Functionality of Nonfunctional Diluent Ligands within Bicomponent

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Functionality of Non-Functional Diluent Ligands within Bicomponent Layers on Nanoparticles Sohyun Seo, Jang Ho Joo, Do Hyun Park, and Jae-Seung Lee J. Phys. Chem. C, Just Accepted Manuscript • Publication Date (Web): 08 Jun 2017 Downloaded from http://pubs.acs.org on June 8, 2017

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The Journal of Physical Chemistry

Functionality of Non-Functional Diluent Ligands within Bicomponent Layers on Nanoparticles Sohyun Seo, Jang Ho Joo, Do Hyun Park, and Jae-Seung Lee* Department of Materials Science and Engineering, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, Republic of Korea.

ABSTRACT: The selective binding of nanoparticles to a specific entity is highly important for building assembly-based superstructures and detecting various biomarkers and targets. Such type of binding can only be achieved by controlling the particle surface properties. Herein, we demonstrate the construction of a solid/liquid interfacial layer comprising multiple components on nanoparticle surfaces in order to obtain the desired binding properties. In addition to the actual binding components, the chemical structures of non-functional diluent ligands play a significant role in determining the overall binding properties of the nanoparticles. We also evaluated the binding properties of the nanoparticles coated with multiple components, and optimized them by varying the interfacial coating conditions. Gold nanoparticles and singlestranded oligonucleotides are chosen as the core nanoparticles and coating functional ligands for model system, respectively, and examined in the presence of various chemical and biological diluents.

1 ACS Paragon Plus Environment


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1. Introduction Controlling the binding properties of nanoparticles is central to the design of building blocks for constructing novel superstructure materials and developing sensing probes for the detection of various targets.1-13 In the field of modern nanoscience and nanotechnology, the fabrication of nanoparticles bearing chemical functionalities that can recognize and bind specific entities is one of the most important research topics. The most general and reasonable method to achieve this goal is to construct solid/liquid interfacial layers on the nanoparticle surfaces in a solution phase using chemical and biological ligands that can recognize and bind other materials of interest. Owing to the combination of the four natural bases in oligonucleotide sequences and the complexity of the three-dimensional protein structures, these ligands including nucleic acids and antibodies naturally possess substantial specificity and affinity, thus leading to successful assay schemes with high sensitivity and selectivity or well-organized nanoparticle superstructures. Primarily, the performance of controlled and desired binding reactions of nanoparticles typically requires (1) molecular design and synthesis of the ligands,14-17 or (2) thermodynamically favored binding reaction conditions.18 In spite of the previously reported successful approaches, however, the complicated and cumbersome organic syntheses, together with the “trial-and-error” type of excessive, lengthy, and laborious reaction optimization, still remain a challenge. One of the main strategies to control the binding properties of nanoparticles involves constructing multicomponent layers on the nanoparticle surfaces using non-reactive chemicals, or “diluents”, in addition to the original functional ligands.19-24 The primary purpose of the diluents is to simply decrease the density of the functional ligands at the interface, while still leading to significant effects for a variety of other functionalities, such as the controlled “valency”


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of nanoparticles for building superstructure assemblies,25-28 kinetic enhancement of nanoparticle conjugation with thiolated DNA,29 decreased steric hindrance of crowding DNA strands on nanoparticles,30 controlled hybridization properties of DNA-nanoparticle conjugates,31-34 and reduced fouling or nonspecific binding of nanoparticles.19, 35-37 These diluents typically include polyethylene glycol (PEG) and non-functional oligonucleotides, which are combined with functional ligands at a specific ratio before conjugation with the nanoparticle surfaces. To date, however, the interfacial conjugation of diluents on the nanoparticle surfaces has been investigated only with respect to the stoichiometry of the diluents and functional ligands.38-39 Since the presence of diluents on the nanoparticle surfaces, even in small proportions, significantly and precisely affects the binding properties of the nanoparticles, a further systematic examination of the nanoparticle-conjugation conditions needs to be thoroughly carried out. Herein, we present an investigation of the factors that can influence the construction of an interfacial bicomponent ligand layer on gold nanoparticle (AuNP) surfaces, and describe the manner in which this bicomponent layer can affect the overall binding properties of the particles. Unlike the conventional co-functionalization methods using two types of reactive ligands, our method is restricted to the combination of reactive and non-reactive ligands (diluents), both bearing the same anchoring group. While the non-reactive diluents indeed did not directly interact with other AuNPs, they exhibited more complicated interfacial interactions with both their neighboring ligands and the core AuNP surfaces, eventually controlling the binding and catalytic properties of the AuNPs. Importantly, we demonstrated that even potentially selfcomplementary DNA can be stably and functionally conjugated with AuNPs by controlling the



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diluents, and that those DNA-AuNPs were used as excellent nanoprobes for the colorimetric detection of a tumor suppressor gene.

2. Experimental 2.1. Materials and instrumentation Two pairs of thiolated complementary DNA sequences (S1: 5´ HS-A10-ATTATCACT 3´; S2: 5´ HS-A10-AGTGATAAT 3´, BRCA1 3´: 5´ CTTTTGTTC-A10-SH 3´, BRCA1 5´: 5´ HSA10-GATTTTCTTC 3´), 5´ HS-A20 3´, 5´ HS-T20 3´, 5´ HS-C20 3´, and BRCA1-target (5´ GAACAAAAGGAAGAAAATC 3´) were purchased from Genotech Inc. (Daejeon, South Korea) and Bioneer Corp. (Daejeon, South Korea). Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H2O; cat# 520918), trisodium citrate dihydrate (cat# S4641), poly(sodium 4styrenesulfonate) (PSSS, m.w. = 70,000, cat# 527483), polypropylene glycol (PPG, m.w. = 725; cat# 202312), polyethylene glycol (PEG, m.w. = 1000; cat# 81188), Tween20™ (cat#: P7949), dithiothreitol (cat# 43815), 4-nitrophenol (cat# 241326), and sodium borohydride (cat# 480886) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Thiolated polyethylene glycol (HS-PEG; m.w. = 1,000) was purchased from SunBio (Anyang, South Korea). Illustra Nap-5 Sephadex columns were purchased from GE Healthcare Life Sciences (Marlborough, MA, USA). Ultrapure water (18 MΩ·cm) was supplied by Millipore (Billerica, MA, USA) and used for all experiments. UV-vis spectroscopic analysis was conducted using an Agilent 8453 spectrophotometer (Santa Clara, CA, USA).


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2.2. Synthesis of AuNPs AuNPs (15 nm in diameter) were synthesized based on the modified Turkevich-Frens method.40-41 To an aqueous HAuCl4 solution (0.254 mM, 50 mL) heated at 100 oC was rapidly added a trisodium citrate solution (38.8 mM, 0.94 mL) upon stirring. The color of the solution turned from pale yellow to dark red in 5 min, indicating the formation of AuNPs that exhibited their surface plasmon resonance (SPR) in the visible range. The resultant AuNP solution was allowed to cool to room temperature upon stirring, and it was then stored at room temperature until use.

2.3. Construction of bicomponent ligand layers on AuNPs The DNA strands with a terminal disulfide group were deprotected into the corresponding thiolated strands by treatment with 0.1 M dithiothreitol in 0.17 M phosphate buffer (pH 8.0) at room temperature for 30 min, which were then purified using NAP-5 columns. The thiolated functional DNA strands (S1, S2, BRCA1 3´, and BRCA1 5´) were combined with a diluent of interest (HS-PEG or HS-X20, X = A, T, or C) at the desired concentration, and eventually added to the AuNP solution. The mixed solutions were incubated at 0.3 M NaCl in a phosphate buffer (pH 7.4, 0.1 M phosphate, 0.01% Tween20) to promote the ligand-AuNP conjugation under various conditions (conjugation time and temperature). The ligand-AuNP conjugates were finally obtained by removing the unconjugated DNA strands and diluents by repeated sets of centrifugation, removal of the supernatant, and addition of buffer for the redispersion of the ligand-AuNP conjugates.



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2.4. Melting transition of two complementary DNA/PEG-AuNP conjugates (DNA/PEG-AuNPs) Two complementary DNA/PEG-AuNPs or DNA/X20-AuNPs were combined to hybridize under the common buffer condition ([NaCl] = 0.3 M, pH 7.4, 0.1 M phosphate, 0.01% Tween20) at 25 oC for 12 h. The melting transitions were measured by monitoring the change in extinction at 525 nm every 1 min from 25 to 80 oC. The melting temperature (Tm) was obtained from the temperature where the maximum of the first derivative of the melting transition occurred.

2.5. Molecular accessibility control by the on-particle bicomponent interfacial layer Four types of DNA/PEG-AuNPs with different proportions of the diluent (0, 60, 80, and 100%) were prepared. Water (100 μL) was added to the DNA/PEG-AuNP solutions (200 μL, 2.5 nM) and the four mixtures were incubated at 25 oC for 10 min. A 4-nitrophenol solution (400 μL, 0.01 M) was added to the mixtures, immediately followed by the injection of an NaBH4 solution (400 μL, 0.2 M). The color change of the mixtures was observed by monitoring the extinction at 410 nm every 1 min.

2.6. Colorimetric detection of the BRCA1-target The DNA-AuNP and DNA/PEG-AuNP nanoprobes (DNA = BRCA1 3´and 5´) were synthesized as described above under the desired conjugation conditions, and then combined with the BRCA1-target (total [DNA-AuNP] and [DNA/PEG-AuNP] = 1 nM, total [BRCA1target] = 10 or 100 nM, [NaCl] = 0.15 M). The nanoprobes and BRCA1-target in the mixtures were allowed to hybridize at 25 oC and monitored over a time course of 1 day until observation of the color change from red to purple. The melting transition of the nanoprobes was measured by monitoring the change in extinction at 525 nm every 1 min from 25 to 80 oC. The Tm was


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obtained from the temperature where the maximum of the first derivative of the melting transition occurred. 3. Results and Discussion We first constructed a bicomponent layer composed of functional DNA sequences and non-charged polyethylene glycol (PEG) diluent ligands on the nanoparticle surfaces. The functional DNA sequences were designed to contain 19 nucleotides, whose length was comparable with that of the PEG diluent (m.w. 1000).42-47 In fact, when a longer HS-PEG (m.w. 2000) was employed, no DNA hybridization was observed (data not shown), probably because the PEG strand was sufficiently long to block the DNA. Since both DNA and PEG possess a monothiol anchoring group in common for binding on the AuNP surface, they form the same Au-S bonds with the similar bonding energy. Once the DNA/PEG mixtures were prepared, they were further combined with the bare AuNPs for conjugation. All these experimental designs, including (1) the length of both DNA and PEG, (2) anchoring group, and (3) preparation of DNA/PEG mixtures prior to the AuNP conjugation, allow to directly correlate the ratios of DNA and PEG before and after their immobilization on the AuNP surfaces. The chemical and physical properties of the on-particle bicomponent layers were thermodynamically investigated by observing the melting transitions of the hybridized DNA/diluent-AuNPs. The thermodynamic analysis of the melting properties provides a clear measure of their binding properties depending on the conditions under which the on-particle bicomponent layer is constructed. Moreover, this analytical method is convenient, cost-effective, rapid, and does not require complicated instrumentation.



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Figure 1. (A and B) Melting profiles of hybridized DNA/PEG-AuNPs (S1 and S2) synthesized at various ratios of DNA to diluent PEG (A: DNA:PEG = 10:0 to 4:6; B: DNA:PEG = 3:7 to 1:9). (C) First derivatives of the melting profiles in Figure 1A. (D) Plot showing the Tms of the melting transitions in Figure 1A and the FWHMs of the first derivatives in Figure 1C as a function of the proportion of PEG. Note that the melting transitions are normalized for clarity, and that the conjugation time and temperature are 9 h and 25 oC, respectively.


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Next, we investigated the effect of the PEG diluent at different proportions on the binding properties of the DNA/PEG-AuNPs. The AuNPs were conjugated with a mixture of PEG and DNA for 9 h at 25 oC; the mol% of PEG varied from 0 to 60. These conjugation conditions (9 h at 25 oC) were considered as standard and could be compared with a series of other conjugation schemes to elucidate the influence of the controlling parameters. Finally, the binding properties of the DNA/PEG-AuNP conjugates were evaluated by obtaining the Tms of their melting transitions (Figure 1A). As the proportion of PEG increased from 0 to 60 mol%, the melting transitions gradually shifted to lower temperatures and became broader. The high density of DNA on the nanoparticle surfaces is essential for the cooperative melting properties of DNAconjugates of various nanoparticles,48 which explains the apparent decreasing cooperativity upon increases of PEG on the nanoparticles. We also analyzed the number of DNA strands of the DNA/PEG-AuNPs, and obtained ~79 strands per particle conjugated with 100% DNA, and ~19 strands per particle conjugated with 60 mol% of PEG, which is in good agreement with the analysis of melting profiles.49-51 The decrease in cooperativity and binding properties was further confirmed at increasing proportions of PEG over 70 mol% by observing a larger broadness of the transitions, which also, exhibited negligible increases in extinction, as low as 10% of the maximum extinction of the originally dispersed DNA/PEG-AuNPs (Figure 1B). The cooperative binding properties of the DNA/PEG-AuNPs were quantitatively analyzed by obtaining the first derivatives of the melting transitions (Figure 1C). As the proportion of the PEG increased, the binding properties became gradually less cooperative, which was clearly demonstrated by the broadening of the first derivatives. To systematically analyze the full-width at half maximum (FWHM) and Tm, we compared them by plotting as a function of the mol% of the PEG (Figure 1D). With the



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increasing proportion of PEG, the Tm linearly decreased from 52.4 to 41.6 oC (slope = -1.82×10-1 o

C/mol%). This consequence is similar with previous literature report, where polyadenine (polyA)

was employed as a diluent.32, 48 Interestingly, the FWHM slowly increased at low PEG ratios (< 40 mol%, slope = 3.65×10-2 oC/mol%), whereas the FWHM rapidly increased at high PEG ratios (> 40 mol%, slope = 2.08×10-1 oC/mol%). The entire observation described in Figure 1 and its analysis clearly demonstrate that the binding properties of the DNA/PEG-AuNPs are highly dependent on the density of the functional DNA on the particle surface, and that the diluents definitely “dilute” the nanoparticle binding affinity depending on their proportions. In addition to the proportion of the diluents, the conjugation time of the diluents on the nanoparticle surface also attracted our attention and was investigated over different time periods ranging from 1 h to 1 week. In order to perform a systematic analysis, we first conjugated the AuNPs with a mixture of DNA and HS-PEG at a 1:1 ratio ([DNA] = [PEG] = 1.75 μM) to build a bicomponent layer on the particle surfaces, and incubated the final mixture for various time periods at 25 oC. The binding properties of the DNA/PEG-AuNPs were further evaluated by obtaining their melting transitions (Figure 2A). Interestingly, the surface density of DNA dramatically increased with increasing longer conjugation time, as evidenced by the increased Tms and sharpened melting transitions, which are the results of the improved cooperative binding properties of the DNA/PEG-AuNPs.18 The first derivatives of the melting transitions were also obtained to further quantitatively analyze the binding properties of the DNA/PEG-AuNPs (Figure 2B). The sharpening of the melting transitions was more clearly demonstrated by a decrease in FWHM of the first derivatives. The Tm and FWHM of the hybridized DNA/PEGAuNPs were plotted as functions of the conjugation time, and compared with those of hybridized DNA-AuNPs synthesized in the absence of HS-PEG (Figures 2C and 2D). Interestingly, the Tms


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of the DNA-AuNPs were overall far higher than those of the DNA/PEG-AuNPs by a difference of 4 to 17 oC, indicating an overall lower DNA density of the DNA/PEG-AuNPs. Importantly, no significant change in Tm was observed in the case of DNA-AuNPs regardless of the conjugation time, which indicates that the conjugation of DNA on the AuNP surfaces was almost immediately completed after a few hours (Figure S1A, see Supporting Information). On the other hand, the presence of HS-PEG during the conjugation led to a gradual time-dependent increase in DNA density over a period of one week, which was demonstrated by a gradual increase in Tm (Figure 2C). This strong contrast between the bicomponent interfacial layers of the DNA/PEGAuNPs and the monocomponent layers of the DNA-AuNPs also resulted in a dramatic difference in the FWHMs of the first derivatives of their melting transitions. The FWHM of the DNAAuNPs remained almost the same at 2.5 oC over a conjugation period of one week, while that of the DNA/PEG-AuNPs kept decreasing significantly over the same period (Figure 2D and Figure S1B, see Supporting Information). The delayed equilibrium of the conjugation mixture in the presence of HS-PEG could be explained as illustrated in Scheme 1. Once the number of the DNA strands on the DNA-AuNPs reaches a plateau, the free thiol DNA strands in the reaction mixture can hardly be in close proximity to the DNA-AuNPs owing to the strong electrostatic repulsion.52 Without any additional increase in ionic strength, this consistent repulsion practically leads to the termination of the conjugation reaction and consequently the consistent Tms. In the presence of HS-PEG at 50% in the reaction mixture, nearly 50% of PEG and 50% of DNA, at a lower density, could initially occupy the AuNP surface. The immobilized PEG on the AuNPs, however, would sterically hinder the free thiol DNA and HS-PEG from anchoring on the AuNP surface. This steric hindrance may reduce the frequency of the effective collision for the conjugation, but can be occasionally ineffective only when the PEG strands are stretched out of



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the particle to expose more available gold surface for the conjugation. As a result, it takes a significant time delay of over 1 week until the equilibrium of the conjugation reaction is reached. Eventually, the AuNP is coated with a layer composed of both PEG and DNA, whose Tm cannot be as high as that of the DNA-AuNPs (Figure 2C). The conjugation equilibrium could be accelerated at a lower pH.53-54


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Figure 2. (A) Melting profiles of the hybridized DNA/PEG-AuNPs (S1 and S2; DNA:PEG = 1:1) whose conjugation time ranges from 1 h to 1 week at 25 oC, (B) their first derivatives, (C) plot showing their Tms, and (D) plot showing the FWHMs of their first derivatives as a function of the conjugation time. For comparison, the Tms and FWHMs of the DNA-AuNPs synthesized without HS-PEG (DNA 100%) were obtained in Figures 2C and 2D. (E) Melting profiles of the hybridized DNA-AuNPs synthesized with DNA at 2 μM and non-thiolated PEG at various concentrations ranging from 2 to 2000 μM at 25 oC. (F) Tms of the melting transitions of the hybridized DNA-AuNPs synthesized with DNA at 2 μM and one of the non-thiolated polymers (PEG, PPG, and PSSS) at various concentrations up to 2000 μM at 25 oC. Note that the melting transitions are normalized for clarity, and that the x-axis is drawn to a log scale in Figures 2C, 2D, and 2F.



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Scheme 1. Schematic illustration of (A) rapid completion of the DNA-AuNP conjugation, and (B) delayed equilibrium of the conjugation mixture for the bicomponent interfacial layer formation in the presence of DNA and the HS-PEG diluent. As the PEG immobilized on the AuNPs unfavorably decreased the conjugation kinetics, the free PEG in the solution can possibly reduce the effective collision between the thiol ligands and AuNPs because of its non-specific adsorption on the AuNP surface.55 To elucidate whether the anchoring of HS-PEG on the particle surface plays an essential role to produce delayed kinetics, we employed non-thiolated PEG of the same molecular weight (m.w. = 1000) instead of HS-PEG, then prepared a 1:1 mixture with thiol DNA, and finally combined the mixture with AuNPs. To maximize the potential effect of non-thiolated PEG, we increased its ratio to 10, 100, and 1000 in comparison with DNA, and limited the conjugation time to 6 h. Two complementary ligand-conjugated particles were prepared and evaluated by obtaining their melting transitions (Figure 2E). Very interestingly, the Tms were obtained almost at 54 oC that is similar to the Tm of the DNA-AuNPs prepared without HS-PEG (Figure 2C), regardless of the added amount of the non-thiolated PEG. This result strongly suggests that the initial fixation of the non-charged diluent on the AuNPs inevitably slows down the thiol DNA-AuNP conjugation reaction. In addition to the non-thiolated PEG, we further investigated the effect of non-thiolated polypropylene glycol (PPG) and poly(sodium 4-styrenesulfonate) (PSSS) on the binding properties the ligand-AuNPs under the same conditions (Figure S2, see Supporting Information). Neither an increased hydrophobicity (PPG) nor the presence of negative charges (PSSS) produced any noticeable difference in Tm (Figure 2F), which provides additional evidence to the pivotal role of the thiol anchoring group of the non-charged diluent.


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Figure 3. (A) Melting profiles of the hybridized DNA/PEG-AuNPs (S1 and S2) synthesized at various conjugation temperatures (10 to 60 oC), (B) their first derivatives, (C) plot showing their Tms, and (D) plot showing the FWHMs of their first derivatives as a function of the conjugation temperature. For comparison, the Tms and FWHMs of the DNA-AuNPs synthesized without HSPEG (DNA 100%) were obtained in Figures 3C and 3D. Note that the melting curves are normalized for clarity and that the conjugation time was 9 h.



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We also investigated the effect of the conjugation temperature on the bicomponent ligand layer formation on the nanoparticle surfaces. Specifically, we controlled the reaction temperature at which the mixed HS-PEG and thiol DNA (50% : 50%) were immobilized on the particle surfaces from 10 to 60 oC, and examined their binding properties by obtaining the corresponding dehybridization profiles (Figure 3A). When the conjugation was conducted at a higher temperature, the dehybridization occurred at a higher temperature, and the shape of the curves became sharper, which was further confirmed by obtaining the first derivatives of the melting transitions (Figure 3B). At high temperatures, the molecular motion of PEG is expected to be more vigorous, leading to a more frequent exposure of the available particle surface for the additional thiol ligand conjugation (Scheme 1). This hypothetical interpretation convincingly supports our observation that the Tm increased from 40 to 49 oC as the conjugation temperature increased (Figure 3C, 50%). On the other hand, the DNA-AuNPs obtained by conjugation under the same temperature conditions without HS-PEG, exhibited a negligible increase in Tm (< 2 oC) (Figure 3C, 100%; Figure S3A, see Supporting Information). The FWHMs of the first derivatives in Figure 3B and Figure S3B (see Supporting Information) show a significant contrast between the DNA/PEG-AuNPs (50%) and the DNA-AuNPs (100%). In the case of the DNA/PEG-AuNPs (50%), the FWHM dropped down dramatically as the temperature increased, while that of the DNA-AuNPs remained almost unchanged regardless of the conjugation temperature (Figure 3D). Although the increased conjugation temperature for the DNA/PEGAuNPs increased the Tms and decreased the FWHMs, their changes became insignificant above 30 oC. Moreover, the Tms and FWHMs of the DNA/PEG-AuNPs are unlikely to reach those of the DNA-AuNPs, indicating that the presence of HS-PEG and the consequent “dilution” of DNA on the AuNPs are based on a thermodynamic equilibrium.


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This unprecedented observation that the chemical equilibrium of the bicomponent layer formation kinetically and thermodynamically shifts in the presence of diluents led us to revisit the conventional chemical design of nanoprobes and their binding functions. Importantly, we found that the conjugation of functional ligands and diluents with a nanoparticle is controlled not only by their ratio, which is known to be a major determinant, but also by the conjugation time and temperature. The conjugation reaction of two ligand components can be far enhanced either at elevated temperatures or after an elongated time period, in contrast to the single-component conjugation whose chemical equilibrium is achieved almost immediately regardless of the conjugation temperature. Therefore, the effect of the diluent on achieving the chemical equilibrium that determines the final binding properties should be essentially and carefully considered for accurate analytical purposes. We further examined the binding properties of the bicomponent layers composed of functional DNA and thiolated oligonucleotide diluents (HS-X20, X = A, T, and C). Unlike the non-charged PEG, oligonucleotide diluents are as negatively charged as the functional DNA, and thus, the DNA/X20-AuNPs exhibit the same total negative charge as that of the DNA-AuNPs. This experimental design allows to precisely examine the role of the diluents without the reduction of the interparticle repulsion, which occurred in the case of PEG.56 In order to prepare the DNA/X20-AuNPs, we combined the AuNPs with DNA (S1 or S2) and HS-X20 at a 1:1 ratio, and allowed them to conjugate over various time periods (1 h to 1 week). Interestingly, the Tms of DNA/A20-AuNPs and DNA/T20-AuNPs almost did not change regardless of the conjugation time (Figure 4), indicating that the conjugation equilibrium was achieved as soon as the ligands and AuNPs were combined. This result indicates that the conjugation of both the functional DNA and diluent HS-X20 to the AuNPs was dramatically accelerated in the presence of Na+



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owing to the electrostatic attraction between Na+ and the negatively charged HS-X20 during the bicomponent layer formation. In fact, at a very low concentration (0.01 M) of Na+, the conjugation between DNA and AuNPs required more than ten days to reach completion, indicating the importance of Na+ for enhanced kinetics (Figure S4, see Supporting Information). Such an accelerated conjugation was hardly observed with non-charged PEG (Figures 2C and 2D), since the conjugation kinetics was mainly governed by the steric hindrance rather than electrostatic interactions (Scheme 1).

Figure 4. Plot showing the Tms of the hybridized DNA/X20-AuNPs (X = A, T, and C) that were conjugated over various time periods (1 h to 1 week). The x-axis is drawn to a log scale.


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Scheme 2. Scheme illustrating the conjugation time-dependent binding properties of the DNA /C20-AuNPs. As the conjugation time increases, the binding strength decreases owing to the increasing i-motif formation of C20 on the AuNPs with free HS-C20.

Surprisingly, we observed the most dramatic change in Tm from 76.6 to 53.2 oC (ΔTm = 23.4 oC) with HS-C20. The unexpectedly high initial Tm of the DNA/C20-AuNPs that were conjugated over a shorter conjugation time is due to their additional binding based on the i-motif formation.57-60 Although the i-motif formation is favored under acidic conditions, it is also known to possibly occur under neutral and even slightly alkaline conditions,61-62 suggesting that our results observed at pH 7.4 are reasonable. As the conjugation time increases, however, the C20 strands on the AuNP surface become blocked by extra HS-C20 via i-motif formation,



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resulting in the weakening of the duplex stability and a Tm decrease to 53.2 oC which is comparable with the Tm of the hybridized DNA-AuNPs (S1 and S2) (Scheme 2). On the other hand, the DNA/A20-AuNPs and DNA/T20-AuNPs exhibited almost the same Tms regardless of the conjugation time, indicating that the on-particle bicomponent layers composed of only oligonucleotides are constructed and equilibrated without delay.

Figure 5. Absorbance changes of the mixtures containing various types of nanoprobes (DNA:PEG = 100:0, 80:20, 60:40, and 0:100) after the catalytic reduction of 4-nitrophenol as a function of time. The mixtures exhibiting different colors at 9 min (540 s) are shown in the inset.


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Although nanoparticles are highly attractive as heterogeneous catalysts, there are two issues that should be considered for their reliable catalytic functionality: (1) stability of the nanoparticles and (2) accessibility to the nanoparticle surface. Because the nanoparticle catalytic activity takes place on the nanoparticle surface, a stable dispersion of the nanoparticles in solution and a resultant large surface area are highly important for their catalytic efficiency. Ironically, the ligand layer formation on the nanoparticle surface is essential for the nanoparticle stabilization, but at the same time it also hinders the substrate molecules from approaching the nanoparticle surface where the actual catalytic reaction occurs.63 The density, charge, hydrophilicity, and length of the surface ligands, and their ability to form secondary bonding with the substrates are all important determinants with respect to the aforementioned two issues. In particular, the bicomponent layer formation on the nanoparticles aids in easy control of these determinants, and thus, enhances the catalytic efficiency of the nanoparticle catalysts. To investigate how the bicomponent layer can control the accessibility of the substrates to the nanoparticle surface, we chose 4-nitrophenol as substrate, because it can be catalytically reduced to 4-aminophenol with a concomitant color change from yellow to colorless in the presence of AuNPs. The AuNPs were coated with bicomponent layers composed of DNA and PEG at various ratios (DNA:PEG = 100:0, 80:20, 60:40, and 0:100). These three different DNA proportions (100, 80, and 60 mol%) were selected based on our results illustrated in Figure 1, according to which the DNA strands were determined to be functional over 40 mol% of the ligand mixture. As demonstrated in Figure 5, the yellow-to-colorless change occurred more rapidly when the proportion of DNA was lower. For example, the AuNPs conjugated with 0% DNA completely reduced 4-nitrophenol at 350 sec, while those with 100% DNA at 650 sec. The difference in time required for the completion of the reduction is directly associated with the



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accessibility of the 4-nitrophenol molecules to the AuNP surface, and can be attributed to the intermittent hydrogen bonding formation of 4-nitrophenol with the nucleobases, which obstructs its transport into the AuNP. This observation indicates that the bicomponent layers on a nanoparticle surface can enhance the kinetics of a catalytic reaction by accelerating the access of the substrate ahead of the reaction. In addition to the target recognition properties and particle stabilization capability, the enhanced catalytic functionality granted by the interfacial bicomponent layers could be particularly important for the development of nanoprobes, as demonstrated in various types of biodetection assays.64-66 For example, we previously developed a highly sensitive and selective colorimetric protein detection system based on the catalytic properties of AuNPs, where the time until the color change gradually increased as the target protein concentration increased.64 In this scheme, however, the assay time for the target protein at only 100 nM was over 4 h, somewhat insufficient for practical applications. The formation of bicomponent layers composed of functional DNA for the protein binding and diluent PEG on nanoparticle probes is promising for reducing the assay time for rapid detection. We finally synthesized two types of nanoparticle probes to conduct a colorimetric assay for the detection of the tumor suppressor gene BRCA1.67 The nanoparticle probes were synthesized by conjugating two batches of AuNPs with two monothiol probe sequences BRCA1 3´ and BRCA1 5´, respectively, at 25 oC for 9 h. Each probe sequence was complementary to one of the two halves of the target sequence (BRCA1-target), which represents a typical strategy to design probe sequences in a binding-based detection scheme. Unexpectedly, however, a significant red-to-purple color change and a spectral broadening with an increase in extinction at longer wavelengths in the UV-vis spectrum occurred after the DNA-AuNP conjugation. These spectral changes indicate the instability of the nanoprobes and their resultant aggregation, even in


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the absence of the target and complementary probes (Figure 6A). When the nanoprobes were synthesized under different conjugation conditions (at 15 oC and at 25 oC for 9 h, and at 25 oC for 1 h), they eventually became unstable and aggregated. This abnormal aggregation was due to the inevitable self-complementarity of BRCA1 3´, as illustrated in Figure 6B. In fact, BRCA1 3´ was designed to employ a spacer composed of 10 nucleotides between the monothiol anchoring group and the target-binding probe sequence to enhance the binding properties and the probe stability.50, 68 In particular, polyadenine (polyA), the most common type of spacer, was chosen because of its high affinity for the gold surfaces, which was sufficient to more stably conjugate thiolated and even non-thiolated DNA sequences to AuNPs.32,

68-72

Unfortunately, however,

polyA has a higher chance to be self-complementary to the T-rich target-binding portion of BRCA1 3´, which would be commonly problematic when A-rich sequences are targeted. For this reason, we schemed out a new nanoprobe design employing a “diluent” to prevent the sequencedependent self-assembly of nanoprobes. As a preliminary examination, we co-functionalized AuNPs with BRCA1 3´ and HS-PEG in 50:50 ratio, which interestingly resulted in very well dispersed stable nanoprobes (Figure 6A, BRCA1 3´ + PEG). This result suggests that the coconjugation of AuNPs with both probe sequences and HS-PEG could be a general strategy to synthesize nanoprobes with probe sequences having potential self-complementary issues.



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Figure 6. (A) UV-vis spectra of the DNA-AuNP and DNA/PEG-AuNP nanoprobes prepared under various conjugation conditions. “Control” is the DNA-free unconjugated AuNPs. (B) Illustration of self-complementarity of BRCA1 3´ and its AuNP conjugates. (C) Colorimetric detection of BRCA1-target employing the DNA/PEG-AuNP nanoprobes synthesized at different conjugation temperatures with different target concentrations: (1) 15 oC, 10 nM, (2) 50 oC, 10 nM, (3) 15 oC, 100 nM, and (4) 50 oC, 100 nM. (D) Tms of the hybridized target with nanoprobes prepared under various conditions ((1)-(4)) described in Figure 6C. Condition (5) corresponds to 25 oC, 10 nM, 1 h.

In addition to the stability of the diluent-containing nanoprobes, we further examined their target-binding properties. In order to confirm that the AuNP-DNA/PEG conjugation


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conditions determine the kinetic properties of the target-binding events, we prepared two different sets of DNA/PEG-AuNP nanoprobe pairs (PEG:BRCA1 3´ = 50:50 and PEG:BRCA1 5´ = 50:50), and combined each set with the BRCA1-target at 10 and 100 nM, respectively. Specifically, the two sets of nanoprobes were synthesized by conjugating either BRCA1 3´ or 5´ with AuNPs at 15 and 50 oC to observe the effect of the conjugation temperature. The targetnanoprobe hybridization was monitored by the consequent assembly formation of the nanoprobes with a concomitant red-to-purple color change. Interestingly, the nanoprobes prepared at 50 oC hybridized with the BRCA1-target much more rapidly than those obtained at 15 oC, indicating a dramatic increase in the hybridization rate of the nanoprobes prepared at a higher temperature (Figure 6C). We also observed that a higher target concentration led to the faster hybridization, demonstrating that the nanoprobes synthesized with both DNA and a diluent exhibited [BRCA1-target]-dependent properties in a quantitative manner (Figure 6C). This quantitative nature of the nanoprobes was further analyzed from a thermodynamic standpoint by comparing their Tms (Figure 6D). For example, the Tms of the hybridized nanoprobes which were prepared at 50 oC appeared higher than those of the ones obtained at 15 oC, indicating that the nanoprobes prepared at a higher temperature exhibited stronger binding properties. The decrease of the conjugation time of the AuNP-DNA/PEG to 1 h also dramatically reduced the binding properties of the nanoprobes (Figure 6D). In summary, the novel nanoprobe design based on the construction of bicomponent layers on the AuNP surface significantly enhances the stability and consequent dispersity of the nanoprobes having self-complementarity. To maximize the kinetic and thermodynamic efficiency of the target-binding reactions, however, we have thoroughly investigated several parameters such as the conjugation time and temperature, providing a comprehensive understanding for the successful design of nanoprobes.



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4. Conclusion In this study, we demonstrated how the conditions employed for the construction of bicomponent ligand layers consisting of diluents and functional entities on nanoparticles can be varied to systematically control the thermodynamic and kinetic binding properties, as well as the catalytic functionality of the nanoparticle-ligand conjugates. Although the conventional stoichiometric approach simply relies on the static status before ligand conjugation, our investigations indicated that the conjugation duration and temperature can cause significant dynamic variations in the resultant bicomponent layers and their corresponding chemical and physical properties. Importantly, this comprehensive analysis of the bicomponent formation at the aqueous interface of metallic nanoparticles provides pivotal guidelines to efficiently design nanoprobes for catalytic and sensing applications as demonstrated in this work. In particular, we reported for the first time that polycytosine requires a specific attention when considered as a diluent owing to the possibility of i-motif formation. In addition to the spherical nanoparticles, oligonucleotides and PEG examined in this work, anisotropic nanoparticles and other types of ligands such as oligopeptides, carbohydrate polymers, and lipids are currently under investigation.22,

73-74

In particular, oligopeptides are expected to exhibit much more various

functions as diluents because their charges are easily controlled from negative to positive, and their hydrophilicity/hydrophobicity can be precisely adjusted by selecting component amino acids with different side chains. Moreover, their 3-dimentionally conformation on the nanoparticle may affect the binding properties of neighboring functional ligands in a favorable way.


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Supporting Information. Melting profiles and their first derivatives of DNA-AuNPs under various conditions in the absence and presence of additives. UV-vis spectra of DNA-AuNPs obtained over various conjugation periods at 0.01 M NaCl.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Phone: +82-2-3290-3267, Fax: +82-2-928-3584

Author Contributions

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the NRF funded by the Korean government, MSIP (NRF2016R1A5A1010148, NRF-2015M3A9D7031015, and NRF-2015R1C1A1A01053865). We thank Dr. Hionsuck Baik at the Korea Basic Science Institute (KBSI; Seoul, Republic of Korea) for their great help with the TEM work for the AuNPs (data not shown).

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TOC Graphic


Functionality of Nonfunctional Diluent Ligands within Bicomponent

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