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J. Phys. Chem. B 2008, 112, 7120–7122
Label-Free Detection of Sequence-Specific DNA with Multiwalled Carbon Nanotubes and Their Light Scattering Signals Li Zhang,† Cheng Zhi Huang,*,† Yuan Fang Li,† Sai Jin Xiao,† and Jian Ping Xie‡ College of Chemistry and Chemical Engineering, MOE Key Laboratory of Luminescence and Real-Time Analysis, CQKL-LRTA, Southwest UniVersity, Chongqing 400715, China, and Institute of Modern Biopharmaceuticals, College of Life Sciences, Southwest UniVersity, Chongqing 400715, China ReceiVed: January 5, 2008; ReVised Manuscript ReceiVed: February 26, 2008
A detection method for DNA sequence-specificity in a homogeneous medium is presented with multiwalled carbon nanotubes (MWCNTs) as optical probes on the basis of the measurements of light scattering signals. ssDNA can prevent MWCNTs from coagulation in electrolyte solution while dsDNA cannot, displaying different light scattering signals. With the light scattering signals, target DNA in the range of 8.6–86.4 nM could be detected and one base pair mismatch could be discriminated. The sequence specificity for the present method has been identified with PCR products. Introduction
Experimental Section 1991,1
there Since the finding of carbon nanotubes (CNTs) in have been a variety of reports concerning their acting as biological transporters and selective cancer cell destructors,2 nanovehicles for drug delivery,3 and biosensors of protein and DNA.4–6 Early research efforts on CNTs and DNA mainly focused on the interesting acting mode and interaction mechanism.7,8 Recently, CNTs have been widely applied to sensitive DNA detection,9,10 which is of central importance to the diagnosis of pathogenic and genetic diseases.11,12 For example, CNTs-modified electrodes in the context of electrochemical methods were employed to detect 16 nM target DNA through hybridization with the advantages of easy miniaturization and possible implementation on-chip.13 The electrochemistrybased methods, however, have drawbacks of fussy modification and washing steps. Although homogeneous optical methods could overcome these obstacles and exhibit high sensitivity, most of them require labeling and involvement in complicated operation procedures.14 From this perspective, a label-free method with simple operations is well-appreciated and urgently required. In this contribution, we report a label-free detection method for DNA sequence specificity in homogeneous solution using multiwalled carbon nanotubes (MWCNTs) as optical probes on the basis of the measurements of light scattering (LS) signals by simultaneously scanning the excitation/ emission monochromators of a common spectrofluorometer. Our method could detect target DNA lower than 8.6 nM with much easier operations and present a high capability to discriminate perfectly complementary targets (perfect CT) and one-base-mismatched DNA, characterizing a diagnostic advantage over the single nucleotide polymorphism (SNP) detection.15 * To whom correspondence should be addressed. E-mail: chengzhi@ swu.edu.cn. Fax: +86 23 68866796. Tel: +86 23 68254659. † CQKL-LRTA. ‡ College of Life Sciences.
Apparatus. The light scattering (LS) and fluorescence spectra and intensities were measured with a F-2500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). A vortex mixer QL901 (Haimen, China) was used to blend the solution. A highspeed TGL-16 M centrifuge (Hunan, China) was used to treat the sample prior to measurements. PCR amplification reactions were carried out in a DNA-Engine thermal cycler (Bio-Rad). Reagents. Oligonucleotide sequences were synthesized by Beijing SBS Genetech Co., Ltd. (Beijing, China), and they were used as received: unlabeled ssDNA probe, 5′-GAA CGA AAC CAT TAT ACG AT-3′; complementary oligomer, 5′-ATC GTA TAA TGG TTT CGT TC-3′; one-base-mismatched oligomer, 5′-ATC GTA TAA TTG TTT CGT TC-3′; two-base-mismatched oligomer, 5′-ATT TTA TAA TGG TTT CGT TC-3′. In addition, there are the following: fluorescein-labeled ssDNA probe, fluorescein-5′-GGT TGG TGT GGT TGG-3′; its complementary, 5′-CCA ACC ACA CCA ACC-3′. Mili-Q purified water (18.2 MΩ) was used throughout. MWCNTs were commercially purchased from Chengdu Organic Chemicals Co., Ltd. (Chengdu, China). The commercial MWCNTs were treated in accordance with the literature,16 and the concentration of final aqueous solution was about 0.122 mg/mL. PCR sequences were derived from genes coding for Bacillus glucanase and Mycobacterium tuberculosis glmS, and the PCR reaction systems involve the use of 10× buffer (without MgCl2) (2.5 µL), MgCl2 (25 mM; 1.5 µL), 4× dNTP (each 25 mM; 4 µL), P1 (10 µmol/ mL; 2 µL), P1 (10 µmol/mL; 2 µL), and pfu DNA polymerase (0.4 µL). Procedures. Hybridization of the ssDNA probe with its CT was conducted at 37 °C for 30 min in 10 mM Tris-HCl (pH 7.4) buffer solution containing 0.25 M NaCl. After the hybridization, dispersed MWCNTs were added into the solution, and the final concentration of NaCl was 0.2 M. The solution then stood for 30 min at room temperature followed by centrifugation (10000 rpm, 17800g) for 10 min. The supernatant was decanted and collected for LS measurements. The LS spectra were obtained by simultaneously scanning the excitation and emission monochromators of the F-2500 fluorescence spectrophotometer from 220 to 700 nm with the slit width for
10.1021/jp800092r CCC: $40.75 2008 American Chemical Society Published on Web 05/20/2008
Label-Free Detection of Sequence-Specific DNA
J. Phys. Chem. B, Vol. 112, No. 23, 2008 7121
SCHEME 1: Schematic of Light Scattering (LS) Detection of DNA Hybridization in Salt Solution:a (A) Stable MWCNTs Homogeneous Solution in the Presence of ssDNA; (B) Aggregation of MWCNTs in the Presence of dsDNAa
a The supernatants of MWCNTs suspensions were collected and transferred for LS measurements.
the excitation and emission of 5 nm. The PCR reaction was routinely performed with the following cycles (with minor changes such as temperature and cycles, if required): 95 °C soak for 5 min, followed by 30 cycles of 95 °C denature for 1 min, 58 °C anneal for 1 min, 72 °C extension for 3 min; after 30 cycles, 72 °C soak for 10 min. The PCR product was confirmed by sequencing (Tiangen Co.).
Figure 1. Concentration of Na+ for complete coagulation of MWCNTs suspension in the presence of ssDNA. As it shows, 0.2 M NaCl could induce the MWCNTs to coagulate completely (curve 1) in the absence of ssDNA, and if 1.08 × 10-7, 2.16 × 10-7, and 2.7 × 10-7 M ssDNA are present, the Na+ concentration for complete coagulation of MWCNTs suspension will increase to be 0.25 M (curve 2), 0.3 M (curve 3), and 0.35 M (curve 4), respectively.
Results and Discussion The protocol of our method is displayed in Scheme 1. MWCNTs were first purified and oxidized according to the literature.16 It has been reported that the suspension of oxidized MWCNTs will coagulate once the concentration of inorganic electrolyte in the medium exceeds the so-called critical coagulation concentration (ccc) owing to the charge screening effects.17 For our oxidization-treated MWCNTs, 0.2 M Na+ could induce them to coagulate completely, indicating that the negative surfaces of MWCNTs are completely screened and the adjacent MWCNTs come into contact because of van der Waals attraction.18 It was found that much higher concentration of Na+ is required to screen the negative charge on the surface of MWCNTs in the presence of single-stranded DNA (ssDNA) so as to make MWCNTs coagulate completely (Figure 1) since the π-π stacking of DNA bases along the sidewall of MWCNTs leads up to ssDNA wrapping on the surfaces of the MWCNTs,19 making the surfaces of MWCNTs much more negatively charged owing to the phosphate group of DNA.20,21 In such case, we could adjust the concentration of Na+ and make the ssDNAwrapped MWCNTs well suspended in the medium. Figure 2 shows the LS signals of the centrifugal supernatant are strong (curves 1 and 5 in Figure 2) after centrifugation at 10 000 rpm for 10 min (Scheme 1A) since the ssDNA-wrapped MWCNTs are well suspended in the supernatant. On the other hand, if stable double-stranded DNA (dsDNA), formed by the hybridization of ssDNA probe with its perfect CT under optimal concentration of Na+, was added into the oxidized MWCNTs suspension, coagulation of MWCNTs were observed visually. The reason is that dsDNA could not wrap along the sidewall of MWCNTs and no π–π stacking interaction exists between dsDNA and MWCNTs.21 Thus, the suspensions of MWCNTs in the presence of dsDNA were not stable in salt medium and formed some ropes that then settled down to the bottom of the vials after centrifugation, leaving behind a transparent supernatant (Scheme 1B). Therefore, the LS signals
Figure 2. LS spectra of supernatants for MWCNTs suspensions in the presence of ssDNA probes (curve 1), ssDNA probes/complementary targets (CT) complex (curve 4), ssDNA probes/one-base-mismatched targets complex (curve 3), and ssDNA probes/two-base-mismatched targets complex (curve 2) as well as salt buffer solution (curve 5). Both ssDNA probes and targets were kept at 1.08 × 10-7 M; the centrifuge rate was 10 000 r/min. The insert shows the LS intensities measured at 310 nm versus the CT DNA concentration, which could be expressed as I ) 2216.6 – 13.7cCT over the range of 8.6–86.4 nM with the correlation coefficient of –0.9988 and the limit of detection (3σ) around 2.9 nM.
of the centrifugal supernatant decrease greatly (curve 4 in Figure 2), though always a little higher than the blank (curve 5 in Figure 2), which could be ascribed to the error and is unavoidable. It was found that the intensities of LS signals of the centrifugal supernatants decrease linearly with the concentration of perfect CT DNA in the range 8.6–86.4 nM with the limit of detection around 2.9 nM (3σ) (the insert in Figure 2). Figure 2 also shows that the LS signals of the centrifugal supernatants of MWCNTs suspensions in the presence of probes/base-mismatched targets are stronger than that of the probes/perfect CT DNA and those of the probes/two-base-mismatched DNA are stronger than that of the probes/one-base-mismatched DNA (curves 2–4 in Figure 2). From these results, we could conclude that ssDNA has a dispersion effect on MWCNTs different from that of dsDNA,
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Zhang et al. same conditions for further testing. It was found that fluoresceinlabeled ssDNA probes could wrap around MWCNTs, resulting in remarkable fluorescence quenching due to the close proximity of fluorescein to the surface of MWCNTs. The fluoresceinlabeled ssDNA probes, if interacting with perfect CT and forming dsDNA, could not wrap around MWCNTs again, and fluorescence emission can only be weakly quenched (Figure 4). From the experiment, we can also conclude that even after modification by oxidization, MWCNTs have the property to form π–π stacking with bases of ssDNA and not with those of dsDNA. Conclusions
Figure 3. Difference of LS intensity at 310 nm after the addition of ssDNA probes to the PCR amplicons (red column) or to the mixture of PCR amplicons and complementary target DNA (green column).
In summary, ssDNA can prevent MWCNTs from coagulation in electrolyte solution while dsDNA cannot, displaying different LS signals that could be applied for the detection of DNA hybridization, even if in the case of one base pair mismatch. By employing thermally denatured PCR amplicons as noncomplementary target DNA, we could identify the sequencespecificity capability. Furthermore, the present assay provides a way for fluorescence change as a result of hybridization using labeled ssDNA probes, which could further confirm the mechanism proposed. Acknowledgment. This work was supported by the National Natural Science Foundation of China (NSFC, Grant Nos. 20425517, 20675065) and the Ministry of Education of China (Grant No. 20060635003). References and Notes
Figure 4. Fluorescence detection of hybridization: curve 1, fluoresceinlabeled ssDNA probes; curve 2, mixture of MWCNTs, ssDNA probes, and complementary targets; curve 3, mixture of MWCNTs and ssDNA probes. All DNA was kept as 7.0 × 10-7 M.
just like previous reports that ssDNA could disperse singlewalled carbon nanotubes (SWCNTs), while dsDNA makes SWCNTs coagulate completely.19–21 To demonstrate the capability of this detection method for sequence specificity, two PCR amplicons derived from Mycobacterium tuberculosis glmS and genes coding for Bacillus glucanase were employed. These PCR amplicons were exposed to thermal denaturation to obtain single-stranded species and then used as samples of noncomplementary target DNA. In the presence of the resulted PCR amplicons, LS signals of centrifugal supernatant of MWCNTs were strong due to its wrapping around MWCNTs and increase with the addition of ssDNA probes (positive ∆I value in Figure 3), showing that the dissolution of MWCNTs improves due to the increase of ssDNA content. On the other hand, the LS intensity of the centrifugal supernatant of MWCNTs suspension decreases with addition of ssDNA probes if it is the complementary target DNA contained in the PCR amplicons (negative ∆I value in Figure 3), indicative of the successful detection of target DNA, demonstrating its sequence-specificity capability. It should be noted that all above experiments were conducted on the basis of the fact that ssDNA and dsDNA have different dispersion effects on MWCNTs. To confirm the mechanism, fluorescein-labeled ssDNA probes were employed under the
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