Peroxidase mimicking DNA–gold nanoparticles for fluorescence detection of the lead ions in blood

Abstract

Oligonucleotide (T30695) modified gold nanoparticles (T30695–Au NPs) have been prepared and employed for quantification of lead ions (Pb²⁺) in blood. The detection of Pb²⁺ ions is through the formation of Au–Pb alloys and oligonucleotide–Pb²⁺ complexes that catalyze the H₂O₂-mediated oxidation of non-fluorescent Amplex UltraRed (AUR) to form a highly fluorescent oxidized AUR product. Surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF MS) and inductively coupled plasma mass spectrometry (ICP-MS) revealed the formation of Au–Pb alloys on the surfaces of the 40T30695–Au NPs (i.e., the system featuring 40 molecules of T30695 per Au NP) in the presence of Pb²⁺ ions, leading to increased catalytic activity for the H₂O₂-mediated oxidation of AUR. The fluorescence intensity (excitation/emission maxima: ca. 540/584 nm) of the oxidized AUR product is proportional to the concentration of Pb²⁺ ions over the range 0.1–100 nM, with a linear correlation (R² = 0.99). The 40T30695–Au NP/AUR probe is highly selective toward Pb²⁺ ions (by at least 200-fold over other tested metal ions). The 40T30695–Au NPs/AUR probe provided limits of detection (LOD, at a signal-to-noise ratio 3) for Pb²⁺ ions of 0.05 and 0.1 nM, in Tris–acetate solution (5 mM, pH 8.0) without and with salt (150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 1 mM CaCl₂), respectively. Without conducting tedious sample pretreatment, the approach allows detection of Pb²⁺ ions in blood samples, showing the potential of the 40T30695–Au NPs/AUR assay for on-site and real-time detection of Pb²⁺ ions in biological samples.

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Introduction

Because of their unique optical properties and high catalytic activity, metal nanomaterials are being studied extensively for their applications in many fields.¹ In the past five years, we have witnessed the development of several gold nanoparticle (Au NP)-based sensors for the detections of many important analytes (e.g., DNA, proteins, small organic analytes, metal ions).² Most of these sensing systems have taken advantage of the unique optical properties (e.g., high absorption coefficient, high quenching efficiency, high quantum yield, long Stokes shift) of Au NPs (sizes > 10 nm) or luminescent Au nanodots (sizes < 3 nm).^2,3 In addition, some Au NPs and nanoclusters (NCs) have been demonstrated to exhibit peroxidase-like activities.⁴ Fe₃O₄ magnetic NPs, sheet-like FeS nanostructures, spherical CeO₂ NPs, single-walled carbon nanotubes, graphene oxide, AgM (M = Au, Pd, Pt) NPs, and metallic nanocomposites that possess peroxidase- or oxidase-like activities have been used for the detection of H₂O₂, glucose, melamine, proteins, or DNA through their catalytic oxidations of various substrates, including 2,2′-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) and 3,3,5,5-tetramethylbenzidine (TMB).⁵

Recently, we developed a fluorescence assay for the detection of lead ions (Pb²⁺) at concentrations as low as 1.0 nM by taking advantage of the effect of Pb²⁺ ions on the AGRO100 (a G-quadruplex DNAzyme)-catalyzed hemin/H₂O₂-mediated oxidation of Amplex UltraRed (AUR).⁶ Monitoring the concentration of contaminating Pb²⁺ ions in aquatic ecosystems is an important issue because it can have potentially severe effects on human health and the environment.⁷ Children are more vulnerable to lead exposure than are adults because of their higher rates of intestinal absorption and retention.⁸ The action level for lead in drinking water, as set by the US Environmental Protection Agency (EPA), is 15 ppb (15 ng mL⁻¹; 72.4 nM). The World Health Organization (WHO) has announced that whole blood lead concentration of greater than 300 ppb in adults is indicative of significant exposure. The analytical methods that are used commonly for the detection of Pb²⁺ ions in blood include graphite furnace atomic absorption spectrometry, anodic stripping voltammetry, and inductively coupled plasma mass spectrometry (ICP-MS).⁹ Alternative techniques based on fluorescent probes using DNAzymes have been demonstrated for the detection of Pb²⁺ ions.¹⁰ Unlike those techniques, our AGRO100 probe does not allow the detection of Pb²⁺ ions in very complicated samples, mainly because the catalytic activity of DNAzyme is suppressed by high concentrations of salt.⁶

In this paper, we describe a simple assay—employing T30695, a G-quadruplex (G4) oligonucleotides possessing a sequence of (GGGT)₄ with T₆ linker at 3′-terminal, modified Au NPs, and AUR (T30695–Au NPs/AUR)—for the highly selective and sensitive detection of Pb²⁺ ions. G-rich oligonucleotide T30695 has been reported as an inhibitor of HIV integrase, acting as a viral enzyme responsible for the integration of viral DNA into the host–cell genome.^11a,b Metal ions such as K⁺ or Pb²⁺ ions induce T30695 to fold into stable intramolecular G-quadruplex structures that exhibit anticancer activity through the inhibition of signal transducer and activator of transcription 3 (Stat3), blocking ligand-induced Stat3 activation, and Stat3-mediated transcription of anti-apoptotic genes.^11c The sensing mechanism of the T30695–Au NPs/AUR probe is based on the formation of Au–Pb alloys and oligonucleotide–Pb²⁺ complexes that catalyze the oxidation of non-fluorescent AUR to form a highly fluorescent oxidized AUR product (Scheme 1). We have evaluated the roles that the pH and the DNA density on the Au NP surface play in determining the sensitivity of the T30695–Au NPs/AUR probe for the detection of Pb²⁺ ions. We validated the practicality of this approach through the analysis of the Pb²⁺ levels in blood samples.

Scheme 1 Cartoon representation of the catalytic ability of H₂O₂-mediated oxidation of non-fluorescent Amplex UltraRed (AUR) to form a highly fluorescent oxidized AUR product by (a) T30695, (b) Au NPs and (c) T30695–Au NP.

Experimental

Chemicals

Acetic acid, tris(hydroxymethyl)aminomethane (Tris), trisodium citrate, and all of the metal salts used in this study were purchased from Aldrich (Milwaukee, WI). Chloroauric(III) acid dehydrate was obtained from Sigma (St. Louis, MO). All of the oligonucleotides were purchased from Integrated DNA Technology (Coralville, IA). AUR was purchased from Invitrogen (Eugene, OR). Whole blood (SRM 955a-4) was obtained from the National Institute of Standards and Technology (NIST, Gaithersburg, MD). Milli-Q ultrapure water was used throughout the experiments.

Synthesis of Au NPs

Au NPs were prepared through citrate-mediated reduction of HAuCl₄.¹² Aqueous 1 mM HAuCl₄ (250 mL) was brought to a vigorous boil with stirring in a round-bottomed flask fitted with a reflux condenser and then 38.8 mM trisodium citrate (25 mL) was added rapidly. The mixture was heated under reflux for another 3 min, during which time its color changed from pale yellow to deep red. The solution was cooled to room temperature with continuous stirring. A Cintra 10e double-beam UV-vis absorption spectrophotometer (Cintra 10e, Dandenong, VIC, Australia) was used to measure the absorption of the Au NP solution. The dimensions of the Au NPs were measured using a transmission electron microscope (TEM, H7100, Hitachi High-Technologies Corporation, Tokyo, Japan); they appeared to be nearly monodisperse, with an average size of 13.6 ± 0.3 nm. The particle concentration of the Au NPs (ca. 15 nM) was determined according to Beer's law, using an extinction coefficient of ca. 2.54 × 10⁸ M⁻¹ cm⁻¹ at 520 nm for the 13.6 nm Au NPs.

Preparation of 40T30695–Au NPs

5′-Thiolated oligonucleotides were reacted directly with the Au NPs through attachment of both the HO(CH₂)₆S and oligo-S units onto the Au NP surfaces.¹³ Aliquots of the aqueous 13.6 nm Au NP solution (15 nM, 990 μL) in 1.5 mL tubes were mixed with the thiol-oligonucleotides (100 μM, 10 μL) to obtain final concentrations of 14.9 nM Au NPs and 1 μM thiol-oligonucleotides. The mixtures were centrifuged [relative centrifugation force (RCF) 30 000g; 20 min] to remove the excess thiol-oligonucleotides. The supernatants were separated and the oily precipitates washed with Tris–acetate (5.0 mM, pH 8.0). After three centrifuge/wash cycles, the DNA–Au NPs were resuspended separately in Tris–acetate (5.0 mM, pH 8.0) and stored in a refrigerator (4 °C). For the quantification of the surface coverage of DNA molecules on the DNA functional Au NPs, we used oligonucleotides with an alkanethiol group at the 5′-end and a fluorescein at the 3′-end to prepare functional Au NPs.¹⁴ The mixtures were centrifuged to remove the excess oligonucleotides by centrifugation for 20 min at 30 000g. The fluorescence of the supernatant containing the fluorescein modified oligonucleotides was measured. A calibration curve of fluorescence intensity (518 nm) against the fluorescein-DNA concentration was used to calculate the number of oligonucleotides linked to the Au NP surface. We estimated that there were 40 ± 3 molecules of T30695 per Au NP in the mixtures of Au NPs (14.9 nM). Characterization of 40T30695–Au NPs by surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF MS) generally followed the procedure reported earlier.¹⁵ Circular dichroism (CD) spectra of Tris–acetate solutions (pH 8.0) containing 1 μM T30695 or random DNA (rDNA) in the absence and presence of Pb²⁺ (10 μM) ions were recorded separately using a JASCO 720 spectropolarimeter (Easton, MD).

T30695–Au NP/AUR probe for Pb²⁺ ions

Equal volumes of different concentrations of Pb²⁺ ions in Tris–acetate buffer (5 mM, pH 8.0) were added separately to individual 1.5 nM T30695–Au NP solutions (100 μL). Solutions of H₂O₂ (4 mM, 50 μL) and AUR (100 μM, 50 μL) were added and the mixtures left at 27 °C for 2 h. The fluorescence of the oxidized AUR products in each solution was measured using a Cary Eclipse fluorescence spectrophotometer (Varian, CA) with excitation at 540 nm.

T30695–Au NPs were also prepared using four different concentrations (250, 500, 1000, and 2000 nM) of T30695. The first three mixtures were prepared without salt aging; the latter was prepared with salt aging in the presence of 200 mM NaCl. After incubation for 24 h at room temperature, the mixtures were centrifuged (RCF 30 000g; 20 min) to remove the excess DNA. The interactions of AUR (300 nM) with T30695–Au NPs (0.3 nM) having various numbers of T30695 molecules per Au NP were also investigated. After incubation for 1 h at room temperature, the mixtures were centrifuged (RCF 30 000g; 20 min) to remove excess AUR. The supernatants (400 μL) were mixed with horseradish peroxidase (HRP; 1 mU mL⁻¹, 50 μL) and H₂O₂ (0.4 mM, 50 μL). After incubation for 30 min, the mixtures were subjected to fluorescence measurements, with excitation and emission wavelengths of 540 and 584 nm, respectively.

Blood sample pretreatment

The blood samples were frozen at −20 °C until required for analysis. Acidic digestion of whole blood samples was performed, according to a sample-digestion method,¹⁶ prior to analysis. Aliquots of the treated blood sample (50 μL, tenfold-diluted) were spiked with standard Pb²⁺ solutions (50 μL, 0–1000 nM). The spiked samples were then diluted to 500 μL with solutions containing 40T30695–Au NPs (0.3 nM), H₂O₂ (0.4 mM), AUR (10 μM), and 5 mM Tris–acetate (pH 8.0). The final concentrations of Pb²⁺ ions in the mixtures extended over the range 0–100 nM. After incubation for 2 h, the mixtures were subjected to fluorescence measurement with excitation and emission wavelengths of 540 and 584 nm, respectively.

Results and discussion

Sensing strategy

The absorption spectra in Fig. 1a of the 40T30695–Au NPs (ca. 40 ± 3 molecules of T30695 per Au NP) in the absence and presence of Pb²⁺ ions reveal characteristic surface plasmon resonance absorption bands at 520 nm, revealing that both systems were dispersed well in the Tris–acetate (5 mM, pH 8.0) solutions. The slight differences in the absorbance of the two solutions arose mainly from the change in refractive index of the Au NP surfaces in the presence of Pb²⁺ ions, which interacted with the DNA and/or were deposited onto the Au NP surfaces (see below). The mixtures containing the 40T30695–Au NPs and AUR in the presence of Pb²⁺ ions (100 nM) exhibited strong fluorescence at 584 nm when excited at 540 nm (Fig. 1b). In control experiments, T30695 (100 nM) with G-quadruplex structures in the presence of Pb²⁺ ions (100 nM) exhibit negligible catalytic activity (curve a in Fig. S1†). In our previous study, we demonstrated the Pb²⁺–T30695 complex has no obvious catalyzing activity of H₂O₂-mediated oxidation in the absence of cofactor (e.g., hemin).⁶ In the absence of Pb²⁺ ions, the 40T30695–Au NPs displayed negligible catalytic activity for AUR (Fig. 1b). The catalytic activity of the 40T30695–Au NPs was approximately 13.7 times higher than that of the unmodified Au NPs (citrate-capped Au NPs) in the presence of 100 nM Pb²⁺ ions (Fig. S1†). Our results suggested that Pb²⁺ ions might interact with the oligonucleotides and Au NPs, thereby playing a role in affecting the catalytic activity of the 40T30695–Au NPs. The Au NPs reacted with thiolated oligonucleotides in solution to form the DNA functional Au NPs through Au–S bonding. The Au⁺ ions on the surfaces of T30695–Au NPs rapidly reacted with Pb²⁺via strong aurophilic interactions.¹⁷ Improved catalytic activity through the formation of Pb atoms on Au NPs was reported by several groups, mainly due to the synergistic effects provided by Au and Pb atoms and low electronic free energy.¹⁸ In addition, Pb²⁺ can also bind to Au NPs via specific Pb²⁺–T30695 interactions. The Pb²⁺/Pb⁰ and Au⁺/Au⁰ coexisted on the particle surface, which greatly affected the surface properties of Au NPs.¹⁹ In other words, various valance (oxidation) states of Pb (Pb²⁺/Pb⁰) and Au (Au⁺/Au⁰) on the particle surfaces accounted for their high peroxidase-like activity even in the absence of cofactor. Therefore, the formation of the T30695–Pb²⁺ complexes and Au–Pb alloys on the particles play major roles in improving their catalytic activity. As a result, the 40T30695–Au NPs in the presence of Pb²⁺ ions possessed remarkable catalytic activity for the transformation of AUR. The oxidized AUR products has a high quantum yield (>70%); in contrast, AUR is only weakly fluorescent.²⁰ Thus, the fluorescence increased upon increasing the concentration of Pb²⁺ ions.

Fig. 1 (a) UV-vis absorption and (b) fluorescence spectra of Tris–acetate solutions (5 mM, pH 8.0) containing AUR (10 μM), H₂O₂ (0.4 mM), and 40T30695–Au NPs (0.3 nM) in the (i) absence and (ii) presence of Pb²⁺ ions (100 nM). The excitation wavelength in (b) was set at 540 nm.

Characterization of Au–Pb alloys and Pb²⁺–oligonucleotide on Au NPs

To confirm that Pb²⁺ ions induced the increased catalytic activity of the 40T30695–Au NPs and citrate-capped Au NPs for the oxidation of AUR, we use X-ray photoelectron spectroscopy (XPS) to measure the binding energy (BE) of the Au 4f_7/2 electrons in the 40T30695–Au NPs and citrate-capped Au NPs in the absence/presence of Pb²⁺ ions (1 μM);²¹ the values of 84.1/84.4 eV and 84.0/84.3 eV, respectively, revealed the increase of oxidation state of the Au NP surfaces in the presence of Pb²⁺ ions (Fig. S2a†). That is, because of the formation of the Au–Pb bonds, the oxidation state of Au increased. In the presence of Pb²⁺ ions (1 μM), the signal of the Pb 4f_7/2 electrons of citrate-capped Au NP and 40T30695–Au NPs appeared at 139.1 eV and 138.8 eV, respectively, higher than that (137.0 eV) for Pb atoms (Fig. S2c†).²² The presence of a greater amount of Au⁺ ions, metastable metal surface atoms with high energy, and Pb²⁺–oligonucleotide complexes on the Au NP surfaces induced increases in the catalytic activity of the 40T30695–Au NPs.²³ The observed peaks (see Fig. S3a†) of 40T30695–Au NPs in the absence of Pb²⁺ ions at m/z 196.94 is assigned to [Au₁]⁺ ions. The observed peaks (Fig. 2) of 40T30695–Au NPs at m/z 402.92, 403.92, and 404.92 corresponding to [^196.94Au + ^205.98Pb]⁺, [^196.94Au + ^206.98Pb]⁺, and [^196.94Au + ^207.98Pb]⁺ ions, respectively, strongly support the formation of Au–Pb alloys on the Au NP surfaces in the presence of 10 μM Pb²⁺ ions. The citrate-capped Au NP in the presence of Pb²⁺ ions show similar results to that of 40T30695–Au NPs (Fig. S3b†). There was no statistical difference in the average particle diameter or size distribution of 40T30695–Au NPs in the absence and presence of 10 μM Pb²⁺ ions as determined from transmission electron microscopy (TEM) images (Fig. S4a†), revealing that there is only a monolayer or submonolayer of Pb on the surface of Au NPs. Moreover, the lattice d-spacing (Fig. S4b†) and X-ray diffraction (XRD) patterns of 40T30695–Au NPs (Fig. S5b†) in the absence and presence of Pb²⁺ were almost the same, revealing the core structure of Au NPs was not changed. Similarly, citrate-capped Au NP (Fig. S5a†) in the absence and presence of Pb²⁺ also have same XRD patterns.

Fig. 2 SALDI mass spectra of solutions containing Tris–acetate buffers (5 mM, pH 8.0) and 40T30695–Au NPs (7.5 nM) in the presence of Pb²⁺ ions (10 μM). The asterisk (*) represents unknown peaks. Inset: comparison of predicted (left; green) and observed (right; red) isotopic distributions of the Pb atom and AuPb alloy in the SALDI mass spectra. The peaks at m/z 196.94, (205.98, 206.98, 207.98) and (402.92, 403.92, 404.92) are assigned to [Au]⁺, [Pb]⁺, and [Au + Pb]⁺ ions, respectively. In total, 300 pulsed laser shots were applied under a laser fluence of 62.5 μJ. Other conditions were the same as those described in Fig. 1.

Effects of the surface oligonucleotide

To determine whether the presence of T30695 was essential for the detection of Pb²⁺ ions, we also tested citrate-capped Au NPs with/without DNA modification and Au NPs modified with a random DNA (5′-HS-TAC GAG TTG AGA ATC CTG AAT GCG-3′) strand (rDNA–Au NPs) as catalysts for the oxidation of AUR in the presence of Pb²⁺ ions and H₂O₂. Fig. 3a reveals that the catalytic activities of the three catalysts decreased in the order T30695–Au NPs > rDNA–Au NPs ≫ citrate-capped Au NPs. In the presence of Pb²⁺ (100 nM), the catalytic activity of the T30695–Au NPs was approximately 1.6 and 13.7 times greater than those of the rDNA–Au NPs and citrate-capped Au NPs, respectively. Using ICP-MS,²⁴ we estimated the numbers of Pb atoms deposited and Pb²⁺ ions (n = 5) adsorbed on each type of Au NP, obtaining values of 760 ± 15, 720 ± 13, and 430 ± 10 Pb atoms (ions) per T30695–Au NP, rDNA–Au NP, and citrate-capped Au NP, respectively. Relative to rDNA, T30695 interacts more strongly with Pb²⁺ ions.^6,23a Unlike T30695, the rDNA did not form G-quadruplexes with Pb²⁺ ions, as confirmed using CD spectroscopy (Fig. S6†), where an apparent CD band at 265 nm and a small positive peak near at 310 nm are typical CD characteristics of Pb²⁺-stabilized parallel G-quadruplex structures (Fig. S6†).^23a Although rDNA is not able to form G-quadruplexes with Pb²⁺, the high densities of oligonucleotides on the particle show strong complexation with Pb²⁺ ions.²⁵ Our results suggest that the oligonucleotides assist in having more Pb²⁺ atoms (ions) on the surfaces of Au NPs, mainly through electrostatic attractions and/or strong aurophilic interactions. The Pb²⁺–oligonucleotide complexes and Au–Pb alloys on the surfaces of Au NPs induced increased catalytic activity for the H₂O₂-mediated oxidation of AUR. We note that the local concentrations (amounts) of Pb²⁺–oligonucleotide complexes on the surfaces of Au NPs are greater than those in the bulk solution, leading to increased catalytic activity.²⁶ The relatively low catalytic activity of citrate-capped Au NPs when compared to the other two DNA functional Au NPs is due to their lower stability in solution. In addition, the selectivity of Au–Pb alloys in the absence of DNA is quite poor; Hg²⁺, Ag⁺, and Cu²⁺ ions affected the determination of the concentration of Pb²⁺ ions.

Fig. 3 (a) Fluorescence responses of solutions containing AUR (10 μM), H₂O₂ (0.4 mM), one of the citrate-capped Au NPs (0.3 nM) or DNA-conjugated Au NPs (0.3 nM), and Pb²⁺ (0–100 nM). (b) Effect of the T30695 density per Au NP in the presence of Pb²⁺ ions (0–100 nM) on the catalytic reaction of AUR. Other conditions were the same as those described in Fig. 1.

By using oligonucleotides that had been modified with an alkanethiol-group at the 5′-end and a fluorescein at the 3′-end, we determined the number of T30695 molecules on each Au NP. By establishing a calibration curve of the fluorescence intensity (518 nm) against the fluorescein–DNA concentration, the numbers of oligonucleotides linked to the Au NP surfaces were determined to be 10 ± 2, 20 ± 4, 40 ± 3, and 120 ± 8 molecules of T30695 per Au NP in the mixtures of Au NPs (14.9 nM) and thiolated-T30695 at the concentrations of 250, 500, 1000, and 2000 nM, respectively. The amount of surface T30695 increased upon increasing the thiolated-T30695 concentration, reaching a plateau at 2000 nM thiolated-T30695. For simplicity, we denote the T30695–Au NPs with n molecules of T30695 per Au NP as nT30695–Au NPs; for example, there were 40 molecules of T30695 per Au NP in the 40T30695–Au NPs. To further support our results that the surface DNA density on the Au NP played an important role in determining the sensitivity, we compared the fluorescence intensity of the four different T30695–Au NP (0.3 nM) solutions in the presence of Pb²⁺ ions (0–100 nM). Fig. 3b reveals that the Pb²⁺-induced catalytic activity of the H₂O₂-mediated oxidation of AUR increased upon increasing the number of T30695 molecules per Au NP. From the ICP-MS data, we estimated the number of Pb atoms deposited and Pb²⁺ ions adsorbed on each 10, 20, 40 and 120T30695–Au NP to be 440 ± 3, 720 ± 8, 760 ± 15, and 770 ± 10, respectively. This result supports the notion that increased numbers of Pb ions and atoms (Au–Pb alloys and T30695–Pb²⁺ complexes) accounted for the increased catalytic activity of the Au NPs for the oxidation of AUR. However, no significant enhancement in the catalytic activity was observed in the 120T30695–Au NPs when compared to that produced by the 40T30695–Au NPs. Therefore, to avoid the excessive increment in the surface density of DNA and steric effects, we used the 40T30695–Au NPs sample for the following experiments.

Next, we also investigated the interactions between the AUR and T30695 molecules on the 10, 20, 40, and 120T30695–Au NPs. At constant concentrations of AUR (300 nM) and the four T30695–Au NPs (0.3 nM), we determined that the number of AUR molecules per 10, 20, 40, and 120T30695–Au NP were 530 ± 2, 590 ± 5, 700 ± 3, and 710 ± 6, respectively. As a control, we found that there were 320 ± 3 AUR molecules bound to each Au NP capped with citrate, mainly through hydrophobic interactions. The electrostatic and π–π stacking play the role of interactions between surface oligonucleotide and AUR substrates. At a low surface density, the T30695 molecules were present on the Au NPs as flattened structures; therefore, they possessed weak affinity toward AUR. In addition, at low T30695 content, the number of Pb atoms and ions on the surfaces of the T30695–Au NPs was low; as a result, the catalytic activities were low. In control experiments, we found that 3-mercaptopropionic acid-modified Au NPs and non-thiolated T30695-adsorbed Au NPs (14 ± 1 molecules of T30695 per Au NP) both exhibited very low catalytic activity toward AUR in the presence of H₂O₂ (0.4 mM). Relative to the thiolated T30695 molecules, which are bound to the Au NPs through Au–S bonding, the non-thiolated T30695 molecules bound to the Au NPs merely through electrostatic interactions and were relatively unstable.²⁷

Effect of pH

Fig. S7† reveals the effect of pH on the catalytic activity of the 40T30695–Au NP/AUR probe in the absence and presence of 100 nM Pb²⁺ ions. The value of (I_F − I_F0)/I_F0 for the 40T30695–Au NP/AUR probe reached a maximum at pH 8.0, in which I_F0 and I_F are the fluorescence intensities of the solutions in the absence and presence of Pb²⁺ ions, respectively. Similar to horseradish peroxidase (HRP)/H₂O₂ systems, the catalytic activity of the 40T30695–Au NP/AUR probe increased upon increasing the pH from 6.0 to 8.0.²⁸ At values of pH greater than 8.0, Pb(OH)₂ and PbO likely formed to great extents, minimizing the formation of Au–Pb alloys and 40T30695–Pb²⁺ complexes. In addition, oxidized AUR products are unstable at values of pH greater than 8.5.²⁸

Sensitivity and selectivity of 40T30695–Au NP/AUR probe toward Pb²⁺ ions

Fig. 4a displays the fluorescence response of the 40T30695–Au NP/AUR probe toward Pb²⁺ ions (0–1.0 μM). This probe exhibited a linear response (R² = 0.99) of its fluorescence intensity at 584 nm with respect to the concentration of Pb²⁺ ions over the range 0–100 nM (inset). At an S/N ratio of 3, the LOD for Pb²⁺ ions was 0.05 nM—at least one order of magnitude lower than those of other NP and oligonucleotide-based optical sensors.^10,29 We further tested the sensitivity of our T30695–Au NP/AUR probe for Pb²⁺ ions in Tris–acetate (5 mM, pH 8.0) solutions containing 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 1 mM CaCl₂ (Fig. S8a†) or containing 10 μM cysteine (Fig. S8b†), providing LODs of 0.1 and 0.3 nM, respectively. These results indicated that our approach holds great potential for the detection of Pb²⁺ ions in biological samples.

Fig. 4 (a) Fluorescence spectra of the 40T30695–Au NP/AUR probe in the presence of different concentrations of Pb²⁺ (0–100 nM). (b) Selectivity of the 40T30695–Au NP/AUR probe toward Pb²⁺ ions. The concentration of Pb²⁺ ions was 100 nM; the concentration of each of the other metal ions was 1.0 μM in (b). Other conditions were the same as those described in Fig. 1. Inset of (a): plot of the value of I_F of the solutions against the concentration of Pb²⁺ ions.

To investigate the selectivity of the 40T30695–Au NP/AUR probe (0.3 nM) toward Pb²⁺ ions under the optimal conditions (10 μM AUR, 0.4 mM H₂O₂, 5 mM Tris–acetate, pH 8.0), we added 100 nM Pb²⁺ and one (1.0 μM) of the following ions—Na⁺, K⁺, Mg²⁺, Ca²⁺, Sr²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Mn²⁺ Cr³⁺, Hg²⁺, Cd²⁺, Ag⁺, Pt²⁺, Al³⁺, or Fe²⁺—into the probe solutions. Fig. 4b reveals that the 40T30695–Au NP/AUR probe was specific toward Pb²⁺ ions over the tested metal ions. We also performed a series of competition experiments to test the practicality of our 40T30695–Au NP/AUR probe for the selective detection of Pb²⁺ ions. The tolerance concentrations of common metal ions for the detection of Pb²⁺ ions were high; positive errors occurred in the detection of 100 nM Pb²⁺ ions in the presence of 10 mM K⁺, 1 mM Mg²⁺, or 1 mM Ca²⁺ were all less than 3%.

Detection of Pb²⁺ ions in blood

To test the practicality of our developed approach, we used a standard addition method to determine the concentrations of Pb²⁺ ions in reference blood sample (NIST SRM 955a-4) and collected blood sample (male, 25 years old). Fig. 5 displays a linear correlation (R² = 0.99) between the response and the concentration of Pb²⁺ ions spiked in the diluted (100-fold) blood sample (SRM 955a-4) over the range 0–100 nM. When using the 40T30695–Au NP/AUR probe, we determined the concentration of Pb²⁺ ions in this blood sample (n = 5) to be 2.48 ± 0.18 μM (certified value: 2.63 ± 0.018 μM). Using a t-test (the t-test value is 2.78 at a 95% confidence level), the 95% confidence interval for Pb²⁺ ions was 2.25–2.71 μM, revealing that no significant differences existed between the values measured using our new approach and the certified value. A blood sample collected from a healthy adult male was diluted (3-fold) with 5 mM Tris–acetate (pH 8.0) and spiked with standard Pb²⁺ (0–50 nM), which were then analyzed separately by using the 40T30695–Au NP/AUR probe (n = 5) and ICP-MS (n = 5). The concentrations of Pb²⁺ obtained by ICP-MS and the 40T30695–Au NP/AUR probe (Fig. S9†) were 84.5 ± 9.2 and 79.1 ± 15.7 nM, respectively. The Student's t-test and F-test value for the correlation between the two methods were 0.59 and 2.91 (the t-test and F-test values are 2.31 and 6.39 at a 95% confidence level, respectively), suggesting that the two methods did not provide significantly different results. In comparison with other NP- and oligonucleotide-based optical methods, our assay for Pb²⁺ ions is relatively simple, cost-effective, selective, and sensitive. Notably, most reported NP-based optical sensors rarely applies the determination of Pb²⁺ ions concentrations in complex blood samples.^10,29

Fig. 5 Analysis of a representative blood (NIST SRM 995a-4) sample using the 40T30695–Au NP/AUR probe. Aliquots of the diluted (100-fold) blood sample were spiked with Pb²⁺ ions at concentrations in the range 0–100 nM. Other conditions were the same as those described in Fig. 1.

Conclusions

The T30695–Au NPs possess ultrahigh peroxidase-mimicking activity for the oxidation of AUR in the presence of Pb²⁺ ions. The increased catalytic activity of the T30695–Au NPs was due to the formation of Au–Pb alloys and Pb²⁺–oligonucleotide complexes. The surface density of T30695 units on Au NP surface played an important role in controlling the formation of the Au–Pb alloys and T30695–Pb²⁺ complexes and, therefore, the catalytic activity of the T30695–Au NPs. Under the optimal conditions, the 40T30695–Au NP/AUR probe was highly sensitive (LOD = 0.05 nM) and selective toward Pb²⁺ ions, with a linear detection range of 0.1–100 nM. Common metal ions such as Na⁺, K⁺, Mg²⁺, and Ca²⁺ in biological samples did not cause interference in the detection of Pb²⁺ ions when applying this highly sensitive and selective assay. To the best of our knowledge, this simple, rapid, and cost-effective system is the first example of a DNA/Au NP-based sensor for the detection of Pb²⁺ ions in real blood samples.

Acknowledgements

This study was supported by the National Science Council of Taiwan (contract NSC 98-2113M-002-011-MY3), the National Taiwan Health Research Institutes Taiwan (contract NHRI-EX100-10047NI), and the Institute of Nuclear Energy Research (contract 1002001INER082).

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Footnote

† Electronic supplementary information (ESI) available: Fig. S1–S8. See DOI: 10.1039/c2an35599j

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