Research Article www.acsami.org
Rapid, Surfactant-Free, and Quantitative Functionalization of Gold Nanoparticles with Thiolated DNA under Physiological pH and Its Application in Molecular Beacon-Based Biosensor Qing Xu,† Xinhui Lou,*,† Lei Wang,† Xiaofan Ding,† Haixiang Yu,‡ and Yi Xiao‡ †
Department of Chemistry, Capital Normal University, Xisanhuan North Road 105, Beijing 100048, China Department of Chemistry and Biochemistry, Florida International University, Miami, Florida 33199, United States
‡
S Supporting Information *
ABSTRACT: The controlled attachment of thiolated DNA to gold nanoparticles (AuNPs) dictates many applications. This is typically achieved by either “aging-salting” processes or low-pH method, where either Na+ or H+ is used to minimize charge repulsion and facilitate attachment of thiolated DNA onto AuNPs. However, the “aging-salting” process takes a long time, and is prone to aggregation when used with larger AuNPs. Surfactants are needed to precoat and thereby enhance the stability of AuNPs. The low-pH method can disrupt the structural integrity of DNAs. We report here an oligoethylene glycol (OEG) spacer-assisted method that enables quantitative and instantaneous attachment at physiological pH without the need for surfactants. The method is based on our finding that an uncharged OEG spacer as short as six EG units can effectively shield against repulsion between AuNPs and DNAs, substantially enhancing both the adsorption kinetics and thermodynamics of thiolated DNAs. We applied this to thiolated DNAs of various lengths and thiol modification positions and to large AuNPs. Importantly, our method also allows for the direct immobilization of thiolated molecular beacons (MB), and avoids particle aggregation due to intermolecular hydrogen bonding. The prepared MBAuNPs were successfully used for the fluorescent detection of target DNA at nanomolar concentrations. The OEG spacer appears to offer a highly effective parameter for tuning DNA adsorption kinetics and thermodynamics besides pH and salt, providing a novel means for highly controllable and versatile functionalization of AuNPs. KEYWORDS: gold nanoparticle, thiolated DNA, DNA-gold nanoparticle conjugates, ethylene glycol spacer, physiological pH, adsorption kinetics, quantitative adsorption
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INTRODUCTION The attachment of thiolated DNA onto gold nanoparticles (AuNPs) via the self-assembly of thiolated DNAs enables the marriage of the unique molecular recognition capabilities of DNA with the outstanding optical properties of AuNPs.1−6 Numerous applications in nanostructure assembly,7−11 biosensors,12−14 and cancer diagnostics and treatment15−17 have been reported since the first demonstration of DNA-AuNP conjugates in 1996.18 The high density of DNAs on AuNPs results in a high local concentration of DNAs and possible steric hindrance, leading to the loading density and conformation-dependent properties. For example, the equilibrium binding constant (Keq) for DNA-AuNPs is a function of DNA loading on the AuNP, and is orders of magnitude higher than the Keq for the corresponding nanoparticle free system.19,20 Aptamer-loaded gold nanostars exhibited enhanced in vitro efficacy in cancer cells by the increased loading of aptamer on the gold surfaces.21 A short internal complement DNA (sicDNA) strand causes up to a 5-fold increase in association rate by inducing a conformational change that extends the DNA away from the surface, making it more available to bind target nucleic acids.22 Therefore, methods that © XXXX American Chemical Society
enable loading density and conformation and well-controlled preparation of DNA-AuNPs are critical for their numerous applications.14,23 However, there are only a few reports on how to load thiolated DNAs onto AuNPs. The most widely used DNAAuNPs are derived from 13 nm AuNPs stabilized by weakly absorbed citrate ions. There are two technical difficulties associated with preparation. First, the strong electrical repulsion force between the negatively charged AuNPs and DNAs seriously restricts DNA adsorption. Second, buffers containing very low concentrations of NaCl (e.g., 30 mM) can cause the irreversible aggregation of AuNPs before attachment occurs. Two elegant methods have been reported to overcome this problem. The first is an “aging-salting” process, where the salt concentration is gradually increased after an overnight aging step in buffer containing a very low concentration of NaCl (Figure 1A).1,18,24 Na+ moderately minimizes the net charge of both AuNPs and DNA (Figure 1D) and thus the electrical Received: July 8, 2016 Accepted: September 22, 2016
A
DOI: 10.1021/acsami.6b08350 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
thiolated DNAs onto AuNPs under physiological pH without the need for surfactant. This method has been successfully applied for thiolated DNAs with various length and thiol modification positions and AuNPs of varying sizes. Working with molecular beacons (MBs),34 we demonstrate rapid immobilization of thiolated functional DNAs with well-defined secondary structures onto AuNPs. This work suggests that the OEG spacer is a highly effective parameter for tuning DNA adsorption kinetics, providing a novel means for the controllable and versatile functionalization of AuNPs.
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EXPERIMENTAL SECTION
Chemicals and Materials. All oligonucleotides (Supporting Information, Table S1) were synthesized and purified by HPLC by Sangon Biotech Co., Ltd. (Shanghai, China). Gold(III) chloride trihydrate (HAuCl4·3H2O), dithiothreitol (DTT), and triethylamine (TEA) were purchased from J&K Scientific LTD (Peking, China). 13 nm AuNPs were synthesized in-house via the standard citrate reduction method.1,39 50 and 100 nm gold nanoparticles (AuNPs) were purchased from BBI Solutions (Cardiff, UK). AuNP sizes were confirmed by transmission electron microscopy (TEM, Figure S1). H2O purified with a Millipore ultrapure water system was used for all experiments. NAP-5 columns (Sephadex G-25 Medium, DNA grade) were purchased from Pharmacia Biotech (GE Healthcare, Uppsala, Sweden). ZEN0117-disposable low volume cuvettes (100 μL) were purchased from Malvern Instruments Ltd. (Malvern, England). Synthesis of 13 nm AuNPs. All glassware was cleaned in aqua regia (HCl:HNO3 of 3:1), rinsed with Millipore water, and then ovendried prior to synthesis. AuNPs were synthesized based on the standard citrate reduction process.18,39 Briefly, an aqueous solution of HAuCl4 (1 mM, 200 mL) was heated and refluxed for 10 min while stirring. 20 mL of 38.8 mM trisodium citrate solution was quickly added, resulting in an immediate solution color change from pale yellow to wine red. After 15 min of refluxing, the solution was slowly cooled to room temperature and filtered through a 0.22 μm cellulose nitrate filter. The final gold nanoparticle solution was ∼10 nM based on UV−vis absorption spectroscopy. The solution was stored at 4 °C. Functionalization of AuNPs with Thiolated DNAs. To generate free thiol groups for surface immobilization, we mixed 40 μL of 100 μM stock thiolated DNAs (Table S1, Supporting Information) with 40 μL of 0.1 M DTT and 1.6 μL TEA at room temperature for 30 min. The freshly reduced DNA sequences were subsequently purified through the desalting NAP-5 columns, preequilibrated with 10 mM phosphate buffer, pH 7.4 (PB, NaH2PO4 /Na2HPO4), and quantified by UV−vis spactroscopy at 260 nm. For the “aging-salting” process, purified DNA probes in 1 mL PB were mixed with 10 nM AuNPs at a molar ratio of 500 (DNA:AuNP) and incubated for 18 h for the “aging” step. Then PB containing 1 M NaCl was added stepwise (every 0.5 h) to gradually increase the concentration of NaCl to 0.3 M from 18 to 28 h for the “salting” step. Finally, the mixture was incubated from 28 to 40 h at 0.3 M NaCl. In our method, DNAs in PB were mixed with AuNPs at designated ratios, followed by the immediate addition of 1 M NaCl in PB to adjust the final concentration of NaCl as needed. Dynamic Light Scattering (DLS) Measurements. To measure the hydrodynamic sizes of unmodified AuNPs and DNA-AuNP conjugates, DLS measurements were conducted at 25 °C using a Zetasizer Nano ZS90 (Malvern Instruments Ltd., England) equipped with a red (633 nm) laser and an avalanche photodiode detector. A ZEN0117-disposable low volume cuvette (100 μL) was used as the sample container. The solution viscosity was set as 0.8872 cP, which is the dynamic viscosity of water at 25 °C. The solution refractive index was set as 1.330, which is the refractive index of water at 25 °C with the light wavelength at 633 nm. The medium used in this study is an aqueous solution with similar viscosity and refractive index to water (see CRC Handbook of Chemistry and Physics). All experiments were performed in the water dispersant state at 25 °C with a 30 s equilibration time and 90° measurement angle. The number of runs
Figure 1. Attaching thiolated DNAs to AuNPs using the (A) agingsalting, (B) low-pH, and (C) OEG spacer-assisted methods. (D) Mechanisms of tuning surface absorption of DNAs by Na+, H+, and OEG spacer, respectively.
repulsion force between them, allowing high-density attachment within 1−2 days. The second is a low-pH strategy, where a citrate buffer at pH 3.0 was used. Three DNA bases, adenine, cytosine, and thymine, are slightly positively charged, and the citrate-capping layer on AuNPs is partially protonated.25,26 Thus, the charge repulsion between the AuNPs and DNA at pH 3 are effectively weakened, which facilitated faster adsorption of DNA onto the AuNPs and enabled quantitative attachment of thiolated DNA in 3 min (Figure 1B,D).26 In both methods, the density of attached DNA can be significantly improved by using higher NaCl concentrations.26−28 However, the “aging-salting” process takes a long time, and is prone to aggregation when used with larger AuNPs. Surfactants (sodium dodecyl sulfate27 or fluorinated surfactant29) or other molecules (bis(p-sulfonatophenyl)phenylphosphine dehydrate dipotassium salt (BSPP),30 dATP,31 high-molecular-weight polyethylene glycol (PEG)32) are needed to precoat and thereby enhance the stability of AuNPs, but this precoating may interfere with downstream applications. On the other hand, the low-pH method can disrupt the structural integrity of the welldefined secondary structures,33 which are required for many functional oligonucleotides, such as molecular beacons,34 aptamers,35,36 and DNAzymes37 to perform their functions. This means that conjugation must be followed by conformational transformation to restore the desired secondary structure of DNAs on the AuNP surface. This process is quite hard to control due to the complicated surface effects. Low pH may also cause depurination of bases, producing abasic sites.38 Therefore, a fast and quantitative immobilization method near neutral pH is highly desirable in order to maintain the structural integrity and stability of oligonucleotides. We have devised a method in which an oligoethylene glycol (OEG) spacer, comprising 6−18 ethylene glycol units located between a terminal thiol group and a DNA sequence, serves as a charge shield to minimize repulsion between DNA and AuNPs and enhance adsorption kinetics (Figure 1C,D). Our method enables the rapid and quantitative attachment of B
DOI: 10.1021/acsami.6b08350 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces and duration of each measurement were set automatically. The intensity weighted harmonic mean size, Z-average size, was used for the whole study due to its high reliability. Three repeated measurements were carried out for each sample to estimate the errors of the measurements. During the aging-salting immobilization process, the concentration of AuNPs was gradually decreased due to the stepwise addition of salt solution, typically from the initial ∼5 nM to around 3 nM. This should have no significant effect on the DLS measurements. The sizes of the DNA-AuNP conjugates were continuously monitored in situ by DLS measurement at the selected time points. Quantification of DNA Loading Capacity. After the loading process, excess DNAs were removed by centrifugation at 17 968 × g for 15 min. The concentration of DNAs in the supernatant was determined by measuring absorption at 260 nm. The total quantity of DNAs on the AuNPs was then calculated by subtracting this amount from the initial input amount. The number of DNAs per particle was calculated by dividing the total amount of DNAs on AuNPs by the quantity of AuNPs. UV−vis spectroscopy was carried out with a SHIMADZU UV-2550. Gel Electrophoresis. DNA-AuNPs were prepared at a DNA:AuNP molar ratio of 500 in PB, followed by the immediate addition of 1 M NaCl to a final concentration of 0.3 M and incubated for 2 h. The same gel conditions used in our previous studies were applied here.39 We prepared a 2% agarose gel in 1 × TBE buffer (89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA, pH 8.3). The 20 μL of concentrated DNA-AuNPs in PB with 50% glycol were added to each lane and the gel was run for 1 h at 100 V in 1 × TBE. The gel image was recorded with a digital camera. Fluorescence Kinetic Experiments. The DNAs (FAM-15-EG18SH, FAM-15-A10-SH, FAM-15-T10-SH) in PB were combined with AuNPs at various molar ratios (Figure 4B), followed by the immediate addition of 1 M NaCl to adjust the final concentration of NaCl. The final concentrations of NaCl we used were 10, 20, 30, 50, 75, 100, and 150 mM, for DNA:AuNP ratios of 25, 50, 75, 100, 125, 150, and 200, respectively. Higher concentrations of NaCl were needed to achieve complete adsorption of DNAs as the DNA-AuNP ratio increased. The concentration of NaCl used in Figure 4 was not optimized. A higher loading percentage could be achieved at the higher NaCl concentration. The fluorescence intensity (F) over time was measured by a fluorescence spectrophotometer (F-7000, HITACHI). The fluorescence intensities of the DNAs that were diluted into a buffer containing a specific concentration of NaCl without AuNP were determined as F0. Then F/F0 as the function of time was plotted in Figure 4A.
Figure 2. Dynamic light scattering (DLS) measurements of the hydrodynamic sizes of DNA-AuNPs during the aging-salting process. The dashed line represents the NaCl concentration of the sample in the procedure. The molar ratio of DNAs to AuNPs was 500 for all samples. Error bars were calculated from three repeated measurements.
Mechanism: OEG Spacer as Short as Six EG Units Effectively Shields against Repulsion between AuNPs and DNAs in a Wide pH Range. To clarify the mechanism, we used DNAs containing OEG spacers of varying lengths (EG18, EG12, and EG6) to prepare DNA-AuNPs by mixing them with AuNPs in PB for 2 h. In contrast to thiolated DNAs without OEG spacers, the loading capacity determined by the UV−vis absorption (Supporting Information) increased as the spacer length increased (Figure 3A). These results strongly support our proposed charge shielding mechanism. The shorter OEG spacer shielded electronic repulsion less effectively, leading to a lower loading capacity. Even so, we still obtained a quite high loading capacity (99 ± 5 strands/particle) with 15EG6-SH, suggesting that the charge shielding effect was quite effective. The charge shielding mechanism was further confirmed by investigating the pH effect. The strong pH effect on the immobilization of thiolated DNAs on AuNPs has been recently reported.26 As the decrease of the buffer pH, the immobilization kinetics of thiolated DNA on AuNP substantially speed up due to the minimized electric repulsion (Figure 1D). Interestingly, we found that pH (from 3 to 10) had no obvious effect on the immobilization kinetics of 15-EG18-SH when the high molar ratio (DNA:AuNP = 500) was used, showing the similar size increase over time (Figure S3). The absorption was quite fast at the beginning of incubation, where ∼70% size increase was achieved in the first 5 min. The results implied that the EG18 spacer indeed effectively shielded the charge of DNA, resulting in the disappear of the pH effect. The speed-up effect of EG18 was so strong that DNAs rapidly attached to AuNPs even under the basic condition (pH > 7). The pH tolerance of our OEG spacer assisted method is beneficial for the preparation of DNA-AuNP complex in an extremely wide pH range. However, pH may have an impact on the conformation of DNAs on AuNPs since pH affects the formation of the hydrogen bonding inside and between DNAs.33 Most biomolecules have the best bioactivity under the physiological pH (7.4), which is near the neutral pH (7.0). Therefore, the preparation of DNA-AuNPs in a desired buffer at the physiological pH can avoid the possible impact of buffer exchange on the conformation of DNAs on the surfaces of AuNPs in the downstream applications. Thus, the most popular
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RESULTS AND DISCUSSION OEG Spacer Substantially Enhances the Adsorption Kinetics of Thiolated DNAs on AuNPs. The effects of various spacers on the immobilization kinetics of three thiolated DNAs (Supporting Information, Table S1) onto 13 nm AuNPs (Supporting Information, Figure S1) was monitored during the “aging-salting” process via dynamic light scattering (DLS) (Figure 2).5,39,40 These thiolated DNAs contained the same 15nt DNA sequence but with a different spacer of about same length: 18 ethylene glycols (15-EG18-SH), 10 deoxyadenosines (15-A10-SH), or 10 deoxythymines (15-T10-SH).27 Interestingly, the size increase of 15-EG18-SH-AuNPs was substantially greater than 15-T10-SH-AuNPs or 15-A10-SH-AuNPs, especially in the first 2 h. By the end of the aging step, the size of 15EG18-SH-AuNPs was similar to that of the 15-A10-SH- or 15T10-SH-AuNPs at the end of the full 40 h “aging-salting” process. This indicated that the EG18 spacer greatly favored the kinetics of attachment. We hypothesized that EG18 therefore serves as an effective shield to minimize the electronic repulsion between AuNPs and DNA (Figure 1D). C
DOI: 10.1021/acsami.6b08350 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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(Figure 3C). The extremely small polydispersity indexes (PDI) was further confirmed no aggregation occurred for 15-EG18SH-AuNPs (Figure 3D). The similar phenomena was also observed for 15-EG12-SH-AuNPs and 15-EG6-SH-AuNPs. Furthermore, we observed that NaCl thermodynamically favored the homogeneous attachment of DNAs as observed in “aging-salting″27 and low-pH method.26 We achieved extremely high loading capacities for 15-EG18-SH, 15-EG12-SH, and 15EG6-SH (267 ± 2, 200 ± 7, and 147 ± 10 strands/particle, respectively) after a 2 h incubation in PB/0.3 M NaCl (Figure 3A). The high loading capacity of 15-EG18-SH was in good agreement with the literature reported values after the whole “aging-salting” process.27 The NaCl concentration can be used to tune the DNA loading capacity on AuNPs, based on the gradually increased hydrodynamic sizes and slower migration rate on agarose gel that we observed as salt concentration increased (Figure 3E, Supporting Information, Figure S4). The sharp red bands41 and small polydispersity index indicated that each AuNP population displayed roughly the same number of DNAs. In addition, NaCl kinetically favored the attachment of DNAs. Noted that the size of 15-EG18-SH-AuNPs immediately after the addition of 15-EG18-SH and 0.3 M NaCl (Figure 3F) was comparable to the final size of 15-EG18-SH-AuNPs prepared via a 40 h “salting-aging” process (Figure 2). The similar effects were also observed for 15-EG12-SH-AuNPs and 15-EG6-SH-AuNPs (Supporting Information, Figures S2, S5). Thus, salt and OEG spacers synergistically enable the rapid attachment of DNAs at high densities. OEG Spacer Allows the Quantitative Attachment of DNAs on AuNP in a Wide Loading Range. We next determined that thiolated DNAs with OEG spacer could be attached to AuNPs in a quantitative fashion. We first combined fluorescein (FAM)-labeled 15-EG18-SH and AuNPs at a molar ratio of 75. The attachment is expected to quench fluorophore emission, due to Förster resonance energy transfer (FRET)42,43 between the AuNPs and FAM.44,45 Thus, the percentage of DNAs attached to AuNPs can be quantitatively obtained from the value of F/F0, where F0 and F indicate fluorescence intensity before and during incubation.26 F/F0 dropped to around zero (∼0.05) immediately after the addition of FAM15-EG18-SH to AuNPs in PB/30 mM NaCl (Figure 4A), suggested that virtually all of the DNA was adsorbed. In contrast, the F/F0 plateaued at 0.24 and 0.1 for FAM-15-A10SH and FAM-15-T10-SH, respectively, after 40 min. We observed complete adsorption of FAM-15-EG18-SH onto AuNPs (F/F0 < 0.10) over a wide molar ratio range from 25
Figure 3. (A) DNA loading capacity of AuNPs after 2 h incubation respectively with 15-EG18-SH, 15-EG12-SH, and 15-EG6-SH in PB with or without 0.3 M NaCl. The data reported are averages of three individual experiments. (B) Photographs of DNA-AuNPs after 2 h incubation. Characterization of 15-EG 18-SH-AuNPs after 2 h incubation using (C) UV−vis spectroscopy and (D) DLS. (E) 2% agarose gel analysis of 13 nm AuNPs before and after 2 h incubation with 15-EG18-SH in PB containing various concentrations of NaCl. All conjugates were concentrated in PB before loading onto the gel. (F) Kinetic size measurements of 15-EG18-SH-AuNPs during incubation in PB containing 0, 0.1, 0.2, or 0.3 M NaCl. For all samples, DNA:AuNP ratio was 500. The first measurements were conducted 3 min after the addition of 15-EG18-SH and salt.
used buffer system, PB with or without NaCl at physiological pH, was used in the following study. NaCl Kinetically and Thermodynamically Favors the Attachment of DNAs on AuNPs. Next, we explored the effects of salt concentration, as this can affect the formation of DNA secondary structures. We mixed the 15-EG18-SH, 15EG12-SH, and 15-EG6-SH DNAs with AuNPs in PB, then added 1 M NaCl to achieve various final concentrations of NaCl, followed by a 2 h incubation. We observed that NaCl up to 0.3 M did not cause aggregation of AuNPs when we used DNAs with OEG spacers (Figure 3B), as the AuNP solution retained its red color throughout the incubation period. In sharp contrast, the AuNP solution turned blue when the DNAs were either 15-A10-SH or 15-T10- SH, indicating that extensive aggregation had taken place. The UV−vis spectrum of 15-EG18SH-AuNPs essentially overlapped that of unmodified AuNPs, which indicated that great stability of the 15-EG18-SH-AuNPs
Figure 4. (A) Kinetics of fluorescence decrease indicating DNA adsorption as a function of spacer component at the molar ratio of 75 (DNA-to-AuNP). (B) Percentage of absorbed DNA (FAM-15-EG18SH) as a function of DNA to AuNP ratio, where the concentrations of NaCl in PB were 10, 20, 30, 50, 75, 100, and 150 mM for DNA to AuNP ratio of 25, 50, 75, 100, 125, 150, and 200, respectively. D
DOI: 10.1021/acsami.6b08350 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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MB1-EG18-SH generally retained its stem-loop structure prior to attachment onto AuNPs, with minimal intermolecule hybridization. Therefore, the excellent stability of MB1-EG18SH-AuNPs was obtained during the preparation process (Figure 5A, #4). To further demonstrate the dependence of stem-loop formation on the salt concentration, we respectively prepared MB1-EG18-SH-AuNP in PB with or without 0.15 M NaCl. DLS measurements confirmed rapid attachment under both conditions, with a respective size increase of 6.5 and 14.5 nm in 0 and 0.15 M NaCl immediately after the addition of DNAs (Figure 6, S10). Interestingly, denaturation in 15 mM NaOH
to 200 (Figure 4B). This eliminates the need to quantify the number of DNAs attached to AuNPs and enables multiplexed functionalization with precise control over probe density as demonstrated in the low-pH method.26 OEG Spacer-Assisted Method Is a General Method. Our OEG spacer-assisted method was generally applicable across different DNA sequences (Table S1, Supporting Information) and AuNPs of varying sizes (13, 50, and 100 nm in diameter). In all cases, the DNA-AuNP solution retained its red color in the high salt buffer (Figure 5A,B),
Figure 5. Functionalization of 13 nm (A) and 50 nm (B) AuNPs with various DNAs (1, 15-EG18-SH; 2, HS-EG18-15; 3, 30-EG18-SH; 4, MB1-EG18-SH; 5, FAM-MB2-EG18-SH; 6, MB1-T10-SH). (C) Schematic drawing of molecular beacons active on the AuNP surface. (D) Fluorescence spectra of FAM-MB2-EG18-SH- modified AuNPs at various target DNA concentrations. Figure 6. DLS measurements of MB1-EG18-SH-AuNP prepared in PB without (A) and with 0.15 M NaCl (B) before (red) and after (blue) the addition of 15 mM NaOH. DLS measurements of MB1-T10-SHAuNP prepared by low-pH method before (red) and after (blue) the buffer exchange in PB with 0.15 M NaCl (C) or 15 mM NaOH (D).
demonstrating a lack of aggregation relative to the DNA-free sample. The UV−vis absorption spectra and DLS measurements before and after DNA attachment further confirmed successful adsorption (Supporting Information, Figures S6−8). Importantly, as demonstrated with a thiolated molecular beacon (MB) construct (Figure 5A, #5), the prepared MBAuNPs were fully functional, based on the mechanism shown in Figure 5C. The fluorescence of FAM-MB2-EG18-SH was almost completely quenched after being attached to AuNPs, but gradually recovered (Figure 5D) as the concentration of target DNA (Supporting Information, Table S1) increased. We could detect as little as 1 nM target, with a dynamic range from 1 to 1000 nM that is comparable to the value reported in the literature (0.5−200 nM).45 Indeed the different DNA spacers and immobilization conditions were used in our and others’ studies. OEG Spacer-Assisted Method Enables the WellControlled Secondary Structure of DNAs on AuNPs. More importantly, our method can immobilize the MBs on AuNPs with a well-confined stem-loop structure. To demonstrate the great structure integrity of DNAs by our OEG spacerassisted method, a direct comparison of our method with “aging-salting” process and low-pH method was conducted by using MB1-T10-SH. First, in keeping with prior observations,45,46 we found that AuNPs combined with MB1-T10-SH underwent aggregation during the aging-salting process (Figure 5A, #6; Supporting Information, Figure S9). Ideally, the conformation of AuNP-conjugated MBs should transition from linear to a stem-loop structure as buffer salinity increases. However, increasing salinity also enables intermolecular hybridization, leading to aggregation. With the OEG spacer,
produced no observable change in the size of MB1-EG18-SHAuNP prepared without NaCl, indicating the expected linear structure (Figure 6A). In contrast, we observed a 6 nm increase in size for MB1-EG18-SH-AuNP prepared with 0.15 M NaCl, matching the estimated size change upon the open of the stemloop structure under denatured condition (Figure 6B). The narrow DLS peaks were observed not only during the preparation process, but also upon incubation with 15 mM NaOH (Figure 6A,B), suggesting the excellent stability MB1OEG18-SH-AuNP. The results confirmed that the structure integrity of MB1-EG18-SH on AuNPs highly depended on the buffer salinity. With the OEG spacer, a buffer with high salinity was able to be used from the beginning of the immobilization process to ensure the stem-loop structure of MB1-EG18-SH prior to attachment onto AuNPs. Thus, the intermolecule hybridization was minimized and the MB1-EG18-SH-AuNPs kept an excellent stability. MB1-T10-SH-AuNPs were then prepared by following the low-pH method reported in the literature.26 Mild aggregation was observed during the preparation at pH 3.0 (Figure 6C), showing the obviously broadened major peak at 21.04 nm and a small broad peak centered at 458.67 nm. In addition, the 5.35 nm size increase from 15.69 to 21.04 due to the immobilization of MB1-T10-SH on AuNP was much smaller than that observed in our OEG spacer-assisted method, suggesting the significantly E
DOI: 10.1021/acsami.6b08350 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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lower loading capacity of MB1-T10-SH by the low-pH method. Upon buffer exchange into PB containing 0.15 M NaCl, serious aggregation occurred, revealed by significantly shifted and broadened DLS peaks. The similar phenomenon was also observed when the buffer changed to 15 mM NaOH (Figure 6D). The bad stability should be attributed to either the hardto-control conformational transformation of MB1-T10-SH on AuNP or the release of DNAs upon buffer change. For example, PB/0.15 M NaCl at pH 7.4 favors the hybridization than citrate/0.15 M NaCl at pH 3.0, and thus MB1-T10-SH with the linear conformation on AuNP would hybridize with each other, resulting in aggregation. On the other hand, it has been reported that the low pH also favored the immobilization of nonthiolated DNAs on AuNPs.25 Therefore, it is highly possible that some thiolated DNAs were immobilized on AuNPs via electrostatic interaction. When the buffer was changed into 15 mM NaOH, the electrostatic interaction between DNAs and AuNPs was minimized, probably leading to the release of DNAs from AuNPs and then the decreased stability. Overall, the results indicated that MB1-T10-SH-AuNP prepared by the low-pH method was less stable than that prepared by our OEG spacer-assisted method due to the coexistence of the multiple conformation of MB1-T10-SH on AuNP prepared by the low-pH method.
CONCLUSIONS In summary, we report a facile method for rapid and quantitative attachment of thiolated DNAs to AuNPs at physiological pH condition. Compared to the classical “agingsalting” process and recently reported low-pH method, our OEG spacer-assisted method provides many advantages (Table S2). There are two unique features of our method. First, attachment occurs at physiological pH, avoiding the stability and structural integrity problems caused by low pH. Second, our method enables complete attachment of DNAs in a very broad surface density range, potentially eliminating the need to quantify the extent of DNA attachment. This approach should be especially appealing for applications requiring DNAs with well-defined secondary structures such as duplex, G-quadruplexes, or aptamers. Our work also suggests that the OEG spacer offers a novel parameter for controlling the adsorption kinetics and functionalization of AuNPs. ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b08350. DNA sequences used in this study, Methods, TEM images, UV−vis spectra, and DLS (PDF)
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REFERENCES
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AUTHOR INFORMATION
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[email protected]. Tel: +86-10-68902491 ext. 808. Fax: +86-10-68902320. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful for financial support from the National Natural Science Foundation (21305093, 21675112) and Yanjing Young Scholar Program of Capital Normal University. F
DOI: 10.1021/acsami.6b08350 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
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DOI: 10.1021/acsami.6b08350 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
