Zijlmans, H.; Li, S.; Vail, T.; Raap, A. K.; Niedbala, R. S.; Tanke, H. J. Nat. Biotechnol. 2001, 19, 273. (13) Corstjens, P. L. A. M.; Zuiderwijk, M.; Nilsson, M.; Feindt, H.; Niedbala, R. S.; Tanke, H. J. Anal. Biochem. 2003, 312, 191. (14) Zhang, P.; Rogelj, S.; Nguyen, K.; Wheeler, D. J. Am. Chem. Soc. 2006, 128, 12410. (15) Kumar, M.; Guo, Y.; Zhang, P. Biosens. Bioelectron. 2009, 24, 1522.
Published on Web 4/28/2009
Langmuir 2009, 25(11), 6024–6027
Letter Table 1. Probe and Target DNA Sequences oligonucleotide DNA_probe Tar_match Tar_mis
sequence 0
5 -/AmineC6/TTC TCC +ACA GGA-30 50 - GGT GCA CCT GAC TCC TGT GGA GAA G -30 50 - GGT GCA CCT GAC TCC TGA GGA GAA G -30
purchased from Integrated DNA Technologies Inc. (Coralville, IA). All chemicals were used as received without further purification. TEM grids were from Electron Microscopy Sciences, PA. Oligonucleotide Sequences. The sequences of several singlestranded DNAs involved are listed in Table 1. DNA_probe was based on the target sequence of Homo sapiens mutant hemoglobin beta chain (HBB) gene (AY356351), available at NIH’s NCBI GenBank (NCBI). The sequence of Tar_match was a segment of the HBB gene. The sequence of Tar_mis was a segment of the variation of HBB, responsible for the sickle cell disease. DNA_probe is amine-modified at the 50 -end, with its sequence complementary to a portion of the target DNA, Tar_match. Tar_mis differs from Tar_match by only a single base, at the eighth-base position from the 30 -end as indicated by the underline. The strand of DNA_probe includes a locked nucleic acid (LNA)-modified oligonucleotide (+A), as indicated by the ‘+’ sign in the front of the nucleotide.
Synthesis of Photon Upconverting Nanoparticles and Their Attachment at DNA Probe. The NaYF4:Yb3+,Tm3+ nanoparticles were synthesized using hydrothermal method. In brief, 407.5 μL of YCl3, 85 μL of YbCl3, and 7.5 μL of TmCl3 (all of 0.2 M) were mixed together in a Teflon container along with 500 μL of 0.2 M DTPA and 5 mL of DI water. After 25 min 1.5 mL of 0.83 M NaF was added under continuous stirring. The Teflon container was then placed inside an autoclave, and heated at around 160 °C for 19 h. The resulting nanoparticles were collected by adding large volume of acetone and then centrifuging at 14 000 rpm for 15 min. Collected nanoparticles were washed several times with acetone and dried before further use. The NaYF4:Yb3+,Tm3+ nanoparticles are well-dispersed in aqueous solution. Procedures to attach the amine-modified DNA (DNA_probe) to the photon upconverting nanoparticles were as follows: 55 mg of the nanoparticles were mixed with 54 mg of EDC in 550 μL of MES buffer (100 mM, pH 4.5) under stir. 1080 μL of 50 μM of DNA_ probe was then added to the mixture. The mixture was continuously stirred for 2 h before 500 μL of 100 mM phosphate buffer (pH 8.0) was added. A large amount of acetone was next added to the mixture to precipitate the nanoparticles out of the solution and collected via centrifuge. The conjugated nanoparticles were subsequently washed with 100 mM phosphate buffer (pH 8.0) for three to four times, before being left to dry in the oven overnight and weighed. The weighed conjugated particles were then suspended in 600 μL of 100 mM phosphate buffer solution (pH 7.0, 1 mM EDTA) and stored at 4 °C for later use. TEM Characterization. The NaYF4:Yb3+,Tm3+ nanoparticles conjugated with DNA probes were first suspended in methanol through sonication to ensure the extraction of representative dispersion volumes. Then, a drop of suspension was deposited on a Formvar-covered carbon-coated copper grid and let to dry at room temperature. TEM images were taken on a JEOL 2010 high-resolution transmission electron microscope. Photoluminescence Measurements. The photoluminescence measurements using DNA_probe/nanoparticle complex were carried out at 27 °C using a spectrofluorometer (Photon Technologies International) equipped with a Hamamatsu R928 photomultiplier tube (PMT) operating at 1.1 kV. Instead of using the built-in Xenon lamp as the illumination source, a 975 nm diode laser (CrystaLaser, NV) was attached to the side port of the spectrofluorometer, and used to excite the sample. The output of the laser was set at 400 mW for all measurements. The emission slit was set at 4 nm for all measurements. Langmuir 2009, 25(11), 6024–6027
For each measurement, 200 μL of aforementioned solution was taken into a cuvette after sonicating the stock solution to ensure homogeneous suspension of nanoparticles in phosphate buffer. Then, 8.5 μL of 740 nM SYBR Green I solution was added to the cuvette, leading to a final dye concentration of 30 nM. The mixture was treated as blank, and photoluminescence spectrum under IR illumination was taken. The SYBR Green I emission at 512 nm (denoted as Iblank) was measured. An increment of 1 μL of the target DNA (50 nM of Tar_match or Tar_mis) was then added to the blank, and SYBR Green I emission (denoted as Isignal) was again measured while the sample was being illuminated by the IR laser. There was a 10 min waiting period between every addition of the DNA targets and the measurement, to allow for complete hybridization. All samples were under constant stirring while photoluminescence measurements were carried out.
Results and Discussion Characterization of the Photon Upconverting Nanoparticles. The homemade photon upconverting nanoparticles were characterized both spectroscopically and microscopically. Figure 1 shows a typical photoluminescence spectrum of the NaYF4:Yb3+,Tm3+ nanoparticles, upon the excitation by a
Figure 1. Photoluminescence spectrum of the photon upconverting nanoparticles containing Tm3+ excited at 975 nm and excitation/emission spectra of SYBR Green I.
Figure 2. TEM image of photon upconverting nanoparticles conjugated with DNA probes. DOI: 10.1021/la900936p
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Figure 3. Schematic of the nucleotide sensor design.
diode laser of around 975 nm. The excitation and emission spectra of SYBR Green I are also included in the figure for comparison. Figure 2 is a TEM image of the nanoparticles conjugated with DNA probes, which shows the particle size ranging from 20 to 100 nm, with an average of 45 nm (calculated by averaging 50 nanoparticles randomly selected in the TEM image). Scheme of the Detection. The schematic of this design is shown in Figure 3. The underlying principle for the detection is luminescence resonance energy transfer. The selection of the photon upconverting nanoparticles and the corresponding intercalating dye is based on the fact that the emission wavelength of the nanoparticle (energy donor) overlaps with the absorption of the intercalating dye (energy acceptor), while the emission wavelength of the dye does not overlap with any of the emission wavelength of the nanoparticle. The photon upconverting nanoparticles used in this study are NaYF4:Yb3+,Tm3+ nanoparticles, emitting blue color (477 nm) when excited by an infrared laser of 975 nm. NaYF4:Yb3+,Tm3+ nanoparticles can pair with a commonly used intercalating dye, SYBR Green I, as energy donor/acceptor. As shown in Figure 1, the strong blue emission band of Tm3+-doped photon upconverting nanoparticles (when excited at 975 nm) falls well within the excitation band of SYBR Green I, while there is a window of wavelength (
