Information Derived from Cluster Ions from DNA-Modified Gold

Furthermore, we applied this pDNA–Au NP probe to the detection of single-nucleotide polymorphisms (SNPs) of the Arg249Ser unit in the TP53 gene. To ...
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Information Derived from Cluster Ions from DNA-Modified Gold Nanoparticles under Laser Desorption/Ionization: Analysis of Coverage, Structure, and Single-Nucleotide Polymorphism Yin-Chun Liu,† Yu-Jia Li,† and Chih-Ching Huang*,†,‡,§ †

Institute of Bioscience and Biotechnology and ‡Center of Excellence for Marine Bioenvironment and Biotechnology (CMBB), National Taiwan Ocean University, Keelung, Taiwan § School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan S Supporting Information *

ABSTRACT: In this study, we employed laser desorption/ionization mass spectrometry (LDI-MS) to explore the interactions between thiolated DNA (HS-DNA) and gold nanoparticles (Au NPs). Under nanosecond-pulsed laser irradiation (Nd:YAG, 355 nm), the efficiency of Au cluster ion formation from the Au NPs decreased in the presence of HS-DNA. At the optimal laser power density (2.1 × 104 W cm−2), the intensity of the Au cluster signal was sensitive to the DNA coverage and the length of the DNA strands on the Au NPs (diameter: 13 nm). Using this information, we developed a simple and specific DNA sensor that operates through analysis of the Au cluster ions formed from the fragmentation of Au NPs under LDI conditions. The coverage of the thiolated probe DNA (pDNA) on the Au NPs increased in the presence of its perfectly matched DNA (DNApm). As a result, the intensity of the signal of Au cluster ions decreased upon increasing the concentration of DNApm. Coupling these pDNA−Au NPs with LDI-MS allowed the detection of DNApm at concentrations down to the nanomolar regime. Furthermore, we applied this pDNA−Au NP probe to the detection of single-nucleotide polymorphisms (SNPs) of the Arg249Ser unit in the TP53 gene. To the best of our knowledge, this paper provides the first example of the use of LDI to analyze the coverage and structure of DNA strands on metal NPs. This simple, rapid, high-throughput detection system, based on the coupling of biofunctional Au NPs with LDI-MS, appears to hold great practicality for bioanalyses of oligonucleotides and proteins.

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surrounding molecules. Moreover, vaporization and ionization of surrounding molecules are also possible through a thermally driven process, because the heat diffusion length of the NPs is much larger than the average NP diameter.6 On the basis of these phenomena, considerable attention has been drawn to surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using Au NPs as matrixes for the analyses of various molecules of interest.7 Au NPs prepared with and without surface functionalization can act as selective probes and/or matrixes for the SALDI-MS analyses of metal ions, aminothiols, peptides, proteins, and carbohydrates.7 Despite the advantages of and the promising results achieved with Au NP-assisted laser desorption/ ionization (LDI), thermally driven energy transfer from the Au NP substrates to the analytes is generally accompanied by the production of many Au cluster ions.7 As a result, the signals of the sample ions were suppressed dramatically, forming

he optical properties of gold nanoparticles (Au NPs) are attractive because their extremely high surface plasmon resonance (SPR) absorptions, efficient interband transitions, and scattering cross sections are unlike those of their organic and inorganic counterparts.1 The applications of Au NPs are rapidly developing in many fields, including roles as tailor-made nanostructured materials for medicine and biosciences, drug delivery, and phototherapy.2 The photothermal and photomechanical behavior of Au NPs under short pulsed laser excitation has drawn increasing attention in recent years.3 Irradiation of Au NPs with intense pulsed laser light can induce nanobubbles and nanoholes as well as variations in size and morphology. 3 Two kinetic mechanisms, the Coulomb explosion model (electron ejection from particles inducing particle explosion) and the photothermal evaporation model (thermal heating followed by evaporation), have been proposed to explain the fragmentation of Au NPs under pulsed laser light.3,4 The high amount of laser energy absorbed by the NPs can contribute to sudden and dramatic increase in the temperature of the particles and their surrounding medium,3−5 potentially resulting in lattice expansion, melting, and fragmentation and inducing nonthermal desorption of © 2012 American Chemical Society

Received: October 1, 2012 Accepted: December 18, 2012 Published: December 18, 2012 1021

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matched DNA (DNApm). As a result, the intensity of the signals of the Au cluster ions decreased upon increasing the concentration of DNApm. Finally, we applied this probe to the analysis of single-nucleotide polymorphisms (SNPs).

complicated mass spectra and, thereby, lowering the analytical sensitivity. In addition, the abundance of higher-order Au clusters generally increases upon increasing the sizes of the Au NPs.8 Moreover, surface capping can suppress the generation of Au cluster ions.9 For example, we found the efficiency of Au cluster ions formation from aptamer-modified Au NPs on membrane under LDI was decreased in the presence of the target proteins.9b Because the unique properties of Au NPs are closely related to the nature and density of their surface ligands, there is great interest in characterizing their compositions in detail. For example, the binding affinity and specificity of DNA-functionalized Au NPs toward target molecules (e.g., DNA, proteins) are highly dependent on the structure and density of the DNA.10 In this present study, to provide additional chemical information regarding the surface structures of Au NPs, we employed SALDI-MS with Au NPs as matrixes to study the interactions between thiolated DNA (HS-DNA) and Au NPs. The Au cluster ions signals in the mass spectra allowed quantitation of the coverage of DNA on the Au NPs (Scheme 1A). In addition, the length of the DNA strands also strongly

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EXPERMENTAL SECTION Materials. All DNA and thiol-modified DNA samples listed in Table 1 were purchased from Integrated DNA Technologies (Coralville, IA, USA). Sodium chloride, potassium chloride, magnesium chloride, and calcium chloride were purchased from Mallinckrodt Baker (Phillipsburg, NJ). Trisodium citrate and adenosine triphosphate (ATP) were purchased from Aldrich (Milwaukee, WI). Hydrogen tetrachloroaurate(III) trihydrate was obtained from Acros (Geel, Belgium). Sodium phosphate dibasic anhydrous and sodium phosphate monobasic monohydrate, obtained from J. T. Baker (Phillipsburg, NJ, USA), were used to prepare the phosphate buffer (5.0 mM, pH 7.4). Preparation and Characterization of Au NPs. Spherical Au NPs (diameter: 13.3 nm) were prepared through 4.0 mM citrate-mediated reduction of 1.0 mM HAuCl4.11 Their sizes were verified using a transmission electron microscope (Tecnai 20 G2 S-Twin TEM, Philips/FEI, Hillsboro, Oregon) operated at 125 kV; the Au NPs appeared to be nearly monodisperse with an average diameter of 13.3 (±1.2) nm (from 100 counts). A microplate reader (μ-Quant Biotek Instruments, Winooski, VT, USA) was used to measure the absorptions of Au NP solutions. The Au NP concentration (15 nM) was determined using Beer’s law, with an extinction coefficient of 2.08 × 108 M−1 cm−1 at 520 nm for 13.3 nm Au NPs. The 6.0 nm Au NPs were synthesized through NaBH4mediated reduction of HAuCl4, using a slightly modified reported procedure.12 An aqueous solution (50 mL) was prepared containing 0.25 mM HAuCl4 and 0.25 mM trisodium citrate. Next, 0.01 M NaBH4 (1.5 mL) was added in one portion to the HAuCl4 solution under constant stirring. After stirring for 3 min, the color changed from yellow to brown, indicating the formation of Au NPs. The solution was incubated at 4 °C for 24 h prior to further use. The average size of the as-prepared Au NPs was determined through transmission electron microscopy (TEM) to be 6.0 ± 0.8 nm (from 100 counts). The 32 and 56 nm Au NPs were prepared through citratemediated reductions [using 1% trisodium citrate solutions (0.5 and 0.3 mL, respectively)] of 0.01% HAuCl4 solutions (50 mL). The particle sizes of the 32 and 56 nm Au NPs, determined from 100 counts using TEM, were 32.2 ± 5.9 and 56.4 ± 9.8 nm, respectively; their particle concentrations were 280 and 54 pM, respectively. Dynamic light scattering (DLS) data and zeta potentials of the Au NPs were measured using a Zetasizer 3000HS analyzer (Malvern Instruments, Malvern, UK). Preparation of DNA−Au NPs. Thiol-modified DNA having the sequence 5′-HS-TAG GCT CAC AAT CCT-3′ was attached to the Au NPs using a modified procedure.13 The 5′-thiol-modified DNA (thiol-DNA), received in the disulfide form HOCH3(CH2)5S-S-3′-oligo, was reacted directly with the Au NPs, attaching both the HO(CH2)6S and oligo-S units to the Au NP surface. For example, aliquots of aqueous Au NP solutions (15 nM, 1.0 mL) in 1.5 mL tubes were mixed with thiol-DNA (20 μL). After reacting for 12 h at room temperature, the mixtures were centrifuged at a relative centrifugal force (RCF) of 30 000g for 20 min to remove any unattached thiol-DNA. The supernatants were removed, and the oily precipitates were washed with sodium phosphate (pH

Scheme 1. Schematic Representation of the LDI-MS Analyses of Au Cluster Ions from DNA−Au NPs with Various (A) DNA Densities and (B) DNA Lengths

impacted the efficiency of formation of Au cluster ions under pulsed laser irradiation (Scheme 1B). Furthermore, we have developed a simple, highly specific DNA sensor that functions based on the analysis of Au cluster ions formed from the fragmentation of Au NPs (Scheme 2). The coverage of HSDNA on the Au NPs increased in the presence of its perfectly 1022

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Scheme 2. Schematic Representation of the Use of DNA−Au NPs Coupled with LDI-MS for the Detection of Oligonucleotides

The concentration of the oligonucleotide molecules displaced by 2-mercaptoethanol was determined by adding OliGreen (ssDNA labeling reagent, Molecular Probe, Eugene, OR) and then measuring the fluorescence intensities at 524 nm (480 nm excitation wavelength).14 Because the manufacturer did not provide the OliGreen (OG) concentration, we defined the concentration as 100×. OG is a commercially available asymmetrical cyanine dye that is used to label oligonucleotides. It is weakly fluorescent but exhibits a greater-than-1000-fold enhancement in its fluorescence upon binding to ssDNA. The DNA−Au NPs (10 nM) were incubated with 2-mercaptoethanol (10 mM) in sodium phosphate solution (pH 7.4, 5.0 mM) for 2 h, and then, the mixtures were centrifuged at a RCF of 30 000g for 20 min to collect the displaced oligonucleotide from the supernatant. The 10-fold diluted oligonucleotides were further reacted with 0.025× OG in sodium phosphate solution (pH 7.4, 5.0 mM) for 30 min. The mixtures were transferred separately into 96-well microtiter plates, and their fluorescence spectra were recorded using a Synergy 4 Multi-Mode

Table 1. DNA Sequences Used in This Study name

sequence

HS-DNA (15-mer) HS-DNA (23-mer) HS-DNA (30-mer)

5′-HS-TAG GCT CAC AAT CCT-3′ 5′-HS- TTT TTT TTT AGG CTC ACA ATC CT-3′ 5′-HS-TTT TTT TTT TTT TTT TAG GCT CAC AAT CCT-3′ 5′-HS-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TAG GCT CAC AAT CCT-3′ 5′-HS-TTT TTT TTT TTT TTT GGA TGG GCC TCC GGT-3′ 5′-ACC GGA GGC CCA TCC-3′ 5′-ACC GGA GTC CCA TCC-3′ 5′-ATC GGA CTA GCC AAT-3′

HS-DNA (45-mer) HS-pDNA DNApm DNAmm rDNA

7.4, 5.0 mM, 1.0 mL). After three centrifuge/wash cycles, the colloids of DNA−Au NPs were resuspended separately in sodium phosphate (pH 7.4, 5.0 mM, 1.0 mL). No precipitation or apparent changes in the SPR band of Au NPs were observed, indicating that the DNA−Au NPs were stable in this solution.

Figure 1. Positive-mode mass spectra of Au NPs (5.0 nM) in the (A) absence and (B−E) presence of 5′-thiolated DNA [HS-DNA (15-mer); 5′-HSTAG GCT CAC AAT CCT-3′] at [HS-DNA]/[Au NPs] ratios of (B) 10, (C) 20, (D) 40, and (E) 80. Signals at m/z 196.967, 393.933, 590.900, 787.866, and 984.832 are assigned to [Au1]+, [Au2]+, [Au3]+, [Au4]+, and [Au5]+ ions, respectively. A total of 300 pulsed laser shots were applied to accumulate the signals from five LDI target positions under a laser power density of 2.1 × 104 W cm−2. Peak intensities are plotted in arbitrary units (a. u.). 1023

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Notably, the intensities of the signals of the [Au4]+ and [Au5]+ cluster ions were much lower than those of the [Au1−3]+ ions. It has been proposed that the yield (Yn) of metallic cluster of n atoms is proportional to n−5, where δ is related to the total sputter yield.17 In the presence of DNA, however, the Au NPs transferred their energy to the surface DNA molecules, thereby decreasing their lattice temperature (Tl).18 As a result, evaporation of surface atoms was suppressed and the signals of the Au clusters appeared with low abundance in the mass spectrum. Alternatively, Coulomb explosion was possibly inhibited through lowering of the electron temperature (Te) and electrons ejecting from the surface DNA molecules. In addition, competitive ionization of the DNA and the Au clusters, or reactions of the Au clusters with the DNA ions in the gas phase, might also have resulted in the decreased intensities of the Au cluster signals. We also found that the negative mode resulted in decreased intensities of the negatively charged Au ([Aun]−; n = 1−5) cluster ions, similar to the behavior of the positive cluster ions in the positive mode (Supporting Information, Figure S2). Accordingly, we could use the decrease in the signal intensities of the Au clusters as an indicator of the surface coverage of DNA. The relative signal intensities of the Au cluster ions ([Aun]+; n = 1−3) decreased upon increasing the [DNA]/[Au NPs] ratio to 25 (Supporting Information, Figure S3A). In addition, the [Au3]+/[Au1]+ and [Au3]+/[Au2]+ signal ratios also decreased upon increasing the [DNA]/[Au NPs] concentration ratio (Supporting Information, Figure S3B), revealing that the fragmentation of Au clusters of larger size was more sensitive to the density of the surface ligands. The dense coverage of highly negatively charged DNA molecules on the Au NPs strongly inhibited electron ejection and sharply decreased the electron temperature.3 Therefore, explosive fragmentation, inducing the formation of relatively large Au cluster ions, was strongly inhibited. Although the clusters of larger size (e.g., [Au3]+) were more sensitive to the DNA coverage, the reproducibility was poor [relative standard deviation (RSD): ∼20% from 300 shots; the RSD of [Au1]+ was ca. 7%]. Thus, we used the singly charged ion [Au1]+ for our following study of the coverage and structure of DNA on the Au NPs. Effect of the Power Density of Laser. Surface melting and evaporation of Au NPs can occur regardless of the excitation wavelength of the laser, but it is highly related to the power density.4,19 From a simple light-to-heat energy conversion model [Q = (CλabsI)/(ρVp); Q: absorbed laser energy; Cλabs: absorption cross section at wavelength λ; I: laser power density; ρ: density of bulk Au; Vp: particle volume],20 we would expect the lattice temperature and electron temperature (and, thus, the degree of fragmentation) to increase upon decreasing the laser power density. In Figure S4 (Supporting Information), we observed an increase in the intensity of the signals for the Au clusters upon increasing the laser power density from 2.0 × 104 to 2.2 × 104 W cm−2. Because of fragmentation and ionization of the surface molecules and formation of Au cluster ion adducts, the mass spectra were more complicated at higher laser power densities. Figure 2 reveals that the peak intensity of the [Au1]+ ion decreased upon increasing the [DNA]/[Au NPs] ratio from 0 to 30 under various laser power densities. When the laser power density was less than 2.0 × 104 W cm−2, the boiling temperature (ca. 3000 K) and the value of Tefrag (electron ejection-induced fragmentation at ∼8000 K) of the 13 nm Au NPs were not exceeded;4 therefore, the low efficiency of fragmentation of the

Microplate Reader. We quantified the displaced oligonucleotides using an oligonucleotide calibration curve (10−250 nM). The results indicated that ca. 70 oligonucleotide molecules were attached to each Au NP. LDI-MS Analysis of Au Cluster Ions. A portion of the DNA−Au NPs samples (ca. 2.0 μL) was cast onto a stainlesssteel 384-well MALDI target and dried in air at room temperature prior to SALDI-time-of-flight (TOF) MS measurements. MS experiments were performed in the reflectron positive- or negative-ion mode using an AutoflexIII MALDI TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). The samples were irradiated with a SmartBeam laser (Nd:YAG, 355 nm) at 100 Hz. Ions produced by laser desorption were stabilized energetically during a delayed extraction period of 30 ns and then accelerated through the TOF chamber in the reflection mode before entering the mass analyzer. The available accelerating voltages ranged from +20 to −20 kV. The instruments were calibrated with Au clusters using their theoretical mass values ([Aun]+; n = 1−3). A total of 300 pulsed laser shots was applied to accumulate signals from five MALDI target positions under a power density in the range from 2.0 × 104 to 2.2 × 104 W cm−2. Assays of DNA. Mixtures (300 μL) of 40 nM probe DNA (pDNA, 5′-HS-TTT TTT TTT TTT TTT TAG GCT CAC AAT CCT-3′; a T15 linker and a 15-base sequence for hybridization) and perfectly matched DNA (DNApm; 0−500 nM) were incubated in 5 mM sodium phosphate containing 200 mM NaCl at room temperature for 10 min and then reacted with 30 μL of ATP (2.0 μM)-protected Au NPs (1.0 nM) for another 30 min. The ATP-stabilized AuNPs were prepared by simply mixing the AuNPs (1.0 nM) with ATP (2.0 μM) and incubating for 10 min. ATP can bind to citrate-capped AuNPs, displacing weakly bound citrate ions through metal− ligand interactions.15 The ATP-stabilized AuNPs are stable in the buffer solution in the presence of high concentrations of salt.16 The mixture of DNA and Au NPs was centrifuged at an RCF of 30 000g for 20 min to remove excess pDNA and DNApm, and then, the pellet was resuspended in 5 mM sodium phosphate (pH 7.4). After three centrifuge/wash cycles, the pDNA−Au NPs was concentrated to 5-fold and resuspended in 5 mM sodium phosphate (pH 7.4). A portion of the sample (ca. 2.0 μL) was cast onto a MALDI plate and then subjected to LDI-MS measurement.

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RESULTS AND DISCUSSION LDI-MS Analyses of Au Cluster. Figure 1 displays the mass spectrum of 13 nm Au NPs in the absence and presence of thiolated DNA (HS-DNA; 5′-HS-TAG GCT CAC AAT CCT-3′) at concentration ratios of [HS-DNA]/[Au NPs] of 10, 20, 40, and 80. Under pulsed laser irradiation (355 nm Nd:YAG; 100 Hz; 2.1 × 104 W cm−2; pulse width: 6 ns), the produced Au cluster ions ([Aun]+; n = 1−5) were detected. A scanning electron microscopy (SEM) image of the Au NP sample reveals holes having an average diameter of approximately 10 μm after pulsed laser irritation (Supporting Information, Figure S1). For nanosecond pulsed-laser excitation, τP (pulse width) is longer than τph‑ph (phonon− phonon relaxation time: 50−600 ps for Au NPs) and much longer than τe‑ph (electron−phonon relaxation time: 2−3 ps for Au NPs).3,4 Therefore, the fragmentation of Au NPs is favored through the photothermal evaporation mechanism, because the boiling temperature is easily reached, whereas a fragmentation temperature that exceeds the Rayleigh instability limit is not.3,4 1024

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Au NPs led to low signal decreasing ratios [(I0[Au1]+ − I[Au1]+)/ I0[Au1]+] (Figure 2B), where I0[Au1]+ and I[Au1]+ are the signal intensities of the [Au1]+ ions in the absence and presence of thiolated DNA, respectively. On the other hand, at higher laser power densities, the complicated competitive desorption and ionization processes of the Au atoms and surface molecules and the reactions of the Au cluster ions with the desorbed fragment molecules or ions also resulted in low signal decreasing ratios and complicated mass spectra. To avoid the need for an excessively high laser power density, leading to a higher level of background noise and a loss in resolution, we chose a value of 2.1 × 104 W cm−2 for our following experiments. Determination of the Surface Coverage of DNA. Next, we studied the impact of the surface DNA molecules on the formation of Au cluster ions from Au NPs having sizes of 6, 13, 32, and 56 nm. For these four types of Au NPs, the values of [(I0[Au1]+ − I[Au1]+)/I0[Au1]+] increased upon increasing the [HSDNA]/[Au NPs] ratio, plateauing at 5 ± 2, 23 ± 4, 120 ± 18, and 510 ± 65 (n = 3) for the 6, 13, 32, and 56 nm Au NPs, respectively (Figure 3). From the measurements of free (unbound) DNA using a single-stranded labeling dye (OliGreen), we determined the maximum binding ratios of the thiolated DNA to the 6, 13, 32, and 56 nm Au NPs to be 5 ± 1, 27 ± 5, 110 ± 12, and 525 ± 15 (n = 3), respectively. Thus, the surface coverage of DNA units on the Au NPs could be determined by measuring the signal decreasing ratio [(I0[Au1]+ − I[Au1]+)/I0[Au1]+]. We also found that the length of the DNA strand has a strong impact on the signals of the Au cluster ions formed from the DNA−Au NPs (13 nm). The signal intensities of the Au cluster ions [Au1]+, [Au2]+, and [Au3]+ all decreased upon increasing the DNA length from 15-mer to 45-mer (Supporting Information, Figure S5A). For example, the intensities of the signal of [Au1]+ were 72, 57, 44, and 29% for the 13 nm Au NPs modified with the 15-mer, 23-mer, 30-

Figure 2. (A) [Au1]+ intensities (I[Au1]+) and (B) relative values of [(I0[Au1]+ − I[Au1]+)/I0[Au1]+] of [Au1]+ ions obtained from Au NPs at various [HS-DNA]/[Au NPs] concentration ratios after irradiation with light from a pulsed Nd:YAG laser (power densities from 2.00 × 104 to 2.20 × 104 W cm−2). I0[Au1]+ and I[Au1]+ represent the signal intensities of [Au1]+ ions in the absence and presence of HS-DNA, respectively. Error bars represent standard deviations from three repeated experiments.

Figure 3. Relative signal intensities of [Au1]+ ions [(I0[Au1]+ − I[Au1]+)/I0[Au1]+] plotted with respect to the [HS-DNA]/[Au NPs] concentration ratio for (A) 6, (B) 13, (C) 32, and (D) 56 nm Au NPs. Error bars represent standard deviations from four repeated experiments. Other conditions were the same as those described in Figure 1. 1025

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(1.0 nM) in the presence of thiolated pDNA (40 nM), relative to those from the Au NPs in the absence of thiolated pDNA [panel (a) in Figure 4A]. The single-strand DNA was sufficiently flexible to partially uncoil its bases, exposing them to the Au NPs.21 As a result, the degree of attachment of pDNA to the Au NPs was low within a short incubation time (30 min). Upon the interaction of pDNA with its perfectly matched DNA (DNApm), partial duplex DNA structures were formed. Because the duplex structure does not permit uncoiling, the interactions of their bases with the Au NPs were relatively weak.21 As a result, the coverage of pDNA on the Au NPs was higher in the presence of DNApm and, therefore, the intensities of the signals of the Au cluster ions decreased in the presence of DNApm [panel (c) in Figure 4A]. In the control experiment [panel (d) in Figure 4A], we found that the presence of random DNA (rDNA) did not affect the efficiency of formation of the Au cluster ions. Figure 5 reveals that the value of [(I0[Au1]+ −

mer, and 45-mer thiolated DNA, respectively (Supporting Information, Figure S5B). We suspect that electron ejectioninduced particle explosion was suppressed by the presence of long-chain DNA on the Au NP surfaces. In addition, a very similar trend was observed for the values of [(I0[Au1]+ − I[Au1]+)/ I0[Au1]+], which increased with [HS-DNA]/[Au NPs] for the HS-DNA with lengths from 15-mer to 45 mer (Supporting Information, Figure S6) under an optimum laser power density of 2.1 × 104 W cm−2. To the best of our knowledge, this paper provides the first example of the use of LDI to analyze DNA coverage and structure on metal NPs. This simple, rapid, highthroughput detection system, based on the coupling of DNAconjugated Au NPs and LDI-MS, appears to hold great practicality for bioanalyses. Detection of Target DNA. We applied our findings to develop a simple and rapid MS-based assay, employing probe DNA (pDNA, 5′-thiolated DNA having a T15 linker and a 15base sequence for hybridization; sequence listed in Table 1) and 13 nm Au NPs, for the detection of targeting DNA, based on control of the interactions of thiolated pDNA and Au NPs (Scheme 2). Panel (b) in Figure 4A reveals slight decreases in the intensities of the Au cluster ions derived from the Au NPs

Figure 5. Values of [(I0[Au1]+ − I[Au1]+)/I0[Au1]+] for the pDNA−Au NP (1.0 nM) probe plotted with respect to the concentration of DNApm (0−500 nM). Inset: linear plot of [(I0[Au1]+ − I[Au1]+)/I0[Au1]+] against the concentration of DNApm. Other conditions were the same as those described in Figure 1. Error bars represent standard deviations from four repeated experiments.

I[Au1]+)/I0[Au1]+] (where I0[Au1]+ and I[Au1]+ are the signal intensities of [Au1]+ in the absence and presence of DNApm, respectively) of the pDNA−Au NPs (1.0 nM) probe was linearly sensitive to DNApm concentrations in the range of 5.0− 75 nM (the inset of Figure 5), with a correlation efficient (r) of 0.96. The limit of detection (LOD) for DNApm was 2.5 nM, based on a signal-to-noise (S/N) ratio of 3. Although a multistep procedure is required for the detection of target DNA in our system, the conjugation of oligonucleotides to Au NPs (Au−S interaction) and the centrifugation/washing process is relatively simple. Please note that the preparation of molecularbeacon-based probes for DNA analysis usually involves tedious labeling of expensive fluorophores and quenchers and laborious separation processes.22 However, our LDI-MS detecting system provides the advantages of rapid analysis (