Colorimetric Detection of DNA Using Unmodified Metallic

Anal. Chem. 2009, 81, 6122–6129

Colorimetric Detection of DNA Using Unmodified Metallic Nanoparticles and Peptide Nucleic Acid Probes Roejarek Kanjanawarut and Xiaodi Su* Institute of Materials Research and Engineering, ASTAR (Agency for Science, Technology and Research), 3 Research Link, Singapore 117602 We have developed a colorimetric assay for DNA detection based on the aggregation of unmodified metallic nanoparticles. Charge neutral peptide nucleic acids (PNA) are used as a “coagulant” of citrate anion-coated particles and as hybridization probe. In the absence of a complementary target DNA, free PNA molecules in solution induce aggressive particle aggregation because of the removal of charge repulsion as a result of PNA coating on nanoparticles. When a complementary DNA is present and PNA-DNA complexes are formed, the particles remain stable because the negative charges of the DNA strands in the complexes adsorbed on the particle surface ensure sufficient charge repulsions. In this method, no probe immobilization is needed and PNA-DNA hybridization occurs in a homogeneous phase. The assay results are displayed as rapidly as the changes in color and/or in UV-vis adsorption spectra of the colloidal solutions. We have validated the assay principle using gold- and silvernanoparticles (AuNPs and AgNPs), with the involvement of a shorter (13 mer) and a longer (22 mer) probe sequences. A specific DNA can be detected in the presence of at least 10 times of interference DNA, and the detection limit is at a DNA/PNA ratio of 0.05. When NaCl is added to accelerate the particle aggregation, the selectivity is further improved, and single-base-mismatch discrimination is achieved. A two-component assay using a mixture of AuNPs and AgNPs has also been constituted, aiming to improve the result accuracy by making use of the multiple aggregation signatures from the two types of particles. For single-base-mismatch discrimination, the AgNPs offer a higher sensitivity than AuNPs by showing more obvious spectra and color alternation, and the twocomponent assay offers three parameters in the UV-vis adsorption spectra. Colorimetric assays based on aggregation of metallic nanoparticles have received considerable attention in (bio)chemical analysis because of their simplicity, high sensitivity, and low cost.1,2 Numerous assays have been developed for detecting a wide * To whom correspondence should be addressed. E-mail: xd-su@ imre.a-star.edu.sg. Phone: 65-68748420. Fax: 65-68720785. (1) Zhao, W.; Brook, M. A.; Li, Y. ChemBioChem 2008, 9, 2363–2371. (2) Thaxton, C. S.; Georganopoulou, D. G.; Mirkin, C. A. Clin. Chim. Acta 2006, 363, 120–126.

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range of analytes (e.g., DNA,3-13 metal ions,14 small molecules,14-17 proteins,18 etc.), for studying enzymatic activities,19-23 and for measuring affinity interactions (e.g., protein-protein24 and peptide-heparin25). The innovation in this field includes development of new assay schemes using unmodified nanoparticles,10-13,17,21-25 exploitation of nanoparticles of various materials (e.g., silver5,9,22 and core-shell nanoparticles6) to achieve better sensitivity, and preparation of nanoparticle bioconjugates having a better stability for use in physiological conditions.9,24,25 Colorimetric DNA detection using metallic nanoparticles was started by Mirkin and co-workers upon their discovery and development of DNA-functionalized gold nanoparticles (AuNPs).3 They modified two sets of AuNPs with different single-stranded DNA probes and mixed them with a target DNA. If the target DNA contains sequences complementary to both the probes, it (3) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078–1081. (4) Cao, Y. C.; Jin, R.; Thaxton, C. S.; Mirkin, C. A. Talanta 2005, 67, 449– 455. (5) Thompson, D. G.; Enright, A.; Faulds, K.; Smith, W. E.; Graham, D. Anal. Chem. 2008, 80, 2805–2810. (6) Liu, S.; Zhang, Z.; Han, M. Y. Anal. Chem. 2005, 77, 2595–2600. (7) Sato, K.; Hosokawa, K.; Maeda, M. J. Am. Chem. Soc. 2003, 125, 8102– 8103. (8) Sato, K.; Hosokawa, K.; Maeda, M. Nucleic Acids Res. 2005, 33, e4. (9) Lee, J.; Lytton-Jean, A. K. R.; Hurst, S. J.; Mirkin, C. A. Nano Lett. 2007, 7, 2112–2115. (10) Li, H.; Rothberg, L. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14036–14039. (11) Li, H.; Rothberg, L. J. J. Am. Chem. Soc. 2004, 126, 10958–1096. (12) Cho, K.; Lee, Y.; Lee, C.; Lee, K.; Kim, Y.; Choi, H.; Ryu, P.; Lee, S. Y.; Joo, S. J. Phys. Chem. C 2008, 112, 8629–8633. (13) Rho, S.; Kim, S. J.; Lee, S. C.; Chang, J. H.; Kang, H.; Choi, J. Curr. Appl. Phys. 2009, 9, 534–537. (14) Zhao, W.; Chiuman, W.; Lam, J. C. F.; McManus, S. A.; Chen, W.; Cui, Y.; Pelton, R.; Brook, M. A.; Li, Y. J. Am. Chem. Soc. 2008, 130, 3610–3618. (15) Han, M. S.; Lytton-Jean, A. K. R.; Oh, B.; Heo, J.; Mirkin, C. A. Angew. Chem., Int. Ed. 2006, 45, 1807–1810. (16) Liu, J.; Lu, Y. Angew. Chem., Int. Ed. 2006, 45, 90–94. (17) Wang, J.; Wang, L.; Liu, X.; Liang, Z.; Song, S.; Li, W.; Li, G.; Fan, C. Adv. Mater. 2007, 19, 3943–3946. (18) Tessier, M.; Jinkoji, J.; Cheng, Y.; Prentice, J. L.; Lenhoff, A. M. J. Am. Chem. Soc. 2008, 130, 3106–3112. (19) Zhao, W.; Lam, J. C.; Chiuman, W.; Brook, M. C.; Li, Y. Small 2008, 4, 810–816. (20) Guarise, C.; Pasquato, L.; Filippis, V. D.; Scrimin, P. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3978–3982. (21) Zhao, W.; Chiuman, W.; Lam, J. C. F.; Brook, M. A.; Li, Y. Chem. Commun. 2007, 3729–3731. (22) Wei, H.; Chen, C.; Han, B.; Wang, E. Anal. Chem. 2008, 80, 7051–7055. (23) Oishi, J.; Asami, Y.; Mori, T.; Kang, J. H.; Niidome, T.; Katayama, Y. Biomacromolecules 2008, 9, 2301–2308. (24) Chen, Y.; Yu, C.; Cheng, T.; Tseng, W. Langmuir 2008, 24, 3654–3660. (25) Jeong, K. J.; Butterfield, K.; Panitch, A. Langmuir 2008, 24, 8794–8800. 10.1021/ac900525k CCC: $40.75  2009 American Chemical Society Published on Web 07/02/2009

Table 1. Scheme and Principle of Metallic Nanoparticle Based Colorimetic DNA Detection

will cause the particles to aggregate through sandwich hybridization. The aggregation is detectable as color change of the colloidal solution and red shift of the surface plasmon peak. This principle has been further adopted in the development of silver-,5,9 Ag/Au core-shell,4 and Ag/SiO2 core-shell6 nanoparticle-based DNA detection methods. Alternatively, if the DNA probes on two sets of DNA-AuNPs conjugate are designed to contain complementary sequences, direct conjugate-conjugate hybridization (no DNA cross-linker needed) will also induce particle aggregation, being useful for DNA detection.9 In general, all the above assays use a “interparticle cross-linking” aggregation mechanism.1 Another assay scheme involving DNA-AuNPs conjugates, but “non-crosslinking” aggregation mechanism, was developed by Sato et al.7,8 In their method, only one type of DNA-AuNPs conjugate is used. When a target DNA, being perfectly complementary to the probe in sequence as well as chain length, hybridizes to the DNA on AuNPs, the nanoparticles will alter their ability against salt induced aggregation. This is an example of using salt induced aggregation, driven by the London-van der Waals attractive force, as sensing principle. In the aforementioned assays, preparation of DNA-nanoparticle conjugates with well-controlled DNA surface coverage and stability is a critical step. Despite a large extent of success in this aspect,3,6,9,26 attempts to use unmodified metallic nanoparticles for colorimetric assays have been made, aiming to further increase the simplicity and robustness. Li and Rothberg10 found that singleand double-stranded oligonucleotides (ssDNA and dsDNA) have different electrostatic properties to protect AuNPs from salt induced aggregation that can be used for DNA detection without

covalent immobilization of DNA onto AuNPs. This method not only introduces additional simplicity but also eliminates DNA-DNA recognition on nanoparticles, which is slow and has a low efficiency, a higher hybridization efficiency and a faster color change. This assay principle has been further adopted for PCR product detection,11 single-base-mismatch detection,12 and ssDNA detection.13 Peptide nucleic acids (PNAs) are DNA analogues in which the entire sugar-phosphate backbone is replaced by a charge neutral polyamide backbone. The use of PNA for gold nanoparticle assembly has been reported by Chakrabarti and Klibanov.27 In their study, substantial efforts were made to prepare stable PNAAuNPs conjugates through optimization of PNA structures. The PNA-AuNPs conjugates were further used for DNA detection because the colloidal stability increases significantly upon DNA hybridization. Table 1 summarizes the assay schemes and aggregation mechanisms of existing metallic nanoparticle-based colorimetric DNA detection methods, covering so-called type I assays (using DNA- or PNA-modified nanoparticles) and type II assays (using unmodified particles), involving a “interparticle cross-linking” or “non-crosslinking” aggregation mechanism. In this study, we report a DNA colorimetric assay using unmodified metallic nanoparticles (type II) and moreover charge neutral PNA as probes based on our discoveries that citrate ionsprotected gold- and silver-nanoparticles undergo immediate aggregation in the presence of PNA molecules, and the aggregation can be retarded if PNA-DNA hybrids are formed. In this method, no PNA immobilization is needed and free PNA molecules serve as a “coagulant”. Two randomly selected PNA sequences of different chain lengths and sequence (13 mer and 22 mer) and

(26) Shorhoff, J. J.; Elghanain, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959–1964.

(27) Chakrabarti, R.; Klibanov, A. M. J. Am. Chem. Soc. 2003, 125, 12531– 12540.


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Table 2. PNA and DNA Sequences length

name

sequences

13 mer

PNA DNAcomp DNAm1 PNA DNAcomp DNAnc DNAm1

N′-TTCCCCTTCCCAA-C′ 5′-TTGGGAAGGGGAA-3′ 5′-TTGGGAGGGGGAA-3′ N′-AACCACACAACCTACTACCTCA-C′ 5′-TGAGGTAGTAGGTTGTGTGGTT-3′ 5-CAAAACAAAGATCTACATGGAT-3′ 5′-TGAGGTAGTAAGTTGTGTGGTT-3′

22 mer

their DNA targets are involved to validate the assays for detecting a specific DNA sequence and for single-base-mismatch discrimination. Both gold- and silvernanoparticles are used to construct the assays, of which the sensitivity is compared. Besides, a twocomponent assay using a mixture of gold- and silvernanoparticles is constructed for single-base-mismatch detection. In contrast to Li and Rothberg’s method using the distinct ability of ssDNA and dsDNA to protect unmodified gold nanoparticles from salt induced aggregation, using PNA as probe in our method allows for detection of a specific DNA sequence using the particles’ intrinsic stability in the absence of salt. Salt is only added to accelerate particles aggregation for improving the selectivity and for singlebase-mismatch discrimination. The distinct backbone properties of PNA and DNA have been extensively exploited in chip-based DNA detection, where PNA serves as probe, being immobilized on solid-substrates for DNA hybridization.28-31 Cationic substances with enzymatic activity,28 electrochemical activity,29,30 or optical property31 are often used to report the hybridization as they can selectively adsorb on the negatively charged DNA strands. In the current study we report an example of how the distinct backbone property of PNA and DNA can be used for developing homogeneous phase DNA detection. MATERIALS AND METHODS Reagents. HAuCl4 · 3H2O (99.99%) and AgNO3 (99.9%) were obtained from Alfa Aesar (MA, U.S.A.). Trisodium citrate dihydrate (99.9%) was obtained from Aldrich and NaBH4 from Fluka. Concentrated HCl and HNO3 were all analytical grade and used without further purification. Two PNA probes (13 and 22 mer) (from Eurogentec S.A. Leige, Belgium) and their target DNA (Proligo-Sigma) of fully complementary sequence (DNAcomp), non complementary sequence (DNAnc), and single-base-mismatch sequence (DNAm1) were used in this study (for the sequences, see Table 2). Colloidal Preparation and Characterization. Citrate-stabilized Au nanoparticles (AuNPs) were prepared by thermal reduction of HAuCl4 with sodium citrate.32 Spherical AuNPs of 13.2 ± 1.1 nm were observed under TEM. Citrate-stabilized Ag nanoparticles (AgNPs) were synthesized by the reduction of (28) Su, X. D.; Teh, H. F.; Lieu, X.; Gao, Z. Q. Anal. Chem. 2007, 79, 7192– 7197. (29) Kerman, K.; Vestergaard, M.; Nagatani, N.; Takamura, Y.; Tamiya, E. Anal. Chem. 2006, 78, 2182–2189. (30) Steichen, M.; Decrem, Y.; Godfroid, E.; Buess-Herman, C. Biosens. Bioelectron. 2007, 15, 2237–2243. (31) Raymond, F. R.; Ho, H. A.; Peytavi, R.; Bissonnette, L.; Boissinot, M.; Picard, F. J.; Leclerc, M.; Bergeron, M. G. BMC Biotechnol. 2005, 5, 10–15. (32) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735–743.

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silver nitrate using sodium borohydride and sodium citrate.22 Spherical AgNPs with an average size of 16.1 ± 0.6 nm were observed under TEM (detailed synthesis conditions and TEM characterization see Supporting Information). The concentration of the AuNPs and AgNPs were 7.3 and 0.39 nM, respectively, calculated using Beer’s law and extinction coefficients (ε) of 2.7 × 108 M-1 cm-1 for 13 nm AuNPs33 and 9.4 × 109 M-1 cm-1 for 16 nm AgNPs (the extinction coefficient for the silver particles is calculated using the following equation ln ε ) 1.4418 ln D + 18.955, D is the diameter in nm34,35). Colorimetric Assay Procedure. The colorimetric assays were conducted in 96 well microplates. In 150 µL AuNPs solutions, 1.5 µL of stock PNA (100 µM) was added (the final PNA concentration was 1.0 µM). After 10 min incubation at room temperature, the color of the solutions was recorded using a camera, and the absorption spectrum was recorded using a TECAN infinite M200 (Tecan Trading AG, Switzerland) in a wavelength range of 400-800 nm. To detect a specific DNA sequence, PNA probe and DNA targets (fully complementary or noncomplementary) of varied amount (i.e., varied DNA/PNA ratio) were mixed in 10 mM phosphate buffer (pH 7.2), containing 100 mM NaCl, and 0.1 mM EDTA, to attempt hybridization, prior to addition to AuNPs solutions. After 10 min incubation at room temperature, color and absorption spectrum were recorded. To improve selectivity and to detect and distinguish single-base-mismatch DNA, 3 µL of 5.0 M NaCl was added to the nucleic acid and nanoparticle mixtures (the final concentration of NaCl in the mixture was 0.1 M) to accelerate the aggregation. The same experiments were repeated with AgNPs solutions and a mixture of AuNPs-AgNPs solutions. Absorption spectra for AgNPs and the mixture of AuNPs-AgNPs were recorded at a wavelength range from 300 to 800 nm. RESULTS AND DISCUSSION Detection of the Presence of a Specific DNA Sequence Using PNA-Induced AuNPs Aggregation. The idea of using PNA probes and unmodified metallic nanoparticles to detect a specific DNA sequence is based on our discoveries that the citrate anions-protected AuNPs undergo immediate aggregation in the presence of charge neutral PNA, and the aggregation is retarded when a DNAcomp is present to form PNA-DNAcomp complex, but not a DNAnc that forms PNA/DNAnc mixture (Scheme 1). We have validated this concept using the 13 and the 22 mer samples (for the sequences, see Table 2). Results with the 22 mer samples are shown in Figure 1. When PNA is added, the welldispersed AuNPs solution (in red color with a specific surface plasmon peak at 520 nm) turns into dark purple, accompanied with a shift of the adsorption peak to 600 nm. We believe PNAinduced particle aggregation originates from the strong PNAAuNPs interactions, involving both nucleobases and peptide backbone.36 The adsorption of PNA either displaces weakly bound citrate ions (as it happens in AuNPs interactions with DNA (33) Jin, R.; Wu, G.; Li, Z.; Mirkin, C. A.; Schatz, G. C. J. Am. Chem. Soc. 2003, 125, 1643–1654. (34) Yguerabide, J.; Yguerabide, E. E. Anal. Biochem. 1998, 262, 137–156. (35) Yguerabide, J.; Yguerabide, E. E. Anal. Biochem. 1998, 262, 157–176. (36) Gourishankar, A.; Shukla, S.; Ganesh, K. N.; Sastry, M. J. Am. Chem. Soc. 2004, 126, 13186–13187.

Figure 1. Detection of a specific DNA sequence using AuNPs. Photographs and corresponding adsorption spectra of (A) bare AuNPs (7.3 nM, ca.13 nm in diameter) and AuNPs solutions with (B) the 22 mer PNA, (C) PNA-DNAcomp complex, and (D) PNA/DNAnc mixture in the absence of NaCl. Photographs A′-D′ are solutions of A-D containing 0.1 M NaCl (the corresponding absorption spectra are not shown). Final PNA and DNA concentrations in the AuNPs are 1 µM. Scheme 1. Assay Principle

bases,36 nucleoside,37,38 and dsDNA39) or shields the citrate ions (no displacement occurs as reported for single-stranded oligonucleotide-AuNPs interaction10), both causing the loss of charge repulsion and therefore particle aggregation (the confirmation of whether citrate ions are displaced would subject to further studies using Raman spectroscopy, zeta potential, FTIR, etc,). In addition, the presence of positive charges of the N-terminal amines of the PNA at neutral pH may contribute to the immediate aggregation by initiating uptake of the PNA via electrostatic interaction (positive charged amino group toward the negatively charged gold), as similarly reported for peptide-AuNPs interactions.40 In contrast, when complementary PNA-DNAcomp complex is added, the AuNPs solution remains stable. This would be attributable to the adsorption of PNA-DNAcomp complex on AuNPs, with which the negative phosphate backbone of the DNA strands ensure sufficient charge repulsion, essential for AuNPs to remain dispersed. When the solution contains a mixture of unhybridized PNA and noncDNAnc, the particle solution underwent a certain extent of aggregation, showing a color change to purple and the appearance of adsorption at 600 nm. (37) Jang, N. H. Bull. Korean Chem. Soc. 2002, 23, 1790–1800. (38) Storhoff, J. J.; Elghanian, R.; Mirkin, C. A.; Letsinger, R. L. Langmuir 2002, 18, 6666–6670. (39) Gearheart, L. A.; Ploehn, H. J.; Murphy, C. J. J. Phys. Chem. B 2001, 105, 12609–12615. (40) Lvy, R.; Thanh, N. T. K.; Doty, R. C.; Hussain, I.; Nichols, R. J.; Schiffrin, D. J.; Brust, M.; Fernig, D. G. J. Am. Chem. Soc. 2004, 126, 10076–40084.

Originally, it is well-known that single-stranded DNA is able to stabilize AuNPs10-13 because of its flexible, uncoiled structure that allows the nucleosides to be exposed and interact with AuNPs effectively. In our case, the depletion of the ability of the singlestranded DNAnc to protect AuNPs when PNA is present implies that PNA molecules bind predominantly to AuNPs. It must be the charge repulsion between the single-stranded DNAnc and the citrate anions on the particle surface that renders the DNAAuNPs interaction less efficient relative to the PNA-AuNPs interaction. Unlike other colorimetric assays that use salt induced particle aggregation for DNA detection,10-13 using PNA as a “coagulant” and hybridization probe in our method allows for detection of a specific DNA sequence with the nanoparticles’ intrinsic stability in the absence of salt. Further experiments show that adding salt accelerates the aggregation for solutions without the DNAcomp and therefore improve the selectivity, as shown by the further color discrimination between the DNAcomp- and DNAnc-containing solutions (photographs C′ and D′ in Figure 1). The similar color code (blue) of the PNA- and PNA/DNAnc mixture-containing solutions with NaCl indicates a similar degree of particle aggregation. The failure of single-stranded DNAnc to protect AuNPs from salt induced aggregation further proves that charge neutral PNA predominately interact with AuNPs. To further quantify the selectivity of this assay, higher concentrations of DNAnc (2, 5, and 10 times of the PNA Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

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Figure 2. Selectivity of the AuNPs-based assay. Photographs and corresponding adsorption spectra of 22 mer PNA-containing AuNPs solutions (A-E) in the presence of DNAnc of 10, 0, 2, 5, and 10 µM with (A) or without (B-E) DNAcomp (1 µM). Photographs of A′-E′ are solutions A-E containing 0.1 M NaCl (the corresponding spectra not shown). The final PNA concentration in each well was 1 µM.

concentration) were added into PNA-containing solutions with or without the DNAcomp (Figure 2). PNA-induced aggregation (in the absence of DNAcomp,) is gradually reduced with the increase of DNAnc (photographs B-E in Figure 2), indicating that single-stranded DNA tends to compete with PNA to interact with AuNPs at higher concentrations. But the aggregation remains largely detectable in the presence of DNAnc of 10 times of the PNA concentration (photograph E), relative to the stable colloidal when DNAcomp is present (photograph A). This means that a specific DNA sequence can be detected in the presence of at least 10 times of interference DNA. Again, adding NaCl induced further aggregation for the solutions containing PNA and DNAnc (photographs B′ to E′ in Figure 2) but not the solution also containing DNAcomp (photograph A′ in Figure 2), which further amplifies the stability difference. Thus, adding salt is a strategy to further improve the selectivity to ensure DNAcomp to be detected under large excess amount of interference DNA. The high selectivity of this assay would be appreciated for DNA detection in complicated media, like PCR products, in which primers and other fragments are often present.8,11 Quantification of Specific DNA Sequence Using PNAInduced AuNPs Aggregation. To assess the sensitivity of the current method for quantifying DNA having a complementary sequence to the PNA probe, the DNAcomp target was mixed with 1 µM PNA at ratios of 0, 0.05, 0.08, 0.1, 0.2, 0.5, and 1.0 for hybridization prior to addition to AuNPs solutions. The degree of PNA-induced AuNPs aggregation gradually decreases as the DNA concentration increases (Figure 3). This is because the more the PNA-DNAcomp hybrids are present, the more charge repulsion is introduced to the particles because of the capping of the particles with the PNA-DNAcomp complex. In the color photographs, a noticeable color difference is observed between AuNPs solutions with PNA alone (deep blue) and the PNA-DNAcomp solution at a DNA/PNA ratio of 0.1 (deep purple). In the corresponding UV-vis adsorption spectra, a gradual increase in the absorbance around 520 nm for solutions containing increasing amount of DNAcomp indicates a gradual increase in the popularity of dispersed particles under protection of the PNA-DNAcomp complex. At the DNA/PNA ratio as small as 0.05, the absorbance is still distinguishable from that with PNA alone. At this ratio, the absorbance at the second peak around 600 nm, representative of the popularity of 6126


Figure 3. Sensitivity of the AuNPs-based assay. Photographs and corresponding adsorption spectra of 22 mer PNA-containing AuNPs solutions in the presence of increasing amount of DNAcomp at DNA/ PNA ratio of 0, 0.05, 0.08, 0.1, 0.2, 0.5, and 1.

aggregated particles, is also largely distinguishable from the case with PNA alone. The spectrum detection limit of ratio 0.05 and the visual detection limit (from the color) of ratio 0.1 are lower than those previously reported by Rho et al.13 (ratio 0.5) and Li et al.10 (ratio 0.2), who use unmodified AuNPs but different electrostatic properties of ssDNA and dsDNA for target quantification. We attribute the lower detection limit of the current method to the extraordinary coagulating property of PNA probes, which is greatly distinguishable from the corresponding PNA-DNAcomp complex. The higher affinity of PNA-DNA hybridization than DNA-DNA hybridization may also contribute to the lower DNA detection limit. It is acknowledged that metal nanoparticle-based colorimetric DNA assays, especially when non-crosslinking aggregation is used, have a moderate sensitivity in the nanomolar range.2,7,10,13 However, DNA samples at this concentration range can be obtained through a standard PCR protocol;7,8,11 thus we and others7,11 believe that this aspect will not impose a severe limitation on the practical application of the colorimetric assays, and the merit of the simplicity should be appreciated.

Figure 5. AgNPs-based assay. UV-vis adsorption spectra and photographs (inset) of (A) bare AgNPs (4.1 nM, ca. 16 nm in diameter) and AgNPs solutions containing (B) 22 mer PNA, (C) PNA-DNAcomp complex, and (D) PNA/DNAnc mixture. The inset adsorption spectra show single-base-mismatch discrimination, in the presence of 0.1 M NaCl. Final PNA and DNA concentrations in the AgNPs are 1 µM.

Figure 4. Single-base-mismatch discrimination in (A) 13 mer and (B) 22 mer sequences using AuNPs. UV-vis adsorption spectra and corresponding photographs (inset) of PNA-containing AuNPs solutions in the presence of fully complementary target DNA (comp) and singlebase-mismatch DNA (m1), in the presence of 0.1 M NaCl.

Single-Base-Mismatch Detection Using AuNPs Aggregation in the Presence of Salt. In the previous section, we have shown that adding salt accelerates particle aggregation and leads to a better selectivity for detecting DNAcomp. In this section, salt accelerated aggregation is further utilized to distinguish singlebase-mismatch in the complementary targets (Figure 4). For both the 13- and 22-mer samples, the absorbance at longer wavelengths around 600 nm is increased for the mismatched target (DNAm1). We believe, because of the presence of the single-base-mismatch, that PNA-DNA hybridization efficiency is reduced, leaving some PNA and DNAm1 target unhybridized. The free PNA molecules will then coagulate the nanoparticles, resulting in an increase of absorbance at longer wavelengths. It is generally known that single-base-mismatch is more difficult to detect in a longer sequence41 because the contribution of one base mismatch to the affinity depletion is minor. Our results with the 13 mer and 22 mer samples coincide with this notion very well by showing a larger spectrum difference between fully complementary and single-mismatch targets in the shorter sequence than in the longer sequence. Specifically, a larger increase in the absorbance at 600 nm (51.6%) is obtained for the 13 mer sample than that (17.5%) for the 22 mer sample. In the color photographs, the color difference is only noticeable for the 13 mer sequence. To confirm that the spectrum shifts with the single-basemismatch DNA (Figure 4) are a true reflection of AuNPs’ stability difference rather than a variation in the UV-vis spectrum measurement, we have evaluated the reproducibility of the UV-vis spectrum measurement. The spectra obtained from multiple scans for an AuNPs solution aliquot in different wells overlap extremely well (see Supporting Information, Figure S3). The relative standard (41) Lao, A. I. K.; Su, X. D.; Khin, M. M. A. Biosens. Bioelectron. 2009, 24, 1717–1722.

deviation for the absorbance at 600 nm is 1.03% (Supporting Information, Table S1). We thus are very confident that the measured A600 increase for single-base-mismatch DNA (51.6% and 17.5% for the 13 and 22 mer samples, respectively) are more than account for the stability difference. Detection of Specific DNA Sequence and Single-BaseMismatch Using Silver Nanoparticles. AgNPs are known to have a higher extinction coefficient relative to AuNPs of the same size,5,9,22 and thus a higher sensitivity for use in colorimetric assays. In this study, the ability of PNA to coagulate AgNPs is tested; the concept of using retarded AgNPs aggregation caused by PNA-DNA hybridization for DNA detection is validated; and finally, the sensitivity of single-base-mismatch detection is compared between the AgNPs- and the AuNPs-based assays. In Figure 5, we show that the citrate anion-coated AgNPs (yellow color) aggregate drastically (turn to deep yellow or pale brown) in the presence of PNA probe (22 mer) or a mixture of PNA/DNAnc; whereas in the presence of PNA-DNAcomp complex, the particles remain stable. In the corresponding UV-vis adsorption spectra, a remarkable decrease in the absorbance at 400 nm and the appearance of a second peak at a longer wavelength around 550 nm are observed for PNA- and PNA/DNAnc solutions, as typical AgNPs aggregation signatures.42 Similar to the case with gold nanoparticles, a fully complementary PNA-DNAcomp complex and a single-base-mismatched complex (PNA-DNAm1) exert no distinguishable effects on the silver nanoparticles’ stability in the absence of NaCl. To enhance the stringency for detecting single-base-mismatch, NaCl (final concentration 0.1 M) was added following the incubation of PNA-DNA complexes with AgNPs solutions. For the 22 mer sample (we intentionally chose this sample, rather than the 13 mer sample, because it presents a bigger challenge in the gold nanoparticle-based assay, Figure 4B), successful discrimination between fully complementary and single-basemismatch DNA is achieved. Compared to the single-basemismatch discrimination using gold nanoparticles that shows only a slight spectrum shift to longer wavelength (Figure 4B), the spectrum alternation of the silver nanoparticles is more obvious, (42) Schofield, C. L.; Hainer, A. H.; Field, R. A.; Russell, D. A. Langmuir 2006, 22, 6707–6711.


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Figure 6. Two-component assay using AuNPs-AgNPs mixture. (A) UV-vis absorption spectra and corresponding photographs of bare AuNPs (2 nM), AgNPs (0.2 nM), and their mixture (2 nM and 0.2 nM, respectively). (B to D) Detection of single-base-mismatch in the 13 mer sample using AuNPs-AgNPs mixture, pure AuNPs, and pure AgNPs, respectively. PNA and DNA concentrations are 1 µM and NaCl concentration is 0.4 M.

showing both a drop of absorbance at the original peak wavelength (400 nm) and an increase in absorbance at longer wavelengths (>600 nm). A 55.2% increase in the A650/A400 value (a quantitative measure of the spectrum shift) is obtained for the AgNPs containing single-base-mismatch DNA relative to that containing fully DNAcomp (variation of A650/A400 from multiple scans for a given AgNPs solution is 0.97%. See Supporting Information, Figure S4 and Table S1). With this result we infer that silver nanoparticles are more sensitive than gold nanoparticles when used for single-base-mismatch detection in our method. This result complements a previous discovery by Thomsen et al., who showed that oligonucleotide-AgNPs conjugate offers a higher sensitivity for quantification of a specific DNA than its AuNPs counterpart in a type I assay using sandwich hybridization.5 As mentioned earlier, single-base-mismatch discrimination is difficult for hybridizations with longer probes. For the 22 mer sequence used in this study (a sequence specific for microRNA let-7b),43 single-base-mismatch discrimination using surface plasmon resonance (SPR)41 and electrochemical sensors44 must rely on serious stringency control, for example, using hybridization suppressors, electric field assisted dehybridization, or high temperature; whereas, in this study single-base-mismatch in this sequence can be easily detected based on room temperature hybridization under normal buffer conditions. Construction of Two-Component Assay Using a Mixture of AuNPs and AgNPs. In a previous study, Cao et al.4 developed a two-color-change DNA assay using Ag/Au core-shell NPs and AuNPs selectively functionalized by different DNA probes. By monitoring two color changes available through two types of (43) Lau, N. C.; Lim, L. P.; Weinstein, E. G.; Bartel, D. P. Science 2001, 294, 858–862. (44) Fan, Y.; Chen, X.; Trigg, A. D.; Tung, C. H.; Kong, J. M.; Gao, Z. Q. J. Am. Chem. Soc. 2007, 129, 5437–5443.

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nanoparticle, their results for single-base-mismatch detection become more reliable. In a separate study, Schofield et al.42 reported the use of a mixture of AuNPs and AgNPs modified with different carbohydrate species for selective detection of their cognate ligands by looking at the shifts of surface plasmon bands of Au or Ag nanoparticles in the mixture. Inspired by these studies, we have constituted an assay using a mixture of unmodified AuNPs and AgNPs, with the aim to evaluate what additional merits this two-component assay offers relative to the assays with pure AuNPs or pure AgNPs. Figure 6A shows the original color of 2 nM AuNPs, 0.2 nM AgNPs, and a mixture of the two types of particles (2 nM and 0.2 nM, respectively). As expected the spectrum of the mixture is a simple summation of the two individual spectra of the silver and the gold nanoparticles. Figures 6B, 6C to 6D shows the single-base-mismatch discrimination in the 13 mer sequence measured using the AuNPs-AgNPs mixture, AuNPs, and AgNPs, respectively, in the presence of 0.4 M NaCl. We found that (1) the AuNPs-AgNPs mixture offers three signatures for single-base-mismatch target, that is, absorbance decreases at 400 nm, absorbance decrease at 520 nm, and spectrum shift to longer wavelengths; (2) AgNPs offers two signatures, that is, decrease in 400 nm absorbance and significant spectrum shift to longer wavelengths; and (3) the AuNPs offers only one signature, that is, a slight bump on the spectrum at 600 nm wavelength (points 2 and 3 have been consistently observed early on with the 22 mer sequence, Figure 4B and inset of Figure 5). With these results we infer that single-base-mismatch detection using a mixture of AuNPs and AgNPs is more reliable because of the presence of multiple signatures. When looking at the color contrast of the solutions, we found the AgNPs-based assay provides the best visual discrimination (yellow to pale yellow), followed by the AuNPs-AgNPs mixture-based assay (orange to

pale orange). Comparatively, the color difference of AuNPs is the least significant for this particular application. CONCLUSION We have developed a metallic nanoparticle-based colorimetric DNA detection method, using the aggregation behavior of unmodified nanoparticles as sensing element. The innovation of this method is the use of charge neutral PNA as probe, which can induce drastic particle aggregation in the absence of DNAcomp. The concept has been proved with AuNPs, AgNPs, and a mixture of AuNPs/AgNPs, with the involvement of two randomly selected PNA sequences to show that the assay is generally valid without sequence and chain length constraint. Compared to the large body of colorimetric DNA detection methods that involve DNA-nanoparticle conjugation, our method offers additional robustness. The use of charge neutral PNA as probe, whose electrostatic property is tremendously different

from the corresponding PNA-DNA complexes and ssDNA of the same sequence, ensures a higher sensitivity for the detection of a specific DNA in the presence of high concentration of interference DNA. With this method, single-basemismatch can be detected at room temperature without additional stringency control. Constitution of the assay using a two component nanoparticle mixture can enhance the confidence for single-base-mismatch discrimination. SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review March 12, 2009. Accepted June 16, 2009. AC900525K


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Colorimetric Detection of DNA Using Unmodified Metallic

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